Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Greuel, Bridget Kaytie
Master of Science - MSc
University of British Columbia
Alzheimer’s disease (AD) is the sixth leading cause of death worldwide and is characterized by chronic neuroinflammation. Microglia are the brain immunocytes, which normally support and protect neurons; however, they can become adversely activated contributing to onset and progression of neuroinflammation. In addition, defective phagocytic activity of microglia has been suggested to contribute to AD pathogenesis. Astrocytes maintain brain homeostasis but can also become adversely activated leading to the production of excessive pro-inflammatory cytokines and alteration of astrocytic phagocytic activity. Nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor (NLRP) 3 inflammasomes are intracellular protein complexes responsible for the activation and production of pro-inflammatory cytokines by immune cells, including microglia and astrocytes. The inflammasome inhibitors, MCC950, shikonin, and oridonin, have previously been shown to suppress inflammasome activation through several different mechanisms and reduce inflammatory activity of peripheral immune cells; however, the full spectrum of the effects of these drugs on the brain cells is still unknown. I hypothesized that MCC950, shikonin, and oridonin regulate a broad range of immune responses by microglia and astrocytes. Therefore, I studied the effects of these inflammasome inhibitors on murine and human microglia-like cells and human astrocytic cell lines that were challenged with established immune stimulants to induce pro-inflammatory responses. MCC950, shikonin, and oridonin decreased the secretion of nitric oxide and glutamate by murine microglia-like cells; however, none of the inflammasome inhibitors had a significant effect on their secretion of interleukin (IL)-1β or tumor necrosis factor-α (TNF). In human microglia-like cells, MCC950 significantly reduced the secretion of IL-1β, but not TNF, while shikonin and oridonin did not influence the secretion of either of these cytokines. Shikonin and oridonin reduced overall toxicity of microglia towards neuronal cells. In human astrocyte-like cells, MCC950, shikonin, and oridonin significantly reduced the secretion of IL-1β. Shikonin also significantly reduced the overall toxicity of astrocytes towards neuronal cells. Phagocytic activity of both resting and immune-stimulated microglia and astrocytes was upregulated by the inflammasome inhibitors, further supporting their potential neuroprotective effects. These results indicate that MCC950, shikonin, and oridonin should be investigated further as possible therapeutic agents for targeting neuroinflammation in AD.
Potential Chemical Inhibitors of Inflammasomes Modulate Microglial and Astrocytic Functions By Bridget Kaytie Greuel B.Sc., Colorado State University, 2019 A THESIS SUBMITTED In PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) April 2023 © Bridget Kaytie Greuel, 2023 ii Committee Recommendation Form The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: POTENTIAL CHEMICAL INHIBITORS OF INFLAMMASOMES MODULATE MICROGLIAL AND ASTROCYTIC FUNCTIONS submitted by Bridget K. Greuel in partial fulfillment of the requirements of the degree of Master of Science (M.Sc.) . Dr. Andis Klegeris, Department of Biology Supervisor Dr. Julien Gibon, Department of Biology Supervisory Committee Member Dr. Wesley Zandberg, Department of Chemistry Supervisory Committee Member Dr. Stephen McNeil, Department of Chemistry University Examiner iii Abstract Alzheimer’s disease (AD) is the sixth leading cause of death worldwide and is characterized by chronic neuroinflammation. Microglia are the brain immunocytes, which normally support and protect neurons; however, they can become adversely activated contributing to onset and progression of neuroinflammation. In addition, defective phagocytic activity of microglia has been suggested to contribute to AD pathogenesis. Astrocytes maintain brain homeostasis but can also become adversely activated leading to the production of excessive pro-inflammatory cytokines and alteration of astrocytic phagocytic activity. Nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor (NLRP) 3 inflammasomes are intracellular protein complexes responsible for the activation and production of pro-inflammatory cytokines by immune cells, including microglia and astrocytes. The inflammasome inhibitors, MCC950, shikonin, and oridonin, have previously been shown to suppress inflammasome activation through several different mechanisms and reduce inflammatory activity of peripheral immune cells; however, the full spectrum of the effects of these drugs on the brain cells is still unknown. I hypothesized that MCC950, shikonin, and oridonin regulate a broad range of immune responses by microglia and astrocytes. Therefore, I studied the effects of these inflammasome inhibitors on murine and human microglia-like cells and human astrocytic cell lines that were challenged with established immune stimulants to induce pro-inflammatory responses. MCC950, shikonin, and oridonin decreased the secretion of nitric oxide and glutamate by murine microglia-like cells; however, none of the inflammasome inhibitors had a significant effect on their secretion of interleukin (IL)-1 or tumor necrosis factor- (TNF). In human microglia-like cells, MCC950 significantly reduced the secretion of IL-1, but not TNF, while shikonin and oridonin did not influence the secretion of either of these cytokines. Shikonin and iv oridonin reduced overall toxicity of microglia towards neuronal cells. In human astrocyte-like cells, MCC950, shikonin, and oridonin significantly reduced the secretion of IL-1. Shikonin also significantly reduced the overall toxicity of astrocytes towards neuronal cells. Phagocytic activity of both resting and immune-stimulated microglia and astrocytes was upregulated by the inflammasome inhibitors, further supporting their potential neuroprotective effects. These results indicate that MCC950, shikonin, and oridonin should be investigated further as possible therapeutic agents for targeting neuroinflammation in AD. v Lay Summary Neurodegenerative disorders, including Alzheimer’s disease, progressively impair memory and other important brain functions over time. Current treatment options are available to improve quality of life; however, there is no cure for Alzheimer’s disease, which makes the search for one imperative. In this thesis work, I tested three inflammasome inhibitors; MCC950, shikonin, and oridonin, on brain cells to determine their ability to impede neurodegeneration. Inflammasomes are protein complexes that are found inside cells and, when activated, can lead to the release of pro-inflammatory molecules, and contribute to the pathogenesis of Alzheimer’s disease by causing neuronal death. I determined that these inhibitors reduced the secretion of pro-inflammatory molecules by glial cells, the supportive cells of the brain, and therefore could alleviate the progression of neurodegeneration. Since the research presented in this study is on the cellular and molecular level, these drugs should be further investigated in more complex models of Alzheimer’s disease. vi Preface The work described in this thesis was completed at the University of British Columbia, Okanagan campus, in the Laboratory of Cellular and Molecular Pharmacology. I was responsible for the design, experimental work, data analysis, and writing for the research reported in this thesis. I conducted all experiments excluding the blinded cell image analysis which was performed by Anna Bernath, Victora Robert-Gostlin, and Alyssa McElhinney. I co-authored the following publications during my MSc studies: 1. Murray, T. E., Wenzel, T.J., Simtchouk, S., Greuel, B. K., Gibon, J., & Klegeris, A. (2022). Extracellular cardiolipin modulates select immune functions of astrocytes in toll-like receptor (TLR) 4-dependent manner. Mediators of Inflammation, 9946439. https://doi.org/10.1155/2022/9946439 2. Wenzel, T. J., Haskey, N., Kwong, E., Greuel, B. K., Gates, E. J., Gibson, D.L., & Klegeris, A. (2022). Dietary fats modulate neuroinflammation in mucin 2 knock out mice model of spontaneous colitis. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease, 1868 (3), 166336. https://doi.org/10.1016/j.bbadis.2021.166336 vii Table of Contents Committee Recommendation Form ................................................................................................ ii Abstract .......................................................................................................................................... iii Lay Summary .................................................................................................................................. v Preface............................................................................................................................................ vi List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations .................................................................................................................... xv Acknowledgements ...................................................................................................................... xix Chapter 1: Introduction ................................................................................................................... 1 1.1 Background on Alzheimer's Disease (AD) ........................................................................... 1 1.2 Glia ........................................................................................................................................ 2 1.2.2 Astrocytes ....................................................................................................................... 3 1.2.3 Microglia ........................................................................................................................ 4 1.3 Nucleotide-Binding Domain Leucine-Rich Repeat and Pyrin Domain Containing Receptor (NLRP) 3 Inflammasome ............................................................................................................ 6 1.3.2 NLRP3 Inflammasome Assembly .................................................................................. 7 1.4 MCC950 .............................................................................................................................. 12 1.4.2 NLRP3 Interactions ...................................................................................................... 14 1.4.3 NEK7-NLRP3 Interaction ............................................................................................ 14 1.4.4 Phosphoinositide 3-kinase (PI3K) Interactions ............................................................ 15 1.4.5 Secondary Outcomes .................................................................................................... 15 1.4.6 Summary ....................................................................................................................... 16 1.5 Shikonin .............................................................................................................................. 16 1.5.2 NLRP3 and AIM2 Interactions..................................................................................... 19 1.5.3 ASC and Caspase-1 Inhibitor ....................................................................................... 19 1.5.4 NF-κB Inhibition .......................................................................................................... 19 1.5.5 PI3K Interaction ........................................................................................................... 20 1.5.6 Secondary Outcomes .................................................................................................... 20 1.5.7 Summary ....................................................................................................................... 21 1.6 Oridonin .............................................................................................................................. 21 1.6.2 NLRP3 Interactions ...................................................................................................... 24 1.6.3 NEK7-NLRP3 Interaction ............................................................................................ 24 1.6.4 ASC Oligomerization ................................................................................................... 25 viii 1.6.5 NF-κB Inhibition .......................................................................................................... 25 1.6.6 PI3K Interaction ........................................................................................................... 26 1.6.7 Secondary Outcomes .................................................................................................... 26 1.6.8 Summary ....................................................................................................................... 26 1.7 Modeling Brain Immune Cell Activation............................................................................ 28 1.8 Research Hypotheses and Objectives .................................................................................. 31 Chapter 2: Materials and Methods ................................................................................................ 34 2.1 Chemicals and Reagents...................................................................................................... 34 2.2 Equipment and Supplies ...................................................................................................... 34 2.3 Cell Lines ............................................................................................................................ 35 2.4 Cell Seeding ........................................................................................................................ 36 2.4.1 BV-2 Murine Microglia ................................................................................................ 36 2.4.2 NSC-34 Murine Neuronal Cells ................................................................................... 36 2.4.3 THP-1 Human Microglia-like Cells ............................................................................. 37 2.4.4 SH-SY5Y Human Neuronal Cells ................................................................................ 37 2.4.5 HT-22 Mouse Neuronal Cells....................................................................................... 37 2.4.6 HL-60 Human Microglia-like Cells ............................................................................. 38 2.4.7 U-118 MG Human Astrocytic Cells ............................................................................. 38 2.5 Assays.................................................................................................................................. 38 2.5.1 MTT Assay - Cell Viability .......................................................................................... 38 2.5.2 Griess Assay - Quantification of Nitric Oxide Secretion ............................................. 39 2.5.3 Supernatant Transfer Assay - Murine Glia-Mediated Toxicity Towards Neurons ...... 41 2.5.4 Supernatant Transfer Assay - Human Glia-like Cell Mediated Toxicity Towards Neurons .................................................................................................................................. 41 2.5.5 Enzyme-Linked Immunosorbent Assay (ELISA) - Quantification of IL-1 and TNF 42 2.5.6 Assay Measuring the Neurotransmitter (and Neurotoxin) Glutamate .......................... 43 2.5.7 Respiratory Burst Assay - Quantification of Reactive Oxygen Species ...................... 45 2.5.8 Phagocytosis Assay ...................................................................................................... 46 2.5.9 Statistical Analysis ....................................................................................................... 47 Chapter 3: Results ......................................................................................................................... 48 3.1 BV-2 Murine Microglia ...................................................................................................... 48 3.1.1 Determination of the Non-Toxic Concentrations of Inflammasome Inhibitors towards LPS-Stimulated BV-2 Murine Microglia .............................................................................. 48 3.1.2 Effects of Inflammasome Inhibitors on the Secretion of Nitric Oxide by LPS-Stimulated BV-2 Murine Microglia ...................................................................................... 50 3.1.3 Effects of Inflammasome Inhibitors on the Cytotoxicity of LPS-Stimulated BV-2 Murine Microglia towards NSC-34 or HT-22 Murine Neuronal Cells ................................. 52 ix 3.1.4 Effects of Inflammasome Inhibitors on the Secretion of IL-1 and TNF by LPS-Stimulated BV-2 Murine Microglia ...................................................................................... 56 3.1.5 Effects of Inflammasome Inhibitors on the Secretion of Glutamate by LPS-Stimulated BV-2 Murine Microglia ......................................................................................................... 59 3.1.6 Effects of Inflammasome Inhibitors on the Phagocytosis of Latex Beads by Unstimulated and LPS-Stimulated BV-2 Murine Microglia ................................................. 61 3.1.7 Effects of Oridonin in the Presence of PKD and JNK Inhibitors on the Secretion of Nitric Oxide by LPS-Stimulated BV-2 Murine Microglia .................................................... 63 3.2 Human Microglia-Like Cells .............................................................................................. 66 3.2.1 Effects of Inflammasome Inhibitors on the Cytotoxicity of LPS plus IFN--Stimulated THP-1 Human Microglia-Like Cells towards SH-SY5Y Human Neuronal Cells ................ 66 3.2.2 Effects of Inflammasome Inhibitors on the Secretion of IL-1 and TNF by LPS plus IFN--Stimulated THP-1 Human Microglia-Like Cells ........................................................ 70 3.2.3 Effects of Inflammasome Inhibitors on the Secretion of ROS by DMSO-Differentiated HL-60 Cells Primed with LPS and Stimulated with fMLP ................................................... 73 3.3 U-118 MG Human Astrocyte-like cells .............................................................................. 75 3.3.1 Effects of Inflammasome Inhibitors on LPS-Stimulated U-118 MG Human Astrocytic Cell-Mediated Cytotoxicity and IL-1 plus IFN--Stimulated U-118 MG Human Astrocytic Cell-Mediated Cytotoxicity towards SH-SY5Y Human Neuronal Cells .............................. 75 3.3.3 Effects of Inflammasome Inhibitors on the Secretion of Glutamate by LPS-Stimulated U-118 MG Human Astrocytic Cells ...................................................................................... 82 3.3.4 Effects of Inflammasome Inhibitors on the Phagocytosis of Latex Beads by Unstimulated and LPS-Stimulated U-118 MG Human Astrocytic Cells .............................. 84 3.4 Summary of Results ............................................................................................................ 86 Chapter 4: Discussion ................................................................................................................... 88 4.1 Cells ..................................................................................................................................... 88 4.2 Toxins .................................................................................................................................. 90 4.2.1. Nitric Oxide ................................................................................................................. 90 4.2.2. Glutamate..................................................................................................................... 90 4.2.3 Reactive Oxygen Species ............................................................................................. 92 4.2.4 Toxins Summary........................................................................................................... 93 4.3 Pro-inflammatory Cytokines ............................................................................................... 93 4.4 Cytotoxicity ......................................................................................................................... 97 4.5 Phagocytosis ........................................................................................................................ 99 4.7 PKD and JNK .................................................................................................................... 100 4.8 Summary ........................................................................................................................... 101 x Chapter 5: Conclusion................................................................................................................. 103 5.1 Hypotheses Addressed ...................................................................................................... 103 5.2 Limitations of the Study .................................................................................................... 104 5.3 Future Directions ............................................................................................................... 105 5.4 Research Significance ....................................................................................................... 106 References ................................................................................................................................... 108 xi List of Tables Table 1. All three drugs, MCC950, shikonin, and oridonin, are NLRP3 inflammasome inhibitors...................................................................................................................................... 28 Table 2. The effects of MCC950, shikonin, and oridonin on BV-2 murine microglia................ 86 Table 3. The effects of MCC950, shikonin, and oridonin on human microglia-like cells........... 87 Table 4. The effects of MCC950, shikonin, and oridonin on U-118 MG human astrocytic-like cells............................................................................................................................................... 87 xii List of Figures Figure 1. Microglia and astrocytes are activated by stimuli such as LPS and ATP....................... 6 Figure 2. Simplified assembly of the NLRP3 inflammasome complex from the four main components: NEK7, NLRP3, ASC, and pro-caspase-1.................................................................. 9 Figure 3. The canonical NLRP3 inflammasome pathway and the sites of action for the three inflammasome inhibitors: MCC950, shikonin, and oridonin....................................................... 10 Figure 4. MCC950 chemical structure.......................................................................................... 12 Figure 5. Shikonin chemical structure.......................................................................................... 17 Figure 6. Oridonin chemical structure.......................................................................................... 22 Figure 7. Toxicity of inflammasome inhibitors towards LPS-stimulated BV-2 murine microglia....................................................................................................................................... 49 Figure 8. Effects of inflammasome inhibitors on nitric oxide secretion by LPS-stimulated BV-2 murine microglia........................................................................................................................... 51 Figure 9. Effects of inflammasome inhibitors on cytotoxicity of LPS-stimulated BV-2 murine microglia cells towards NSC-34 murine neuronal cells............................................................... 54 Figure 10. Effects of inflammasome inhibitors on cytotoxicity of LPS-stimulated BV-2 murine microglia cells towards HT-22 murine neuronal cells.................................................................. 55 Figure 11. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS-stimulated BV-2 murine microglia........................................................................................................................... 57 Figure 12. Effects of inflammasome inhibitors on the secretion of TNF by LPS-stimulated BV-2 murine microglia........................................................................................................................... 58 Figure 13. Effects of inflammasome inhibitors on the secretion of glutamate by LPS-stimulated BV-2 murine microglia................................................................................................................. 60 xiii Figure 14. Effects of inflammasome inhibitors on the phagocytosis of latex beads by unstimulated and LPS-stimulated BV-2 murine microglia........................................................... 62 Figure 15. Effects of oridonin in the presence of PKD and JNK inhibitors on nitric oxide secretion by LPS-stimulated BV-2 murine microglia................................................................... 65 Figure 16. Effects of inflammasome inhibitors on the cytotoxicity of LPS plus IFN--stimulated THP-1 human microglia-like cells towards SH-SY5Y human neuronal cells............................. 68 Figure 17. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS plus IFN--stimulated THP-1 human microglia-like cells.............................................................................. 71 Figure 18. Effects of inflammasome inhibitors on the secretion of TNF by LPS plus IFN--stimulated THP-1 human microglia-like cells.............................................................................. 72 Figure 19. Effects of inflammasome inhibitors on the secretion of ROS by DMSO-differentiated HL-60 cells primed with LPS and stimulated with fMLP............................................................ 74 Figure 20. Effects of inflammasome inhibitors on the cytotoxicity of LPS-stimulated U-118 MG human astrocytic cells towards SH-SY5Y human neuronal cells................................................ 77 Figure 21. Effects of inflammasome inhibitors on the cytotoxicity of IL-1 plus IFN--stimulated U-118 MG human astrocytic cells towards SH-SY5Y human neuronal cells.............................. 78 Figure 22. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS-stimulated U-118 MG human astrocytic cells.................................................................................................... 80 Figure 23. Effects of inflammasome inhibitors on the secretion of TNF by LPS-stimulated U-118 MG human astrocytic cells........................................................................................................... 81 Figure 24. Effects of inflammasome inhibitors on the secretion of glutamate by LPS-stimulated U-118 MG human astrocytic cells................................................................................................ 83 xiv Figure 25. Effects of inflammasome inhibitors on the phagocytosis of latex beads by unstimulated and LPS-stimulated U-118 MG human astrocytic cells.......................................... 85 xv List of Abbreviations A Amyloid-beta AD Alzheimer’s disease AIM Absent in melanoma Akt Protein Kinase B ANOVA Analysis of variance APP/PS1 Double transgenic mice expressing amyloid precursor protein/chimeric mouse ASC Apoptosis-associated speck-like proteins containing caspase recruitment domains -NAD Beta-nicotinamide adenine dinucleotide hydrate BBB Blood-brain barrier BMDM Bone-marrow derived macrophages BSA Bovine serum albumin C3HeB/FeJ Mouse model of Wilson’s disease C57BL/6J C57 black 6 CARD Carboxy-terminal caspase recruitment domain CBS Calf bovine serum CHL Chemiluminescence CIITA MHC class II transcription activator CNS Central nervous system COX Cyclooxygenase Cys Cysteine DAMP Damage-associated molecular pattern DMEM/F12 Dulbecco’s modified eagle medium F12/Ham DMF Dimethylformamide xvi DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FDA Food and Drug Administration FITC Fluorescein isothiocyanate fMLP N-formylmethionine-leucyl-phenylalanine GFAP Glial fibrillary acidic protein GLT Glutamate transporter HET-E Incompatibility locus protein from Podospora anserina HK Human kidney IFN Interferon IB Inhibitor of nuclear factor-kappa B IL Interleukin JNK c-Jun terminal kinase KD Dissociation constant LOD Limit of detection LPS Lipopolysaccharide LRR Leucine-rich repeat MAPK Mitogen-activated protein kinases MCP Monocyte chemoattractant protein MS Multiple sclerosis MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide xvii NACHT NAIP, CIITA, HET-E, and TP1 NADPH Nicotinamide adenine dinucleotide phosphate NAIP Neuronal apoptosis inhibitory protein NEK NIMA-related kinase NF-B Nuclear factor-kappaB NLRC Nucleotide-binding domain leucine-rich repeat family caspase activation and recruitment domain containing NLRP Nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor OD Optical density PBS Phosphate-buffered saline PD Parkinson’s disease PI3K Phosphoinositide 3-kinase PKD Protein kinase D PMA Phorbol-12-myristate-13-acetate PPAR Peroxisome proliferator-activated receptor PYD Pyrin domain RBC Red blood cell ROS Reactive oxygen species RT-qPCR Quantitative reverse transcription polymerase chain reaction SD Standard deviation SEM Standard error of the mean Ser Serine xviii SLS Sodium lauryl sulfate TLR Toll-like receptor TMB 3,3′,5,5′-tetramethylbenzidine TNF Tumor necrosis factor-alpha TP Telomerase-associated protein WT Wild-type xix Acknowledgements Firstly, I would like to express my gratitude to the University of British Columbia, I am deeply thankful for this opportunity and the ability to work alongside some incredible people. I would like to thank Dr. Klegeris for his guidance and support throughout this thesis. He has taught me more than I could have ever imagined and has made a significant impact on my growth as a scientist. I will always be grateful for his supervision and guidance throughout my master’s. I also want to thank Dr. Zandberg for his expertise into the field of chemistry and for ensuring I maintain focus on the big picture throughout my project. Thank you to Dr. Gibon for teaching me advanced microscopy and for his very informed and thoughtful suggestions in committee meetings. A special thank you to Dr. Gibon and the Barker Lab for allowing me to use their microscope to complete experiments. Second, thank you to my graduate family; this would not have been possible without your support including the many late-night discussions. Thank you to my colleagues at the Laboratory of Cellular and Molecular Pharmacology for shaping me as a scientist both inside and out of the lab. I am grateful to Taryn and Seamus for teaching me everything from experiments to data analysis and more. I also want to thank Christy, Alyssa, Dylan, and Zainab for constantly reviewing my written work and providing excellent suggestions. Thank you to Victoria, Anna, and Alyssa for their countless hours of cell image analysis, and to Ishvin for his ongoing support. Lastly, I would like to thank my family. To my Aunt Sue and Uncle Greg, thank you for taking me in when I moved to the Okanagan and for creating wonderful memories with me every weekend. I am deeply grateful for your kindness and generosity. To my partner, thank you for your encouragement and support throughout my journey, from applying to graduate school to completing my thesis. I am grateful for my grandparents’ belief in me and my ability to achieve this challenging degree. Most of all, I want to thank my parents for their unwavering support. They have always been my biggest cheerleaders, and I will always value their advice. I would not be where I am today without their inspiration. xx For my family 1 Chapter 1: Introduction 1.1 Background on Alzheimer's Disease (AD) Alzheimer’s disease (AD) affects an estimated 44 million people worldwide and, despite decades of research, there is still no cure. There is a lack of knowledge surrounding this disease due to its overwhelming complexity; therefore, viable treatments for AD remain elusive. Patients are limited to a few available drugs, which usually only provide a temporary and moderate improvement in symptoms. Approximately 25 percent of those affected by AD die from disease-related complications, including malnutrition and dehydration, making it the sixth leading cause of death in North America (Alzheimer’s Disease Facts and Figures, 2022). Other AD symptoms include memory loss, impaired cognitive function, reduced attention span, and increased anxiety and depression (Weller & Budson, 2018). AD patients living with severe disease require 24-hour care which can overwhelm their families and the health care systems. As the number of AD patients increases due to the growth in the aging population, so does the toll on health care systems; thus, finding effective treatments is imperative to improving the quality of life of both the patients and the people around them. The central nervous system (CNS) is composed of two main types of cells: neurons and glia, the latter of which are non-neuronal, supportive, cells. Neurons are considered the fundamental units of the brain and function in, for example, sensory transmission and information processing (Ribrault et al., 2011). Transport of signals by neurons is crucial for proper cognitive function, which includes functional memory, emotion, and thought. Glial cells, which include microglia, astrocytes, and oligodendrocytes, are more numerous than neurons and perform a wide range of functions in the CNS, including providing neuroimmune support and maintaining homeostasis (Hanisch & Kettenmann, 2007). Inflammatory processes in the CNS 2 (also known as neuroinflammation) contribute to the pathogenesis of neurodegenerative diseases, such as AD, where glial cells are overactivated and viable neurons deteriorate (Rauf et al., 2022). There are two microscopically visible hallmarks of AD: extracellular amyloid-beta (A) peptide deposits and intracellular neurofibrillary tangles composed of the microtubule binding protein, tau (Hanisch & Kettenmann, 2007). A can directly cause neuronal cell death and induce the release of neurotoxins from glia, such as pro-inflammatory cytokines and chemokines (Serrano-Pozo et al., 2011). It is well known that as part of their protective function, microglia phagocytose (i.e., engulf) and clear A in healthy brains. However, in the brains of AD patients, phagocytosis is impaired; this drastically reduces A clearance, leading to the accumulation of amyloid and contributing to increased neuroinflammation (Fu et al., 2014). Although amyloid deposition has been studied extensively, knowledge of the cellular events involving glia that are responsible for neurodegeneration is limited. 1.2 Glia First described in 1913 by Cajal, astrocytes are star-shaped cells that function to maintain an optimal extracellular environment in the brain (Garwood et al., 2011; Cajal, 1913). Discovered by Virchow in 1856, microglia play a critical role in immune support of the CNS (Garwood et al., 2011; Virchow, 1856). Glial cells become overactivated leading to elevated production of pro-inflammatory cytokines, which subsequently interfere with the normal function of neurons (Acosta et al., 2017; Araki et al., 2021). Because neuronal degeneration can be induced by glial cells, including microglia and astrocytes, they are currently being explored as potential targets for AD therapies. Glial cells release pro-inflammatory cytokines, such as interleukin (IL)-1 and tumor necrosis factor- (TNF), as well as produce toxins, such as reactive oxygen species (ROS), nitric 3 oxide (NO), and glutamate. IL-1 is a key mediator of inflammation and higher expression of this pro-inflammatory cytokine can be associated with chronic glial activation (Dinarello, 2000). TNF plays a role in the immune response by aiding in cell survival and proliferation, but it can also contribute to tissue destruction and loss of function under chronic inflammation. ROS are a product of mitochondrial oxidative metabolism and play an important role in cell signaling and survival; however, high levels of ROS have been implicated in neurodegeneration as they damage DNA, lipids, and proteins (Shields et al., 2021). Respiratory burst is the rapid release of ROS from immune cells, such as microglia. Under normal conditions, NO is an important signaling molecule in the defense against pathogens; however, NO is considered a pro-inflammatory molecule in neurodegeneration once large amounts have been released, which can cause tissue injury (Sharma et al., 2007). Lastly, glutamate is a neurotransmitter both sequestered and released by glial cells that can cause neuronal dysfunction and degeneration when elevated (Lau & Tymianski, 2010). Glial phagocytosis plays an important role in maintaining the extracellular environment by clearing debris, defective synapses, and abnormal proteins, such as A aggregates (Jung & Chung, 2018). Glial cells recognize the target particle by employing their receptors such as, TLR4, complement receptors, and scavenger receptors (Fu et al., 2012; Tremblay et al., 2019). After recognition, the cell engulfs the particle, enclosing it in a phagosome to be degraded by a lysosome (Lancaster et al., 2021). In AD, phagocytosis may be impaired leading to accumulation of A or other toxins contributing to neuroinflammation (Fu et al., 2014). 1.2.2 Astrocytes In a healthy state, astrocytes maintain the integrity of the blood-brain barrier (BBB), play a role in ion homeostasis, and protect neurons from toxins (Araki et al., 2021). Additionally, 4 studies have shown that astrocytes can facilitate inflammation through the release of cytokines (Iravani et al., 2012; Li et al., 2009). Following exposure to toxins or stimuli, such as A or ATP, astrocytes exhibit elevated secretion of cytokines, thereby contributing to neuroinflammatory diseases (Acosta et al., 2017; Garwood et al., 2011). Astrocytes have been considered as a potential therapeutic target for treatment of neurodegenerative diseases due to their contribution to pathogenesis. Aβ inhibits glutamate clearance in rat brains, and excess glutamate can lead to neuronal death by excitotoxicity. However, astrocytes have been shown to maintain glutamate homeostasis, leading to cell survival (Fernández-Tomé et al., 2004; Harris et al., 1996). Additionally, cultured astrocytes have also shown an increase in release of pro-inflammatory cytokines, such as IL-1β, in the presence of Aβ (Hu et al., 1998). The secretion of such factors is a major mechanism of neuronal damage in AD and inhibiting this process could be an effective therapy. Furthermore, astrocytes have been shown to engulf and degrade Aβ, thus preventing the accumulation of Aβ (Iliff et al., 2012; Jones et al., 2013; Pihlaja et al., 2008); ultimately, therapies could be designed to upregulate astrocytic phagocytosis. 1.2.3 Microglia Over the last several decades, microglia have been a major focus in research on neurodegenerative diseases. Microglia are the resident immune cells of the CNS and are responsible for releasing immunomodulatory factors upon stimulation and clearing debris in the brain through phagocytosis (Condello et al., 2015). They also regulate synaptic function; therefore, dysfunctional microglia contribute to neuronal damage, increase synaptic loss, and promote neurodegeneration (Harry & Kraft, 2008). Microglia can become activated following exposure to the bacterial endotoxin, lipopolysaccharide (LPS), damage-associated molecular 5 patterns (DAMPs) such as ATP, or pro-inflammatory cytokines, including IL-18 and interferon (IFN)-. LPS, which is present on the outer membrane of gram-negative bacteria, can be elevated in blood of patients affected by neurodegenerative diseases (Brown, 2019). When injected intravenously into mice, LPS induces microglial activation, resulting in memory deficits and degeneration of neurons (Brown, 2019). This is considered the endotoxin hypothesis which suggests that neurodegeneration can be caused or exasperated by endotoxins. LPS activates the immune response by stimulating toll-like receptor (TLR) 4 leading to the release of pro-inflammatory cytokines (Lu et al., 2008). IFN- has been shown to upregulate TNF in THP-1 microglia-like cells by activating the IFN- receptor (Inagaki et al., 2002). Microglial activation leads to the release of pro-inflammatory cytokines, such as IL-1β and TNF, as well as the production of ROS and NO which ultimately contribute to neurodegeneration (Fig. 1). Reduction of microglial overexpression of pro-inflammatory cytokines, such as IL-1β and TNF, through pharmacological intervention could prove to be beneficial in delaying AD progression. Phagocytosis also plays an important role in microglia-mediated neuroprotection and is effective in the clearance of amyloid (Sierra et al., 2010). As such, upregulation of microglial phagocytosis is a potential treatment strategy for neurodegenerative diseases. The overall objective of this thesis work is to search for novel molecules that inhibit the production of toxins by astrocytes and microglia but enhance their phagocytic activity. 6 Figure 1. Microglia and astrocytes are activated by stimuli such as LPS and ATP. Reactive glial cells release ROS, glutamate, pro-inflammatory cytokines (such as IL-1β), and NO, all of which promote neurodegeneration. Figure was made with BioRender. 1.3 Nucleotide-Binding Domain Leucine-Rich Repeat and Pyrin Domain Containing Receptor (NLRP) 3 Inflammasome First discovered by Jurg Tschopp in 2002, the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor (NLRP) 3 inflammasome plays a key role in AD pathogenesis given that its overactivation leads to chronic neuroinflammation. NLRP3 is an intracellular protein complex in immune cells, which contributes to neuroinflammation by the production of pro-inflammatory cytokines. When immune cells are exposed to pathogens, the NLRP3 inflammasome is activated and initiates a cascade of biochemical reactions, leading to the release of pro-inflammatory cytokines to eliminate the pathogens and infection (Kim & Jo, 2013). Overactivation of NLRP3 is associated with the increased severity of AD (Saresella et al., 2016). Other inflammasomes, such as absent in melanoma (AIM) 2, NLRP2, and NLRP1, have not been well studied in AD. Expression of these complexes varies between the different cell types of the brain, and different species. For instance, neurons, but not microglia nor astrocytes, 7 have been shown to express AIM2 (Chiarini et al., 2020; Freeman et al., 2017). Astrocytes express other inflammasomes, including NLR family caspase activation and recruitment domain containing (NLRC) 4, NLRP3, and NLRP2, whereas microglia express NLRP3 and NLRP1 (Chiarini et al., 2020). In addition, AD brains express NLRP3 but not NLRP2 or AIM2, whereas murine models of AD express all three inflammasomes (Chiarini et al., 2020). In the absence of neuroinflammation, the NLRP3 inflammasome complex is not present within glial cells. However, when immune cells are exposed to A or other stimuli, the inflammasome assembles and in doing so becomes activated (Halle et al., 2008). Ultimately, A-induced NLRP3 inflammasome priming and activation in microglia is one of the pathways that triggers neuroinflammation (Saresella et al., 2016; Heneka et al., 2013; Voet et al., 2019). Accordingly, previous studies using AD model mice have shown that NLRP3 deficiency can have protective effects against this neurodegenerative disease, by preventing the harmful release of excess cytokines (Heneka et al., 2013). Heneka et al. (2013) also found that NLRP3 activation inhibited microglial phagocytosis. Since NLRP3 deficiency led to decreased accumulation of A, inhibiting NLRP3 activity could restore or enhance phagocytic activity of microglia and facilitate A clearance. Therefore, the contributions of the NLRP3 inflammasome to neurodegeneration and AD are substantial, and inhibition of the inflammasome complex formation or its activity would be beneficial. 1.3.2 NLRP3 Inflammasome Assembly The intracellular NLRP3 inflammasome consists of four components: NLRP3 proteins, also known as sensor proteins; apoptosis-associated speck-like proteins containing caspase recruitment domains (ASC), also called adaptor proteins; pro-caspase-1, known as the downstream effector; and NIMA-related kinase (NEK) 7 (Fig. 2) (Saresella et al., 2016; Strowig 8 et al., 2012). The NLRP3 protein consists of the carboxy-terminal leucine-rich repeat (LRR) domain, the central NACHT domain [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)], and the amino-terminal pyrin domain (PYD). The ATPase activity of the NACHT domain is essential for NLRP3 association with ASC, and pro-caspase-1 (Duncan et al., 2007). Specifically, the Walker B motif of the NACHT domain hydrolyzes ATP to ADP and inorganic phosphate, inducing a conformational change in NLRP3 tertiary structure. The ASC component of the NLRP3 inflammasome consists of a PYD, which is analogous to that of the NLRP3 PYD domain, and a carboxy-terminal caspase recruitment domain (CARD). In contrast, caspase-1 consists of an amino-terminal CARD in addition to a large subunit, and a carboxy-terminal small subunit. NLRP3 oligomerization is due to the binding of several NACHT domains to NLRP3 proteins, resulting in ASC recruitment mediated by the interaction between PYD domains of the NLRP3 and ASC components (Lu et al., 2014). ASC/NLRP3 complexes recruit pro-caspase-1 by CARD-CARD interactions. Pro-caspase-1 then self-cleaves and matures into caspase-1. The PYD-PYD and CARD-CARD interactions of NLRP3, ASC, and caspase-1 hold the NLRP3 inflammasome complex together. NEK7 is another component of NLRP3 inflammasome that is essential for inflammasome assembly (Schmid-Burgk et al., 2016). This serine-threonine kinase oligomerizes with the activated NLRP3 by binding to the LRR domain of the NLRP3 protein (Chen et al., 2019). This interaction is necessary for NLRP3-ASC complex formation, ASC oligomerization (also called speck formation), and pro-caspase-1 maturation (Chen et al., 2019). 9 Figure 2. Simplified assembly of the NLRP3 inflammasome complex from the four main components: NEK7 (61 kDa), NLRP3 (118 kDa), ASC (22 kDa), and pro-caspase-1 (45 kDa). Figure was made with BioRender. Activated inflammasomes initiate caspase-1 activity, which leads to the cleavage of pro-IL-1 and pro-IL-18 into mature IL-1 and IL-18, respectively (Yang et al., 2019). This process, shown in Fig. 3, is described as the canonical pathway for NLRP3 inflammasome activation. In general, the inflammasome must first be primed for activation, for instance, through TLR4 by LPS (Liu et al., 2020). A and ROS have been shown to directly activate the NLRP3 inflammasome in macrophages (Nakanishi et al., 2018). The canonical pathway responds to TLR4 activation by LPS, triggering nuclear factor (NF)-κB activity and the oligomerization of NLRP3 (Pellegrini et al., 2017). In addition to the canonical pathway, an alternative and less common, non-canonical pathway for NLRP3 activation exists. In the non-canonical pathway, 10 activation of NF-κB induces caspase-11 activity, further promoting the inflammasome pathway. IL-1 and IL-18 are key products in both the canonical and the non-canonical inflammasome-mediated neuroinflammation. Figure 3. The canonical NLRP3 inflammasome pathway and the sites of action for the three inflammasome inhibitors: MCC950, shikonin, and oridonin. Figure was made with BioRender. Phosphorylation events are necessary for inflammasome activation, for example, protein kinase D (PKD) and c-Jun terminal kinase (JNK) phosphorylate serine (Ser)295 and Ser198, respectively, of the NLRP3 protein (Sandall & MacDonald, 2019). The phosphorylation of Ser295 by PKD is an important regulator of ATPase activity of the NLRP3 protein (Danot et al., 2009; Sandall & MacDonald, 2019), while phosphorylation of Ser198 by JNK activates the NLRP3 inflammasome by inducing a conformational change (Sandall & MacDonald, 2019; Song et al., 2017). As noted above, ATP binding is essential for NLRP3 association with ASC 11 and pro-caspase-1 (Danot et al., 2009; Duncan et al., 2007). Furthermore, SP600125, by inhibiting JNK, has been shown to inhibit inflammasome activation in LPS-stimulated murine and human bone marrow-derived macrophages (BMDM) (Song et al., 2017). Likewise, a study using rat primary microglia stimulated with uridine diphosphate demonstrated that PKD, and subsequently inflammasome activation, can be inhibited by CID755673 (Uesugi et al., 2012). Inflammasomes have previously been shown to contribute to the pathogenesis of neurodegenerative diseases, therefore, inflammasome inhibitors could serve as potential drugs used in the treatment of these pathologies. For example, in AD, NLRP3 activation in microglia has been shown to increase tau pathologies, A deposits, and neuronal toxicity (Ising et al., 2019; Li et al., 2003). In AD brains, microglia have increased caspase-1, causing the release of pro-inflammatory IL-1 (Burguillos et al., 2011). Venegas et al. (2017) hypothesize that the activation of NLRP3 in microglia leads to the release of ASC specks into the extracellular environment. ASC specks facilitate and maximize caspase-1-mediated cleavage of IL-1, which further contributes to the spread of A (Nagar et al., 2021). Phagocytosis of A is another important mechanism to consider when studying inflammasome inhibition. A previous study has determined that NLRP3 or caspase-1 deficiency leads to increased microglial phagocytosis and A clearance (Heneka et al., 2013). Although we would not want to eliminate or completely block the NLRP3 inflammasome due to its role in maintaining immune homeostasis, reducing its activity in AD would be advantageous. It is important to note that NLRP3 inflammasome inhibitors must have effects on its assembly or activity. Some of these inhibitors also suppress expression of inflammasome proteins. MCC950, shikonin, and oridonin are inflammasome inhibitors, and have previously 12 been shown to suppress NLRP3 inflammasome activation and reduce inflammatory activity of immune cells; however, their effects on microglia and astrocytes are still unclear. 1.4 MCC950 MCC950, or N-(1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide sodium salt, as shown in Fig. 4, is a potent and selective small-molecule NLRP3 inflammasome inhibitor that does not act on the other inflammasomes, for example, AIM2 or NLRP4. As one of the first drugs known to inhibit the NLRP3 inflammasome, it has also been shown to block ASC oligomerization and inhibit caspase-1 enzymatic activity (Coll et al., 2015; Dempsey et al., 2017). MCC950 has been previously studied in other inflammatory diseases, including multiple sclerosis (MS) and type 2 diabetes, and does not have known side effects in mouse models of these diseases (Hutton et al., 2016; Perera et al., 2018). Figure 4. MCC950 chemical structure (drawn by Dr. Stephen McNeil, UBC Okanagan, 2023). In the periphery, MCC950 has been shown to abate ischemia-reperfusion related disorders, tumor pathogenesis, and diabetes (Chen et al., 2018; Fauzia et al., 2018; Ward et al., 2019; Zhou et al., 2021). MCC950 decreased the secretion of IL-1, delayed tumor pathogenesis, and improved the anti-tumor immune response in both mouse models and human tissues of head 13 and neck squamous cell carcinoma (Chen et al., 2018). The NLRP3 inflammasome has been implicated in the pathogenesis of type 2 diabetes. In a study using murine tau knock-in model and wild type (WT) mice, MCC950 was shown to improve insulin sensitivity and glucose tolerance (Ward et al., 2019). This study shows that MCC950 partially recovers metabolic and inflammatory phenotypes in these model mice. In the CNS, MCC950 has been shown to attenuate inflammation. Dempsey et al. (2017) used the AD transgenic mouse model, APP/PS1, to determine that intraperitoneal injections of MCC950 attenuated the LPS-induced increase in IL-1. The study reported a MCC950-mediated decrease in intracellular colocalization of caspase-1 and ASC, as well as an increase in phagocytosis by primary murine microglia. Lučiūnaitė et al. (2020) found that MCC950 decreased IL-1 release by LPS-stimulated primary murine microglia; however, no changes in TNF protein secretion were detected. In a murine model of Parkinson’s disease (PD) with deleted microglial autophagy related 5 protein, the effects of MCC950 were found to be very similar to those observed in the AD mouse models (Cheng et al., 2020): MCC950 inhibited caspase-1 and inflammasome activation. MCC950 is yet to be studied in clinical trials. The effects of MCC950 on ROS and NO production, as well as microglial neurotoxicity, are yet to be investigated, while the available data for the effect of MCC950 on IL-1 and TNF secretion are conflicting (Danielski et al., 2020; Dempsey et al., 2017). MCC950 has been shown to block NLRP3 ATPase activity and NEK7, thus preventing the assembly of the NLRP3 inflammasome and downregulating its enzymatic activity, as shown in Fig. 3 (Chen et al., 2019). The final outcomes of MCC950 inhibitory activity include decreased levels of IL-1, IL-18, and caspase-1. 14 1.4.2 NLRP3 Interactions In 2015, Coll et al. found that MCC950 targeted the inflammasome assembly, but not the expression of NLRP3 protein or its downstream components, IL-1 and IL-18, in human embryonic kidney HEK293T cells primed with LPS and stimulated with ATP. This proved that MCC950 targeted the assembly of the inflammasome, but not the expression of its components. Coll et al. (2019) found that MCC950 interacted with the Walker B motif of the NACHT domain of the NLRP3 protein, which was extracted from HEK293T cells. The Walker B motif is involved in ATP hydrolysis and NLRP3 assembly, therefore, inhibition of this motif leads to decreased inflammasome assembly and function. Immunoprecipitation revealed that MCC950 interacts with both the active and inactive conformations of NLRP3, thereby blocking inflammasome assembly (Coll et al., 2019). Additionally, Coll et al. (2019) used surface plasmon resonance to determine that MCC950 bound directly to NLRP3 with high affinity (dissociation constant, KD = 224 nM). In primary rat microglia extracted from the hippocampus and cortex, MCC950 suppressed levels of NLRP3 and NLRP3-related proteins, such as IL-1, and IL-18 (Chang et al., 2020). The study found that MCC950 did not suppress pro-caspase-1 levels, but reduced cleaved caspase-1 activity; therefore, it likely suppresses the activation of caspase-1, a downstream effect of NLRP3 inhibition. In addition, Dong et al. (2021) determined that MCC950 reduces NLRP3, ASC, and caspase-1 activation in unstimulated primary murine microglia. 1.4.3 NEK7-NLRP3 Interaction NEK7 regulates NLRP3 by promoting conformational changes, which facilitate the activation of the NLRP3 inflammasome (He et al., 2016). According to Sharif et al. (2019), binding of NLRP3 to NEK7 is necessary to trigger a conformational change in the NLRP3 15 protein, resulting in assembly of the inflammasome. Zhang et al. (2017) used co-immunoprecipitation to determine that MCC950 inhibited inflammasome activation through direct binding to NEK7-NLRP3. In contrast, another study found that MCC950 did not inhibit NEK7 catalytic activity and therefore did not affect NEK7-NLRP3 binding (He et al., 2016). 1.4.4 Phosphoinositide 3-kinase (PI3K) Interactions MCC950 has previously been shown to inhibit the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway by reducing the phosphorylation of both PI3K and Akt in the liver of aged C57BL/6J mice (Marín-Aguilar et al., 2020). However, it is noteworthy that these inhibitory effects were observed only at high doses of MCC950, such as 20 mg/kg, which can be toxic. 1.4.5 Secondary Outcomes MCC950 has been shown to block ASC oligomerization, resulting in inhibition of caspase-1 enzymatic activity (Coll et al., 2015). In primary murine macrophages isolated from aortic tissue and challenged with angiotensin II, immunoblotting showed a decrease in NLRP3, active caspase-1, and IL-1 production in the presence of MCC950 (Ren et al., 2020). A study by Dempsey et al. (2017) found MCC950 inhibited the caspase-1 activation and IL-1 release in primary murine microglia from APP/PS1 mice. In agreement, Lučiūnaitė et al. (2020) determined that MCC950 inhibited IL-1 and IL-18 release by LPS-stimulated primary murine microglia; however, no changes in TNF protein secretion were detected. Lastly, in a C3HeB/FeJ mouse model of Wilson’s disease, a hereditary condition characterized by the accumulation of copper in the liver and brain, NLRP3 silencing RNA (siRNA) was utilized to demonstrate a reduction in caspase-1, IL-1, and IL-18 levels caused by lower expression of NLRP3 protein (Dong et al., 2021). 16 1.4.6 Summary Overall, direct binding of MCC950 to NLRP3 was demonstrated by Coll et al. (2019), which confirms that MCC950 is an inflammasome inhibitor. Further analysis of MCC950 and NLRP3 interactions in microglia has been completed on multiple occasions and provides further evidence for the direct binding of MCC950 to the NLRP3 inflammasome (Chang et al., 2020; Dong et al., 2021; Zhu et al., 2021). MCC950 may act by binding and blocking the NACHT domain of NLRP3, ASC oligomerization, and NEK7-NLRP3 interactions (Chang et al., 2020; Coll et al., 2015, 2019; Ren et al., 2020; Zhang et al., 2017; Zhu et al., 2021). The final outcomes of MCC950 inhibitory activity include reduced levels of IL-1 and IL-18, which are products of the NLRP3 inflammasome assembly/activation (Zhu et al., 2021). 1.5 Shikonin Shikonin, or (R)-5,8-dihydroxy-2-(1-hydroxy-4-methylpent-3-en-1-yl) naphthalene-1,4-dione (see Fig. 5) is isolated from the dried root of Lithospermum erythrorhizon and is considered an anti-inflammatory compound. Shikonin inhibits the NLRP3 and AIM2 inflammasomes in a similar manner. Shikonin is commonly found in ointments and herbal medicines available to treat inflammatory diseases such as gastritis. At low doses, it has no known side effects and is considered safe and effective (Wiench et al., 2012). Furthermore, it is used in cancer therapy given that at high doses it can trigger apoptosis and induce ROS production (Huang et al., 2014). Shikonin has also been studied in a clinical trial for treatment of buccal (within the mouth) mucosa lesions (Zagorodnyaya et al., 2013). This human trial indicated decreased pain and a reduction in morphological signs of lesions. 17 Figure 5. Shikonin chemical structure (drawn by Dr. Stephen McNeil, UBC Okanagan, 2023). In 15 cancer cell lines, notably HL-60 myelomonocytic leukemia cells, U937 leukemia cells, and MCF-7 breast carcinoma cells, shikonin induced apoptosis at high concentrations ranging from 0.6 to 2.3 M (Wiench et al., 2012). In addition, another cancer study found that shikonin triggered apoptosis and increased ROS production in human lens epithelial cells, HLE B-3 (Huang et al., 2014). A recent study on human nasopharyngeal carcinoma cell lines revealed that shikonin suppressed cell proliferation and induced cell apoptosis (Zhang et al., 2020). In agreement with previous studies, Zhang et al. (2020) observed an inhibitory effect of shikonin on the PI3K/Akt signaling pathway, which is upregulated by active inflammasomes (Giacoppo et al., 2017; Liu et al., 2021). In the CNS, shikonin has exhibited neuroprotective effects and decreased cytokine secretion. Shikonin has been shown to protect dopaminergic PC12 rat neuroblastic cells, used to model PD, from 6-hydroxydopamine-induced toxicity by inhibiting apoptosis (Esmaeilzadeh et al., 2013). Additionally, shikonin protects Kunming mice from cerebral ischemia-reperfusion injury (Wang et al., 2010). In primary neurons from the D-galactose-induced C57BL/6J mouse model of accelerated aging, intragastric administration of shikonin inhibited mitogen-activated 18 protein kinases (MAPK) and NF-κB signaling pathways, resulting in reduced secretion of inflammatory mediators, including TNF, IL-1, and IL-6 (Zhong et al., 2020). Shikonin was found to lower the expression of glial fibrillary acidic protein (GFAP) expression, a marker of astrocytic activation, in primary astrocytes from C57BL/6J mice (Zhong et al., 2020). In immortalized BMDMs from C57BL/6J mice, shikonin inhibited pro-IL-1 maturation to IL-1 (Zorman et al., 2016). Interestingly, when added before LPS-stimulation, shikonin inhibited pro-IL-1 in BMDMs and THP-1 monocytic cells; however, when shikonin was added after LPS-stimulation, it did not inhibit pro-IL-1. Shikonin derivatives, isobutyrylshikonin and isovalerylshikonin, were first studied at high concentrations to determine their toxicity and anti-inflammatory effects on BV-2 microglia. These derivatives were found to have an inhibitory effect on the secretion of NO and ROS by LPS-stimulated BV-2 microglia (Nam et al., 2008). Furthermore, isobutyrylshikonin and isovalerylshikonin inhibited TNF and IL-1 production, decreased Akt activation, and decreased the DNA-binding activity of NF-κB by blocking IκB phosphorylation. In a C57BL/6J murine model of intracerebral hemorrhage, flow cytometry confirmed that shikonin increased microglial phagocytic activity (Nie et al., 2021). Further investigation demonstrated that shikonin increased survival of primary murine neurons when cultured with microglia. The effects of shikonin on microglial ROS production and TNF secretion are well characterized; however, its effects on LPS-stimulated microglial NO production, IL-1 secretion, and glial-mediated toxicity are yet to be determined. In summary, studies have shown shikonin suppresses the NLRP3 inflammasome by direct inhibition of caspase-1 enzymatic activity and ASC oligomerization as exhibited in Fig. 1 19 (Zorman et al., 2016). Shikonin has also been shown to act on NF-κB signaling and the PI3K/Akt pathway. 1.5.2 NLRP3 and AIM2 Interactions In a C57BL/6J mouse model of ulcerative colitis, shikonin reduced the activation of the NLRP3 inflammasome by inhibiting expression of caspase-1, and thus suppressed the secretion of pro-inflammatory molecules, such as IL-1 and TNF (Han et al., 2021). Immunohistochemistry further confirmed these findings as NLRP3 expression was decreased in mouse colonic tissues due to the reduction in caspase-1. 1.5.3 ASC and Caspase-1 Inhibitor Zorman et al. (2016) presented findings that shikonin was unable to directly bind to and inhibit the NLRP3 complex, but instead inhibited ASC oligomerization and caspase-1 activation. These results indicated that shikonin non-specifically inhibited the assembly of the NLRP3 inflammasome, not the production of NLRP3 protein. Zorman et al. (2016) used immunofluorescence and immunoblotting to determine that shikonin specifically inhibited ASC oligomerization. When BMDMs were stimulated with LPS, ASC formed large oligomers, or specks; however, shikonin inhibited the formation of ASC specks in a concentration-dependent manner. Furthermore, Zorman et al. (2016) found a decrease in caspase-1 activation after treatment with shikonin. Flow cytometry and caspase-1 activation assays determined that shikonin directly targeted caspase-1 and thus inhibited inflammasome activity (Zorman et al., 2016). 1.5.4 NF-κB Inhibition Zorman et al. (2016) found that shikonin attenuated the microglial inflammatory responses by inhibiting NF-κB activity and inflammasome activation. In primary rat microglia, 20 shikonin suppressed the LPS-stimulated DNA-binding activity of NF-κB (Nam et al., 2008). Further immunoblotting showed shikonin decreased phosphorylation and degradation of IκB-α, an endogenous inhibitor of NF-κB. In RAW 264.7 macrophage-like cells and primary astrocytes from C57BL/6J mice with spinal cord injury, shikonin downregulated NF-κB activation, resulting in decreased mRNA expression of TNF, IL-6, and IL-1 in these cells (Bi et al., 2017; Zhong et al., 2020). In mouse colonic tissues, shikonin diminished NF-κB protein levels as shown by immunoblotting (Han et al., 2021). 1.5.5 PI3K Interaction Shikonin has previously been shown to inhibit PI3K and Akt. In unstimulated human glioblastoma cell lines, U87 MG and U251 MG, shikonin inhibited the expression and phosphorylation of PI3K and Akt (Zhang et al., 2015). Shikonin suppressed the PI3K/Akt signaling pathway by decreasing protein expression of the p118 subunit of PI3K and phosphorylation of Akt at Ser473 in human lymphoma cell lines (Ni et al., 2018). In addition, in human umbilical endothelial cells, shikonin downregulated PI3K/Akt signaling as shown through decreased protein levels and mRNA expression (Liu et al., 2020). It is important to note that, similar to MCC950, these effects were seen at high concentrations of shikonin, such as one µM. 1.5.6 Secondary Outcomes Inhibition of ASC, caspase-1, and NF-κB by shikonin leads to decreased release of pro-inflammatory cytokines, such as IL-1. Guo et al. (2020) observed that C57BL/6J mice, which were given LPS to induce cardiac dysfunction, exhibited reduced tissue damage when pre-treated with shikonin. Another study using human primary epithelial tissue samples from burn victims determined that shikonin decreased the concentrations of mature IL-1 and cleaved caspase-1 21 (Vinaik et al., 2020). Additionally, Xie et al. (2016) determined that exposure of BMDMs to extracellular ATP activated the NLRP3 and AIM2 inflammasomes. Immunoblots showed that shikonin inhibited caspase-1 cleavage in a dose-dependent manner, inhibiting IL-1 and IL-18 (Xie et al., 2016). Additionally, a study by Han et al. (2021) found that shikonin reduced peak levels of caspase-1, IL-1, and IL-6 in colonic tissues of mice with inflammatory bowel disease induced by dextran sulfate sodium. 1.5.7 Summary The studies outlined above indicate that shikonin is a potent inflammasome inhibitor. Although it appears to act through multiple pathways, there is clear evidence that shikonin decreases NLRP3 inflammasome activation by inhibiting ASC and caspase-1. Unlike MCC950, there is no evidence that shikonin inhibits NEK7-NLRP3 interactions; however, shikonin inhibits NF-κB signaling, PI3K/Akt pathway, and PI3K and Akt protein expression. 1.6 Oridonin Oridonin, also called 7a,20-epoxy-1a,6b,7,14-tetrahydroxy-kaur-16-en-15-one, as shown in Fig. 6, is found in the herb Rabdosia rubescens and has previously been studied for its potent anti-tumor activity. Recent research suggests that oridonin reduces toxicity and side effects of chemotherapy in cancer patients (Liu et al., 2021). Oridonin has been shown to prevent NLRP3 oligomerization, and suppress active caspases and NLRP3 activation, subsequently blocking the conversion of pro-IL-1 to its mature form in LPS-stimulated BMDMs and human peripheral blood mononuclear cells (He et al., 2018). 22 Figure 6. Oridonin chemical structure (drawn by Dr. Stephen McNeil, UBC Okanagan, 2023). Previous research has revealed the inhibitory potential of oridonin in the periphery. In LPS-stimulated human renal proximal tubular epithelial cells (HK-2 cells), oridonin inhibited cyclooxygenase (COX)-2, NO production, ROS production, and JNK phosphorylation (Huang et al., 2020). In human umbilical vein endothelial cells stimulated with TNF, oridonin inhibited NF-κB and MAPK activation and suppressed secretion of pro-inflammatory cytokines, such as TNF, IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 (Huang et al., 2018). The same study also found oridonin regulated JNK signaling. Interestingly, oridonin inhibited RAW 264.7 murine macrophage chemotaxis to epithelial cells in vitro, providing evidence for protective effects of the drug against inflammation (Huang et al., 2020). Oridonin was also found to inhibit mRNA expression of IL-1 and IL-6 in RAW 264.7 murine macrophages (Zhang et al., 2013), as well as inhibited LPS-induced activation of NLRP3 and NF-κB in RAW 264.7 murine macrophages (Yang et al., 2019). In contrast, oridonin did not affect the secretion of TNF or IL-1 by LPS-primed BMDMs (He et al., 2018). 23 In LPS-stimulated human primary gingival fibroblasts modeling gum disease, oridonin has been shown to activate the peroxisome proliferator-activated receptor (PPAR)-𝛾, which is downregulated in AD, thus suppressing the NF-κB/COX-2/IL-1 pathway, which is normally involved in the process of inflammation (Yu et al., 2019; Jia et al., 2019). Through inhibition of NF-κB, oridonin can reduce the production of IL-1, IL-6, IL-8, NO, and TNF by human gingival fibroblasts (Yu et al., 2019). In the CNS, primary rat microglia treated with oridonin have been shown to promote nerve growth factor expression and attenuate A deposition (Xu et al., 2009). In vitro, oridonin decreased NO production and mature IL-1 secretion by LPS-stimulated microglia (Xu et al., 2009). Furthermore, NF-κB protein levels were suppressed in primary rat microglia in the presence of oridonin. A recent study by Yan et al. (2020) used a mouse model of traumatic brain injury and found that oridonin decreased NLRP3, ASC, and caspase-1 mRNA and protein levels in mouse cerebral cortex tissues, further reducing NLRP3 inflammasome activation. The same study observed that oridonin inhibited IL-1 and IL-18 release in mouse cerebral cortex tissues as well as increased neuronal viability and improved neuronal morphology as observed by Nissl-staining (Yan et al., 2020). In C57BL/6 mouse models where AD was induced by A injections, oridonin increased mitochondrial activation in synaptosomes and prevented synaptic loss (Wang et al., 2016). In a study using the APP/PS1 transgenic mouse model, oridonin was found to decrease A and attenuate NO production (Zhang et al., 2013). There have been no previous studies of the effects of oridonin on astrocytes and no clinical trials have been performed. The effects of oridonin on NO production and IL-1 secretion by microglia have been elucidated; however, the effects of oridonin on phagocytic activity, ROS, and microglia-mediated neurotoxicity remain unknown. 24 Oridonin is an NLRP3 inhibitor. It has been shown to bind and block NLRP3 activity, NEK7-NLRP3 signaling, ASC oligomerization, and NF-κB signaling as shown in Fig. 3. Downstream effects of oridonin include inhibition of IL-1, IL-18, and caspase-1 activity. 1.6.2 NLRP3 Interactions Oridonin has been shown to prevent NLRP3 oligomerization and activation, consequently blocking the conversion of pro-IL-1 to its mature form and suppressing active caspases. He et al. (2018) determined that oridonin binds directly to NLRP3 with high affinity (KD = 52.5 nM). Furthermore, oridonin decreased NLRP3 protein levels in LPS-stimulated RAW 264.7 macrophages and primary murine microglia (Jia et al., 2019; Yang et al., 2019). In addition, Yang et al. (2019) demonstrated that intraperitoneal administration of oridonin significantly reduced NLRP3, ASC, caspase-1, and IL-1 in C57BL/6 nuclear factor-erythroid 2-related factor 2 knockout mouse model for acute lung injury. Recently, it was discovered that oridonin administration to LPS-induced C57BL/6 mouse astrocytes directly inhibited NLRP3 mRNA expression as shown by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (Li et al., 2022). 1.6.3 NEK7-NLRP3 Interaction In murine RAW 264.7 macrophages, oridonin inhibited the NLRP3 inflammasome by disrupting NEK7-NLRP3 interactions (Liang et al., 2021). Oridonin covalently binds the NACHT domain of NLRP3 via the thiol group of cysteine (Cys)279 as observed using immunoprecipitation and immunoblotting (He et al., 2018). He et al. (2018) found that upon binding of oridonin to Cys279, oridonin blocks the NLRP3-NEK7 interaction and prevents the activation of inflammasome. Immunoprecipitation of NEK7-NLRP3 in the presence of oridonin confirmed NEK7 inhibition (He et al., 2018). Therefore, oridonin exclusively inhibited NLRP3 25 and does not influence the activation of other inflammasomes, such as AIM2 or NLRP1, both of which lack NEK7 interactions. 1.6.4 ASC Oligomerization He et al. (2018) also determined that oridonin directly suppressed ASC oligomerization. This further led to inhibited IL-1 production in both LPS-primed HEK293T cells overexpressing NLRP3 and LPS-primed macrophages derived from WT mice that overexpress NLRP3 but had no effect in LPS-primed macrophages from NLRP3 knockout mice. Therefore, oridonin suppressed the formation of ASC oligomers and IL-1 in cells that expressed NLRP3, but not in the cells that did not express NLRP3. ASC-NLRP3 inhibition was measured by immunoprecipitation in the presence of oridonin. ASC oligomerization is an important upstream step in NLRP3 activation; therefore, the prevention of this by oridonin inhibited further NLRP3 assembly. 1.6.5 NF-κB Inhibition Oridonin has also been shown to reduce expression of NF-κB in human osteoarthritis chondrocytes as shown by immunoblotting (Jia et al., 2021). In a study using LPS-stimulated microglia, oridonin inhibited NF-κB through direct interference with its DNA-binding activity resulting in the downregulation of pro-inflammatory cytokines such as IL-1 and IL-18 (Xu et al., 2009). Furthermore, Yang et al. (2019) and Zhao et al. (2017) observed that oridonin decreased NF-κB protein levels in LPS-stimulated RAW 264.7 macrophages as shown by immunoblotting. Further analysis using immunostaining determined that oridonin blocked the translocation of NF-κB from the cytosol to the nucleus (Zhao et al., 2017). 26 1.6.6 PI3K Interaction In addition to MCC950 and shikonin, oridonin has also been shown to interact with the PI3K signaling pathway. Oridonin reduced PI3K and Akt protein levels in human oral squamous cell carcinomas as shown by immunoblotting (Yang et al., 2018). Additionally, oridonin blocked PI3K/Akt signaling pathway activation and mimicked the activity of LY294002, a PI3K inhibitor, in the human melanoma cell line, A375 (Li et al., 2018). Similar to MCC950 and shikonin, the concentrations of oridonin used in the above studies were considered toxic in our experiments. 1.6.7 Secondary Outcomes Lu et al. (2020) found that oridonin inhibited the NLRP3 signaling pathway, given that they observed decreased levels of NLRP3, ASC, cleaved caspase-1, and IL-1 in pericardial tissues extracted from C57BL/6 mice seven days after intraperitoneal injection of oridonin. In BV-2 microglia, oridonin induced significant reductions in NLRP3 and caspase-1 protein levels, as shown by immunoblotting (Jia et al., 2021). Further analysis by using enzyme-linked immunosorbent assays (ELISAs) demonstrated a reduction in IL-1 and IL-18 secretion following oridonin treatments. The inhibition of NEK7-NLRP3 signaling by oridonin leads to a decrease in NLRP3 inflammasome assembly and subsequent decrease in production of cytokines such as IL-1 and IL-18 (He et al., 2018; Liang et al., 2021). Inhibition of ASC oligomerization and binding to NF-κB by oridonin also leads to a decrease in IL-1 and IL-18 (He et al., 2018; Xu et al., 2009; Zhao et al., 2017). 1.6.8 Summary Due to the extensive research regarding oridonin and its interactions with the NLRP3 inflammasome, these direct interactions do not need to be pursued further and are indicated in 27 Table 1. He et al. (2018) were thorough in conducting their research and determined the effects of oridonin on the NLRP3 inflammasome complex, including its interaction with Cys279 of NLRP3 protein. Oridonin is also a well-defined inhibitor of NF-κB and NLRP3-NEK7 signaling (He et al., 2018; H. Huang et al., 2020; Huang et al., 2018; Jia et al., 2021; Xu et al., 2009; Yang et al., 2019; Zhao et al., 2017). Similar to shikonin, oridonin inhibited the NF-κB pathway. Notably, a recent review lists oridonin as one of the established pharmacological inflammasome inhibitors (Zahid et al., 2019). 28 Table 1. All three drugs, MCC950, shikonin, and oridonin, are NLRP3 inflammasome inhibitors. MCC950 and oridonin directly inhibit NLRP3 and NEK7-NLRP3 interactions, but there are no studies showing similar mechanism of action for shikonin. MCC950 blocks NLRP3 ATPase activity by inhibiting the Walker B motif. Oridonin inhibits NEK7-NLRP3 by binding to the NACHT domain of NLRP3 via the thiol group of Cys279. Shikonin directly inhibits caspase-1 activity, and ASC oligomerization. Additionally, shikonin and oridonin have been shown to decrease NF-κB protein levels. MCC950, shikonin, and oridonin have also been shown to block PI3K/Akt signaling. All three drugs have downstream secondary outcomes of inactivating the NLRP3 inflammasome, including inhibition of caspase-1 activity, IL-1, and IL-18. Primary Mechanisms Secondary Outcomes References MCC950 1. Blocked NLRP3 ATPase activity by binding to the Walker B motif 2. Inhibited NLRP3 protein expression 3. Inhibited NEK7-NLRP3 interaction 4. Reduced phosphorylation of PI3K and Akt Inhibited: 1. caspase-1 activity 2. IL-1 3. IL-18 (Chang et al., 2020; Coll et al., 2015, 2019; Dong et al., 2021; Lučiūnaitė et al., 2020; Ren et al., 2020; Zhang et al., 2017; Zhu et al., 2021) Shikonin 1. Blocked NLRP3 and AIM2 by directly inhibiting ASC oligomerization and cleaved caspase-1 2. Inhibited NLRP3 protein expression 3. Decreased NF-κB 4. Reduced expression and phosphorylation of PI3K/Akt Inhibited: 1. caspase-1 activity 2. IL-1 3. IL-18 (Han et al., 2021; Xie et al., 2016; Zorman et al., 2016) Oridonin 1. Bound and blocked NLRP3 2. Inhibited NLRP3 protein expression 3. Bound and blocked ASC oligomerization 4. Inhibited NEK7-NLRP3 interaction by mutating Cys279 5. Decreased NF-κB 6. Reduced PI3K/Akt protein levels Inhibited: 1. caspase-1 activity 2. IL-1 3. IL-18 (He et al., 2018; Jia et al., 2021; Li et al., 2022; Liang et al., 2021; Yang et al., 2019) 1.7 Modeling Brain Immune Cell Activation Immortalized cell lines provide advantages to primary research, such as cost efficiency and ease of use (Kaur & Dufour, 2012). Immortalized cell lines proliferate indefinitely and are 29 genetically identical cultures which allows for reproducible results. Primary cells are freshly extracted from an organism, such as mice or humans, and closely represent the tissue of origin. Immortalized cells can display similar functional features to primary cells, which makes them attractive models for pharmacological research. While the use of immortalized cell lines can provide insight into the functions of the target cells, their gene expression differs drastically from primary cells. The following immortalized cell types were used in this study to model brain microglia activation: BV-2 murine microglia, THP-1 human microglia-like cells, and HL-60 human promyelocytic leukemia cells. U-118 MG human astrocytic cells were used to model astrocytes. NSC-34 murine neuronal cells, HT-22 murine neuronal cells, and SH-SY5Y human neuronal cells were used to model neurons. BV-2 murine microglial cells are derived from C57BL/6 mice and are a well-characterized model system (Blasi et al., 1990). Studies have demonstrated that BV-2 microglia are an adequate alternative to primary microglia in complex cell interaction studies (Henn et al., 2009). BV-2 microglial cells elicit similar immune responses to primary murine microglia, such as secretion of a similar set of cytokines, for example, IL-1 and TNF (Blasi et al., 1990). When induced by stimuli, including LPS, BV-2 microglial cells can be used to study NO secretion, murine microglia-mediated toxicity, cytokine secretion, glutamate release, and phagocytic activity (Blasi et al., 1990; Henn et al., 2009; McKenzie & Klegeris, 2018; Spielman et al., 2017). THP-1 human microglia-like cells were derived from a one-year-old male with acute monocytic leukemia. THP-1 cells are considered microglia-like because they have similar receptors and functions to human microglia (Klegeris et al., 2005; Klegeris & McGeer, 2003; Tsuchiya et al., 1980). Because of this, THP-1 cells were used to study human microglia- 30 mediated toxicity and cytokine secretion. HL-60 myelomonocytic cells were established from a 36-year-old female with acute promyelocytic leukemia. HL-60 cells were differentiated with DMSO to acquire a human microglia-like phenotype. When primed with LPS, DMSO-differentiated HL-60 microglia-like cells can be induced to undergo a respiratory burst in which ROS are released (Collins, 1987; Levy et al., 1990). Astrocytes were modeled using U-118 MG human astrocytic cells derived from a 50-year-old male with malignant gliomas. Studies have shown that U-118 MG cells express astrocyte-specific proteins such as glutamate transporter (GLT)-1 and can carry out immune functions, such as phagocytosis. These cells are therefore commonly used as a model of human astrocytes in cell culture studies such as measurements of glutamate and cytokine secretion (Hashioka et al., 2009; Roemeling et al., 2020). Neurons were modeled using the following cell lines: NSC-34 murine neuronal cells, HT-22 murine neuronal cells, and SH-SY5Y human neuronal cells. NSC-34 neuronal cells are a hybrid cell line derived from the spinal cords of mouse embryos and mouse neuroblastoma cells (Hounoum et al., 2016). NSC-34 neuronal cells are similar to motor neurons, since they synthesize and release acetylcholine (Nango et al., 2020). These cells have been confirmed as an acceptable model for studying neurodegenerative diseases and therefore were used in experiments to determine murine microglia-mediated toxicity. HT-22 neuronal cells are subcloned from the HT-4 cell line, which was derived by the immortalization of murine neuronal tissues. HT-22 cells are considered valuable for measuring glutamate-induced toxicity of neuronal cells and have been shown to possess properties of cholinergic neurons; therefore, these cells were used in addition to NSC-34 motor neuron-like cells to determine murine microglia-mediated toxicity (He et al., 2013; Liu et al., 2009). SH-SY5Y human neuronal cells came from 31 the neuroblastoma tissue of a four-year-old female. SH-SY5Y neuronal cells have markers of primary dopaminergic neuronal cells and are considered an adequate model for human neurons (Heusinkveld & Westerink, 2017; Hong-rong et al., 2010). Therefore, SH-SY5Y human neuronal cells were used to determine human microglia- and astrocyte-mediated toxicity. 1.8 Research Hypotheses and Objectives Neuroinflammation is characterized by the overactivation of glial cells, including microglia and astrocytes. This immune response contributes to neurodegenerative diseases. Recent studies have established the NLRP3 inflammasome in glial cells as a major contributor to neuroinflammation (Guo et al., 2015; Heneka et al., 2013; Liu et al., 2020; Nizami et al., 2019; Tejera et al., 2019; Yang et al., 2019). Previous studies have demonstrated that the inflammasome inhibitors, MCC950, shikonin, and oridonin, can downregulate the overactivation of glial cells in inflammatory diseases (Coll et al., 2015, 2019; Dempsey et al., 2017; Li et al., 2022; Liang et al., 2021; Nam et al., 2008; Tapia-Abellán et al., 2019; Xu et al., 2009; Yang et al., 2014; Zorman et al., 2016). However, the effect of inflammasome inhibitors on the secretion of some pro-inflammatory cytokines, such as TNF, have not been well characterized. Additional properties, such as the effects of inflammasome inhibitors on glial phagocytosis, have not yet been explored. I hypothesized that three structurally different inflammasome inhibitors, MCC950, shikonin, and oridonin, regulate a broad range of immune responses of microglia and astrocytes. Hypothesis I: I hypothesized that selective inhibition of inflammasomes decreases the secretion of multiple murine microglia-derived toxins and increases phagocytic activity of murine microglia, thereby demonstrating a neuroprotective effect. This hypothesis was tested in vitro 32 using the immortalized cell line, BV-2 murine microglia, as the cell model. The following specific research objectives addressed this hypothesis: 1. Determine if MCC950, shikonin, and oridonin modulate the secretion of inflammatory mediators by BV-2 murine microglia. I. Examine if MCC950, shikonin, and oridonin modulate the secretion of NO by BV-2 murine microglia. II. Establish if MCC950, shikonin, and oridonin modulate the secretion of glutamate by BV-2 murine microglia. III. Determine if MCC950, shikonin, and oridonin modulate the secretion of pro-inflammatory cytokines, IL-1β and TNF, by BV-2 murine microglia. 2. Establish if MCC950, shikonin, and oridonin modulate BV-2 murine microglia-mediated neurotoxicity. 3. Determine if MCC950, shikonin, and oridonin modulate the phagocytic activity of BV-2 murine microglia. 4. Determine if the anti-inflammatory activity of oridonin is also mediated by its actions on PKD or JNK by using inhibitors of these kinases. Hypothesis II: I hypothesized that selective inhibition of inflammasomes decreases the secretion of multiple human microglia-derived toxins, thereby demonstrating a neuroprotective effect. This hypothesis was tested in vitro using immortalized microglia-like cell lines, THP-1 and HL-60, as human microglial cell models. The following specific research objectives addressed this hypothesis: 33 1. Determine if MCC950, shikonin, and oridonin modulate the secretion of pro-inflammatory cytokines, IL-1β and TNF, by THP-1 human microglia-like cells. 2. Establish if MCC950, shikonin, and oridonin modulate THP-1 microglia-like cell-mediated neurotoxicity. 3. Examine if MCC950, shikonin, and oridonin modulate the secretion of ROS by DMSO-differentiated HL-60 microglia-like cells. Hypothesis III: I hypothesized that selective inhibition of inflammasomes decreases the secretion of multiple astrocyte-derived toxins and increases phagocytic activity of astrocytes, thereby demonstrating a neuroprotective effect. This hypothesis was tested in vitro using a human immortalized astrocytic cell line, U-118 MG, as a cell model. The following specific research objectives addressed this hypothesis: 1. Determine if MCC950, shikonin, and oridonin modulate the secretion of inflammatory mediators by human astrocytes. I. Establish if MCC950, shikonin, and oridonin modulate the secretion of glutamate by U-118 MG astrocytes. II. Determine if MCC950, shikonin, and oridonin modulate the secretion of pro-inflammatory cytokines, IL-1β and TNF, by U-118 MG human astrocytes. 2. Establish if MCC950, shikonin, and oridonin modulate U-118 MG human astrocyte-mediated toxicity. 3. Determine if MCC950, shikonin, and oridonin modulate U-118 MG phagocytic activity. 34 Chapter 2: Materials and Methods 2.1 Chemicals and Reagents The following reagents were obtained from Sigma Aldrich (Oakville, ON, Canada): bisbenzimide (Hoechst 33258), -nicotinamide adenine dinucleotide hydrate (-NAD), diaphorase, iodonitrotetrazolium chloride, N-formylmethionine-leucyl-phenylalanine (fMLP), luminol sodium salt, LPS, MCC950, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N,N-dimethylformamide (DMF), and phosphate-buffered saline (PBS) tablets. Bovine serum albumin (BSA), calf bovine serum (CBS), fetal bovine serum (FBS), Dulbecco's modified Eagle medium: nutrient mixture F12/Ham (DMEM-F12), phenol red-free DMEM-F12, antibiotic solution containing penicillin G (10,000 U/ml), streptomycin (10 mg/ml) and amphotericin B (25 μg/ml), 0.05% and 0.025% trypsin with ethylenediaminetetraacetic acid (EDTA), sodium lauryl sulfate (SLS), Tween-20, and one-step slow 3,3′,5,5′-tetramethylbenzidine (TMB) - ELISA substrate solution were obtained from Thermofisher Scientific (Ottawa, ON, Canada). Human recombinant IFN-γ as well as ELISA development kits for IL-1β and TNF were purchased from PeproTech (Embrun, ON, Canada). Fluorescein isothiocyanate (FITC) externally labeled one μm fluorescent polystyrene latex beads were purchased from Bangs Laboratories (Fishers, IN, USA). L-glutamate dehydrogenase was purchased from Agilent (Santa Clara, CA, USA). Oridonin and shikonin were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). 2.2 Equipment and Supplies A class 2, type IIA biological safety cabinet was used for cell culture experiments. All cells were grown in T75 cell culture flasks (Sarstedt, Montreal, QC, Canada) and placed in a 35 Steri-Cycle High-Efficiency Particulate Air Class 100 CO2 incubator (Model #370, ThermoFisher Scientific). Cells were counted using a hemocytometer (ChangeBioscience, Castro Valley, CA, USA). CELLSTAR tubes (Greiner Bio-one, Frickenhausen, Germany) were used to centrifuge cell suspensions. Sterile 24-well plastic cell culture plates (Corning, NY, USA) were used for seeding BV-2, THP-1, U-118 MG, NSC-34, HT-22, SH-SY5Y, and HL-60 cells for the glia-mediated toxicity, MTT, and respiratory burst assays. 96-well plates (Corning) were utilized during the glutamate, ELISA, MTT, and respiratory bust assays. Four-chambered sterile petri dishes (Greiner Bio-one) were used for the phagocytosis assay. Sterile 10 cm cell culture dishes (ThermoFisher Scientific) were used for HL-60 cell differentiation. Spectrophotometric, chemiluminescent, and fluorescent measurements were performed using the FLUOstar Omega microplate reader (BMG Labtech, Nepean, ON, Canada). 2.3 Cell Lines BV-2 murine microglia, HL-60 human promyelocytic leukemia cells, and THP-1 human monocytic microglia-like cells were all used as models of microglia. U-118 MG human astrocytoma cells were used to model astrocytes. HT-22 mouse hippocampal neuronal cells, NSC-34 murine neuroblastoma cells, and SH-SY5Y human neuroblastoma cells were used to model neurons. BV-2 cells were donated by Dr. G. Garden (Department of Neurology, University of Washington, Seattle, WA, USA). HL-60, THP-1, and U-118 MG cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). SH-SY5Y cells were donated by Dr. R. Ross (Department of Biological Sciences, Fordham University, Bronx, NY, USA) and NSC-34 cells were donated by Dr. A. Milnerwood (Centre for Applied Neurogenetics and Brain Research Centre, University of British Columbia, Vancouver, BC, Canada). HT-22 cells were donated by Dr. T. Wenzel (University of Saskatchewan, Saskatoon, 36 SK, Canada). All cells were stored in the gaseous phase of liquid nitrogen in 1.2 ml cryovials (ThermoFisher Scientific). Cells were cultured in T75 cell culture flasks containing DMEM-F12 supplemented with 10% CBS or FBS and the antibiotics, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in humidified 5% CO2 and 95% air atmosphere. 2.4 Cell Seeding 2.4.1 BV-2 Murine Microglia BV-2 microglial cells were unadhered from T75 cell culture flasks by incubating in three ml of 0.25% trypsin-EDTA until detached. Cells were resuspended in DMEM-F12 medium containing the previously stated antibiotics and 10% CBS. BV-2 microglial cells were counted using a hemocytometer, then centrifuged at 400 g for seven min at room temperature. The supernatant was decanted, and the cells were resuspended in DMEM-F12 medium containing the antibiotics and 5% CBS for a final concentration of 5 x 104 cells/ml for phagocytosis and 2 x 105 cells/ml for all other experiments. BV-2 cell cultures were then seeded in a 24-well plate at 500 μl per well and incubated for 24 h to allow for adherence. 2.4.2 NSC-34 Murine Neuronal Cells NSC-34 neuronal cells were unadhered from T75 cell culture flasks by incubating in three ml of 0.25% trypsin-EDTA until detached. Cells were resuspended in DMEM-F12 medium containing antibiotics and 10% CBS. NSC-34 neuronal cells were counted using a hemocytometer, then centrifuged at 400 g for seven min at room temperature. The supernatant was then removed, and the cells were resuspended at a concentration of 2 x 105 cells/ml in DMEM-F12 medium containing antibiotics and 5% CBS for all experiments. NSC-34 murine neuronal cell cultures were then seeded in a 24-well plate at 400 μl per well and incubated for 24 h to allow for adherence. 37 2.4.3 THP-1 Human Microglia-like Cells Cultures of non-adherent THP-1 microglia-like cells were removed from T75 cell culture flasks and centrifuged for seven min at 400 g. Cells were resuspended in DMEM-F12 medium containing antibiotics and 5% CBS, then counted using a hemocytometer. THP-1 cell cultures were seeded in a 24-well plate at one ml per well to a final concentration of 4 x 105 cells/ml for all experiments. 2.4.4 SH-SY5Y Human Neuronal Cells SH-SY5Y neuronal cells were unadhered from T75 cell culture flasks using three ml of 0.05% trypsin-EDTA until detached. Cells were resuspended in DMEM-F12 medium containing antibiotics and 10% FBS. SH-SY5Y neuronal cells were counted using a hemocytometer, then centrifuged at 400 g for seven min at room temperature. The supernatant was decanted, and the cells were resuspended at a concentration of 2 x 105 cells/ml in DMEM-F12 medium containing the antibiotics and 5% FBS for all experiments. SH-SY5Y cell cultures were then seeded in a 24-well plate at 400 μl per well and incubated for 24 h to allow for adherence. 2.4.5 HT-22 Mouse Neuronal Cells HT-22 neuronal cells were unadhered from T75 cell culture flasks by incubating in three ml of 0.25% trypsin-EDTA until detached. Cells were resuspended in DMEM-F12 medium containing antibiotics and 10% CBS. HT-22 neuronal cells were counted using a hemocytometer, then centrifuged at 400 g for seven min at room temperature. The supernatant was decanted, and the cells were resuspended in DMEM-F12 medium containing the antibiotics and 5% CBS for a final concentration of 2 x 105 cells/ml for all experiments. HT-22 cell cultures were then seeded in a 24-well plate at 400 μl per well and incubated for 24 h to allow for adherence. 38 2.4.6 HL-60 Human Microglia-like Cells Cultures of non-adherent HL-60 cells were removed from T25 cell culture flasks and centrifuged for seven min at 400 g. Cells were then resuspended in DMEM-F12 medium containing antibiotics and 10% CBS, then counted using a hemocytometer. Cell cultures were then diluted to a final concentration of 2 x 105 cells/ml. HL-60 cells were differentiated to microglia-like cells using dimethyl sulfoxide (DMSO) at a concentration of 1.3% v/v by seeding 15 ml of cell cultures in a 10 cm3 culture dish at 37°C for six days. After differentiation, cells were counted and centrifuged for seven min at 400 g. Supernatant was decanted and cells were resuspended at a concentration of 1 x 106 cells/ml in DMEM-F12 medium containing antibiotics and 10% CBS. Cell cultures were seeded at one ml per well in a 24-well plate. 2.4.7 U-118 MG Human Astrocytic Cells U-118 MG astrocytic cells were unadhered from T75 cell culture flasks by incubating in three ml of 0.05% trypsin-EDTA until detached. Cells were resuspended in DMEM-F12 medium containing antibiotics and 10% FBS. U-118 MG astrocytic cells were counted using a hemocytometer, then centrifuged at 400 g for seven min at room temperature. The supernatant was decanted, and cells were resuspended at a concentration of 2 x 105 cells/ml in DMEM-F12 medium containing the antibiotics and 5% FBS. U-118 MG cell cultures were then seeded in a 24-well plate at one ml per well and incubated for 24 h to allow for adherence. 2.5 Assays 2.5.1 MTT Assay - Cell Viability The MTT assay was used to assess cell viability in all experiments, except for those measuring phagocytic activity. The yellow, water-soluble, tetrazolium dye is reduced by the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent mitochondrial 39 oxidoreductase to purple, water-insoluble formazan, which forms crystals (Liu et al., 1997). This enzyme is metabolically active in viable cells only. Therefore, the quantity of formazan crystals is proportional the number of viable cells (Mosmann, 1983). MTT solution was added to each well to reach a final concentration of 0.5 mg/ml, and plates were incubated for one h at 37°C in a 5% CO2 incubator, at which point the formazan crystals were dissolved by adding a volume of SLS (20% w/v)/DMF (50% v/v) (SLS/DMF) solution equal to that of the culture medium present in the well. The plates were then incubated for three h at 37°C to dissolve crystals and 100 μl of well contents were transferred from the 24-well plate to a 96-well plate. Optical densities (OD) were measured at 570 nm using the FLUOstar Omega microplate reader. Cell viability was calculated as a percentage of the viability of untreated, control cells. % 𝑐𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷𝑚𝑒𝑑𝑖𝑎𝑂𝐷𝑙𝑖𝑣𝑒 − 𝑂𝐷𝑚𝑒𝑑𝑖𝑎 × 100 Where ODsample = optical density in wells containing supernatants from cells exposed to treatments; ODmedia = optical density in wells containing cell culture medium without cells; and ODlive = optical density in wells containing supernatants from fully viable cells incubated in fresh cell culture medium only. 2.5.2 Griess Assay - Quantification of Nitric Oxide Secretion NO is an important cellular signaling molecule that is protective in most tissues and required for many cell functions, including vasodilation and the immune response (Gantner et al., 2020). NO is secreted by activated microglia and is toxic to surrounding cells at high concentrations (Chao et al., 1992). NO is released into the extracellular environment and metabolized into nitrite, a compound that can be detected by the Griess assay (Bryan & Grisham, 2007). The concentration of nitrite is measured to assess the production of NO (Ding et al., 1988; 40 Miranda et al., 2001). Consequently, nitrite levels can be used to approximate microglial activation, and its downregulation can be used to determine the efficacy of various anti-inflammatory drugs. After seeding BV-2 cell cultures in a 24-well plate as described above (1.4.1), the cells were treated with MCC950 (final concentrations of 2-50 μM), shikonin (final concentrations of 40-400 nM), oridonin (final concentrations of 1-4 μM), or their vehicle solution, DMSO, for 30 min. Cells were then incubated for 24 h in either the presence or absence of LPS (100 ng/ml final concentration). The final concentration of DMSO in cell cultures was 0.2%, which was shown in preliminary experiments to be non-toxic to cells. Supernatants (50 μl) were collected from each of the 24 wells and mixed with an equal volume of Griess reagent (1% sulfanilamide, 2.5% phosphoric acid, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in H2O) in a 96-well plate. A range of different concentrations of nitrite in cell culture medium was used to construct the standard curve (final concentrations of 0-50 M sodium nitrite). OD was then measured using a spectrophotometer at 570 nm, and nitrite concentrations within treatment wells were calculated using a nitrite standard curve as follows: 𝑛𝑖𝑡𝑟𝑖𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀) =𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷𝑏𝑙𝑎𝑛𝑘𝑚 𝐿𝑂𝐷 (𝑀) = 𝑚𝑒𝑎𝑛 𝑂𝐷𝑏𝑙𝑎𝑛𝑘 + 2(𝑆𝐷 𝑂𝐷𝑏𝑙𝑎𝑛𝑘)𝑚 Where ODsample = optical density in wells containing supernatants from cells exposed to treatments; ODblank = optical density in wells containing cell culture medium without cells; SD = standard deviation; and m = slope of the line produced by the standard curve. The mean OD of 41 the wells containing 0 M sodium nitrite was used to calculate the limit of detection (LOD) as described by Gates (2020). 2.5.3 Supernatant Transfer Assay - Murine Glia-Mediated Toxicity Towards Neurons Supernatant transfer experiments were performed as described previously to study the effects of inflammasome inhibitors on cytotoxicity of glial cells (Murray et al., 2022; Wenzel et al., 2020). Activated glia secrete proinflammatory mediators, which are toxic to neurons at high concentrations. This toxicity can be examined in cell cultures by transferring glial cell supernatant onto neuronal cells. BV-2 murine microglial cell cultures were plated in a 24-well plate as described above (1.4.1), and cells were treated with MCC950 (2-50 μM), shikonin (40-400 nM), oridonin (1-4 μM), or their vehicle solution (DMSO). Cells were incubated for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. Supernatants were collected and kept at -20°C for ELISAs, and BV-2 cell viability was determined by the MTT assay as described in 1.5.1. NSC-34 murine neuronal cell cultures and HT-22 murine neuronal cell cultures were plated in 24-well plates as described above (1.4.2 and 1.4.5, respectively) and incubated for 24 h before their supernatants were aspirated and replaced with 400 μl of cell-free BV-2 cell supernatant. NSC-34 cells and HT-22 cells were then incubated for 72 h. Percent viability of NSC-34 cells and HT-22 cells at the end of this incubation period was determined by the MTT assay as described in section 1.5.1. 2.5.4 Supernatant Transfer Assay - Human Glia-like Cell Mediated Toxicity Towards Neurons THP-1 human microglia-like cells and U-118 MG human astrocytic cells were plated in a 24-well plate as described above (1.4.3 and 1.4.7, respectively), and treated with MCC950 (2-50 μM), shikonin (40-400 nM), oridonin (1-4 μM), or their vehicle solution (DMSO) for 30 min 42 before stimulation with LPS (100 ng/ml) alone or LPS (100 ng/ml) plus IFN- (100 U/ml) for 48 h. Supernatants were collected and stored at -20°C for ELISAs. THP-1 and U-118 MG cell viability were determined by the MTT assay as described in 1.5.1. SH-SY5Y neuronal cells were plated in a 24-well plate as described above (1.4.4) and incubated for 24 h before supernatant was aspirated and replaced with 400 μl of cell-free THP-1 or U-118 MG supernatant and incubated at 37°C for 72 h. Percent viability of SH-SY5Y cells at the end of this incubation period was determined using the MTT assay as described in section 1.5.1. 2.5.5 Enzyme-Linked Immunosorbent Assay (ELISA) - Quantification of IL-1 and TNF ELISAs are used to colorimetrically quantify the presence of various cytokines, such as IL-1 and TNF, in supernatant samples. The cytokine from the supernatant sample attaches to the capture antibody, which itself is bound to the base of a 96-well plate. Then, the labeled detection antibody is added and binds to the cytokine. Next, the wells are treated with an enzyme-linked conjugate that associates with the detection antibody. After the addition of substrate, the signal is detected based on the absorbance at 450 nm using a spectrophotometer and is proportional to the concentration of cytokine in each sample (Reen, 1994). A standard curve of different cytokine concentrations is added on the same plate as samples to determine the LOD and to calculate the cytokine concentrations in cell culture media. Glial supernatants collected from the supernatant transfer assay were used for the detection of IL-1 and TNF. 96-well plates were initially coated with 100 μl of primary antibodies (diluted in PBS 400- and 100-fold for IL-1 and TNF, respectively) for 24 h at room temperature. Plates were then washed with 300 μl 0.05% Tween in PBS (wash buffer) and paper towels were used to fully remove wash buffer. The wells were filled with 300 μl blocking buffer of 1% BSA in PBS for one h at room temperature. Plates were washed again, and 100 μl of 43 standards (diluted 1000- to 64000-fold for both IL-1 and TNF) or samples were added to the wells overnight. Standards were diluted in 0.1% BSA and 0.05% Tween in PBS to a final concentration of 0-2000 pg/ml; zero pg/ml was utilized in calculating the LOD. Plates were washed and 100 μl of secondary antibody (diluted in 0.1% BSA and 0.05% Tween in PBS 400x for both IL-1 and TNF) were added to the wells for two h. Wells were then washed and treated with 100 μl streptavidin-horseradish peroxidase for 30 min before being washed again prior to the addition of 100 μl of undiluted TMB liquid substrate. Plates were monitored for colour development for 20 min. Lastly, 100 μl of one mM hydrochloric acid stop solution were added in a 1:1 ratio to TMB substrate solution and the OD was then measured at 450 nm using a spectrophotometer. The OD of each sample was corrected for the OD of the blank, and cytokine levels were calculated according to a standard curve obtained by using recombinant cytokines. 𝑐𝑂𝐷 = D𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷𝑏𝑙𝑎𝑛𝑘 𝑐𝑦𝑡𝑜𝑘𝑖𝑛𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑛𝑔/𝑚𝑙) =𝑐𝑂𝐷𝑚 Where cOD = the corrected OD; DODsample = the change in OD in samples from cells exposed to treatments; ODblank = optical density in samples containing cell culture medium without cells; and m = slope of the standard curve. The LOD was also calculated using the following formula: 𝐿𝑂𝐷 (𝑛𝑔/𝑚𝑙) =[D𝑂𝐷𝑚𝑒𝑎𝑛 + (2 × 𝑆𝐷 𝑂𝐷𝑏𝑙𝑎𝑛𝑘)] − 𝑏𝑚 Where SD = standard deviation; b = y-intercept; and m = slope of the standard curve. 2.5.6 Assay Measuring the Neurotransmitter (and Neurotoxin) Glutamate Under normal conditions, glutamate is continuously recycled between neurons and glia (Rothman et al., 2003). When glial cells are activated, extracellular glutamate concentration is 44 elevated and it becomes toxic to neurons (Grewer et al., 2008). Glutamate concentrations in BV-2 and U-118 MG cell supernatants were quantified by the assay in which the enzyme, glutamate dehydrogenase, oxidatively deaminates glutamate and NAD+ to NADH and α-ketoglutarate. In the presence of diaphorase and iodonitrotetrazolium chloride, NADH is converted to NAD+ and iodonitrotetrazolium-formazan, resulting in colour development that is proportional to the concentration of glutamate in the sample. BV-2 and U-118 MG cell cultures were plated in a 24-well plate as described above (1.4.1 and 1.4.7, respectively) and incubated overnight. The cells were then treated with MCC950 (2-50 μM), shikonin (40-400 nM), oridonin (1-4 μM), or the vehicle solution, DMSO, 30 min prior to stimulation with LPS (100 ng/ml) and incubated for 24 h. Cell supernatants were collected and stored at -20°C. After all experiments were completed, cell supernatants were thawed and 20 μl were transferred to 96-well plates. Standards were also added to the 96-well plate using 0-3 mM glutamate in DMEM-F12 phenol-free medium containing the antibiotics and 5% CBS. 155 μl of triethanolamine buffer containing 20 mg/ml -NAD, 12 μl/ml iodonitrotetrazolium chloride and 6.2 μl diaphorase were added to each well. An initial absorbance was measured at 490 nm using a spectrophotometer prior to the addition of L-glutamate dehydrogenase to account for background absorbance. Each well then received 25 μl of glutamate dehydrogenase and final absorbance was measured after 10 min. The OD of each sample was corrected for the OD of the blank, and glutamate levels were calculated according to the standard curve. 𝑐𝑂𝐷 = D𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑂𝐷𝑏𝑙𝑎𝑛𝑘 𝐺𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀) =𝑐𝑂𝐷𝑚 45 Where cOD = the corrected OD; DOD = the change in OD in sample wells; ODblank = OD in wells containing cell culture medium without cells; and m = slope of the standard curve. 2.5.7 Respiratory Burst Assay - Quantification of Reactive Oxygen Species ROS are secreted following activation of microglia. The respiratory burst assay was used to quantify the secretion of ROS by HL-60 cells (Levy et al., 1990). Cells were stimulated with the bacterial peptide, fMLP, inducing the release of ROS, which react with luminol to produce a chemiluminescence (CHL) signal (Dahlgren & Karlsson, 1999). Thus, CHL was used to detect the presence of ROS in a sample. HL-60 human microglia-like cell cultures were plated in 10 cm2 culture dishes and differentiated in DMSO as described above (1.4.6). Supernatant was decanted and cells were resuspended in DMEM-F12 medium containing the antibiotics and 10% CBS. Cell cultures were seeded at one ml per well in a 24-well plate and incubated for 30 min before being treated for 30 min with MCC950 (2-50 μM), shikonin (40-400 nM), oridonin (1-4 μM), or their vehicle solution (DMSO), and primed with LPS (500 ng/ml) for 24 h. Following incubation, individual wells were mixed so the cells were resuspended in medium and 500 μl of samples were transferred to microcentrifuge tubes and counted using a hemocytometer. The remaining 500 μl of each sample were used to conduct the MTT assay as described in section 1.5.1. The samples in the microcentrifuge tubes were centrifuged at 12,000 g for 10 min. The supernatant was carefully removed, and each pellet was resuspended to a final concentration of 1 x 106 cells/ml in phenol red-free DMEM-F12 medium containing the antibiotics and 2% CBS. 85 μl of each sample were transferred to a 96-well plate and incubated for 10 min. Luminol sodium salt solution (0.85 mg/ml in PBS) and fMLP solution (20 μM in PBS) were loaded into the first and second injector of the FLUOstar Omega plate reader, respectively. The 96-well plates were 46 placed in the plate reader and CHL was quantified. The program measured the light intensity of each well for 60 cycles at 21 s per cycle. Luminol (10 μl) was added to each well during cycle five and fMLP (5 μl) was added to each well during cycle 15. Mars Analysis Software (BMG Labtech) was used to analyze the curves over 25 min. Data were expressed as CHL using the following formula: 𝐶𝐻𝐿 = 𝐶𝐻𝐿𝑐𝑢𝑟𝑣𝑒 2 (𝑐𝑦𝑐𝑙𝑒𝑠 15−39) − 𝐶𝐻𝐿𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 (𝑐𝑦𝑙𝑐𝑒𝑠 6−15) Where CHLcurve 2 (cycles 15-39) = the area under the second curve elicited by the application of fMLP; and CHLbaseline (cycles 6-15) = the values from cycles 6-15, or the range between curve one (elicited by luminol) and curve two (elicited by fMLP). CHL values were calculated by summing the area under the curve (light measurement vs. time) at each cycle. 2.5.8 Phagocytosis Assay The phagocytosis assay measures the engulfment of fluorescent beads by BV-2 murine microglia and U-118 MG human astrocytes. Phagocytosis is a necessary physiological process for the removal of debris in the extracellular space (Lian et al., 2016). Therefore, enhancing phagocytic activity of microglia and astrocytes may be beneficial in effectively clearing extracellular material. BV-2 and U-118 MG cell cultures were plated in a four-chambered sterile plate as described above (1.4.1) and given 24 h to adhere. They were then treated with MCC950 (50 μM), shikonin (400 nM), oridonin (4 μM), or their vehicle solution (DMSO) for 30 min prior to LPS (400 ng/ml) stimulation and incubated for 24 h. Fluorescent latex beads were added to each well in a 10:1 bead to cell ratio and the plates were incubated for one h. 100 μl of 0.4% trypan blue were added to each well to quench the fluorescence of the extracellular beads. The trypan blue, supernatant, and floating beads were carefully removed from the wells, which were washed 47 with 500 μl sterile PBS. Cells were fixed in place with 400 μl of 70% ethanol for five min before being washed again with 500 μl PBS. Immediately prior to imaging, bisbenzimide (two μg/ml final concentration) nuclear stain was added to all wells in 500 μl PBS. Cells were imaged using a Zeiss AxioObserver.Z1 microscope and Zen image acquisition software (version 2.0) at an excitation/emission of 350/470 nm for bisbenzimide, and 470/520 for the fluorescent beads. For each well, about 40 viable cells (identified by their intact nuclei) were analyzed per experiment. A blind analysis of the images by an investigator not knowing the experimental conditions was then completed using ImageJ software. Fluorescence was quantified by subtracting the background fluorescence from the fluorescence of the beads within single cells. The fluorescence of internalized latex beads was calculated using the following formula: 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑛𝑎𝑙𝑖𝑧𝑒𝑑 𝑙𝑎𝑡𝑒𝑥 𝑏𝑒𝑎𝑑𝑠= (𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑒𝑙𝑒𝑐𝑡𝑒𝑑 𝑐𝑒𝑙𝑙 × 𝑚𝑒𝑎𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑓𝑟𝑜𝑚 𝑤𝑖𝑡ℎ𝑖𝑛 𝑡ℎ𝑒 𝑐𝑒𝑙𝑙 𝑎𝑟𝑒𝑎)− (𝑎𝑟𝑒𝑎 𝑜𝑓 𝒕𝒉𝒆 𝒔𝒆𝒍𝒆𝒄𝒕𝒆𝒅 𝑐𝑒𝑙𝑙 × 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒) 2.5.9 Statistical Analysis Statistical analysis was completed using GraphPad PRISM software (version 9.0, GraphPad Software Inc., La Jolla, CA, USA). Randomized block one-way analysis of variance (ANOVA) was performed to determine the significance of differences in data obtained. Where significant, ANOVA was followed by Dunnett’s or Tukey’s post-hoc test. Data are presented as means ± standard error of the mean (SEM). Statistical significance was established at P < 0.05. 48 Chapter 3: Results 3.1 BV-2 Murine Microglia 3.1.1 Determination of the Non-Toxic Concentrations of Inflammasome Inhibitors towards LPS-Stimulated BV-2 Murine Microglia Preliminary experiments using the MTT assay were conducted with BV-2 murine microglia to determine the highest non-toxic concentrations of MCC950, shikonin, and oridonin under the experimental conditions used in this thesis work. LPS-stimulated BV-2 microglial cell cultures were treated with different concentrations of MCC950 (2-200 M), shikonin (40-2000 nM), oridonin (1-20 M), or their vehicle solution (DMSO), and were incubated for 24 h. The MTT assay was completed, and the results were compared to values obtained from the cells treated with vehicle solution (DMSO) only. Fig. 7 illustrates the highest non-toxic concentrations: 50 M of MCC950, 400 nM of shikonin, and 4 M of oridonin. At concentrations above these thresholds, MCC950, shikonin, and oridonin significantly decreased LPS-stimulated BV-2 microglial cell viability by about 25%. 49 Figure 7. Toxicity of inflammasome inhibitors towards LPS-stimulated BV-2 murine microglia. Increasing concentrations of MCC950 (2-200 M) (A), shikonin (40-2000 nM) (B), oridonin (1-20 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). Following 24 h incubation, the viability of BV-2 microglial cells was measured by the MTT assay. Data from three independent experiments performed on separate days are presented as means  SEM. * P < 0.05, ** P < 0.01, according to the Dunnett’s post-hoc test. P and F values shown were calculated by the one-way randomized block ANOVA. Dotted lines represent the viability of cells incubated in the cell culture medium only. 50 3.1.2 Effects of Inflammasome Inhibitors on the Secretion of Nitric Oxide by LPS-Stimulated BV-2 Murine Microglia Concentrations of nitrite, a metabolite of NO, in BV-2 microglial cell supernatants were measured using the Griess assay. NO is oxidized into nitrite, which can be used to estimate NO secretion by cells. Fig. 8A, C, E demonstrate nitrite concentrations in BV-2 murine microglia supernatants, and Fig. 8B, D, F illustrate the effects of inhibitors on the viability of LPS-stimulated BV-2 cells. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. Fig. 8B, D, F illustrate that the viability of BV-2 microglial cells in the absence of drugs was reduced by LPS stimulation; however, the addition of MCC950, shikonin, or oridonin at different concentrations did not further affect the viability of the LPS-stimulated BV-2 microglial cells and thus the effects of the inflammasome inhibitors on NO secretion by BV-2 cells were not due to their toxicity towards microglial cells. Unstimulated BV-2 cells did not secrete detectable levels of NO (data not shown), but LPS stimulation successfully induced NO release. In the absence of stimuli, the inflammasome inhibitors had no effect on NO secretion (data not shown). MCC950, shikonin, and oridonin reduced the secretion of NO by LPS-stimulated BV-2 murine microglia (Fig. 8A, C, E). MCC950 decreased the secretion of NO at all concentrations tested, while shikonin (400 nM) and oridonin (2 M and 4 M) reduced the secretion of NO at the highest concentrations tested. MCC950 only slightly reduced the secretion of NO, whereas shikonin decreased nitrite concentration by about 50% and oridonin by almost 70%. 51 Figure 8. Effects of inflammasome inhibitors on the secretion of nitric oxide by LPS-stimulated BV-2 murine microglia. Increasing concentrations of MCC950 (2-50 M) (A, B), shikonin (40-400 nM) (C, D), oridonin (1-4 M) (E, F), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). Following 24 h incubation, concentrations of nitrite were measured using the Griess assay (A, C, E) and the viability of BV-2 microglial cells was measured by the MTT assay (B, D, F). Data from four to five independent experiments performed on separate days are presented as means  SEM. * P < 0.05, ** P < 0.01, according to the Dunnett’s post-hoc test. P and F values shown were calculated by the one-way randomized block ANOVA. Dashed lines represent the detection limit of the Griess assay (A, C, E), and dotted lines represent the viability of cells incubated in the cell culture medium only (B, D, F). 52 3.1.3 Effects of Inflammasome Inhibitors on the Cytotoxicity of LPS-Stimulated BV-2 Murine Microglia towards NSC-34 or HT-22 Murine Neuronal Cells Each of the inhibitors regulated levels of NO in BV-2 microglial cell supernatants; therefore, I studied the effects of MCC950, shikonin, and oridonin on the overall cytotoxicity of BV-2 microglia towards neuronal cells using the supernatant transfer experiment. When NSC-34 or HT-22 neuronal cells are exposed to toxins secreted by LPS-stimulated BV-2 microglial cells, the neuronal viability decreases; therefore, inflammasome inhibitors which suppress the release of toxins by BV-2 microglial cells may provide neuroprotection resulting in increased neuronal viability. Fig. 9B, D, F show data obtained from experiments in which MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to BV-2 murine microglial cell cultures for 30 min before stimulation with LPS (100 ng/ml). Following 24 h incubation, BV-2 microglial supernatants prepared in the same manner were transferred onto NSC-34 neuronal cells (Fig. 9A-C), or HT-22 neuronal cells (Fig. 10A-C). After 72 h incubation with supernatants from BV-2 microglia, the viability of NSC-34 or HT-22 neuronal cells were determined using the MTT assay. As noted above (see Fig. 9B, D, F), at the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS-stimulated BV-2 microglial cells according to the MTT assay and thus, the effects of shikonin and oridonin on neurotoxin secretion by BV-2 microglia were not due to microglial cell death. Unstimulated BV-2 microglial cells did not influence the viability of NSC-34 or HT-22 neuronal cells (see dotted lines on Fig. 9A-C, 10A-C), while LPS-stimulated BV-2 microglial cells reduced the viability of NSC-34 and HT-22 neuronal cells. Treatment of BV-2 cells with the inflammasome inhibitors or their vehicle solution in the absence of stimulation did not induce the cytotoxic response of BV-2 microglial cells towards NSC-34 or HT-22 neuronal cells (data not shown). MCC950 and shikonin had no further significant effect on LPS-stimulated BV-2 53 microglial cell toxicity towards NSC-34 neuronal cells (Fig. 9A, B), while oridonin (1 M and 4 M) slightly reduced LPS-stimulated BV-2 microglial cell toxicity (Fig. 9C) indicating small but significant reduction in secretion of toxins by LPS-stimulated BV-2 cells. Experiments using HT-22 neuronal cells instead of NSC-34 demonstrated that MCC950 did not reduce toxicity of LPS-stimulated BV-2 microglial cells towards HT-22 neuronal cells (Fig. 10A). At the highest concentrations tested, shikonin (400 nM) and oridonin (4 M) reduced the toxicity of LPS-stimulated BV-2 microglial supernatants towards HT-22 neuronal cells (Fig. 10B, C). 54 Figure 9. Effects of inflammasome inhibitors on the cytotoxicity of LPS-stimulated BV-2 murine microglia cells towards NSC-34 murine neuronal cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 24 h incubation, BV-2 microglia supernatants were transferred onto NSC-34 neuronal cell cultures and the viability of BV-2 microglial cells was measured by the MTT assay (see Fig. 8B, D, F for corresponding values). Following 72 h incubation with supernatants from BV-2 microglial cells, the viability of NSC-34 neuronal cells was measured by the MTT assay. Data (means ± SEM) from four to five independent experiments performed on separate days are presented as a percent compared to values obtained from NSC-34 neuronal cells not exposed to BV-2 microglia supernatants. * P < 0.05, according to the Dunnett’s post-hoc test. P and F values calculated by the one-way randomized block ANOVA are shown. Dotted lines represent the mean viability of NSC-34 neuronal cells incubated in supernatants from unstimulated BV-2 cells. 55 Figure 10. Effects of inflammasome inhibitors on the cytotoxicity of LPS-stimulated BV-2 murine microglia cells towards HT-22 murine neuronal cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 24 h incubation, BV-2 microglia supernatants were transferred onto HT-22 neuronal cell cultures and the viability of BV-2 microglial cells was measured by the MTT assay (see Fig. 8B, D, F for corresponding values). Following 72 h incubation with supernatants from BV-2 microglial cells, the viability of HT-22 neuronal cells was measured by the MTT assay. Data (means ± SEM) from three to five independent experiments performed on separate days are presented as a percent compared to values obtained from HT-22 neuronal cells not exposed to supernatants from BV-2 microglial cells. *P < 0.05, according to the Dunnett’s post-hoc test. P and F values calculated by the one-way randomized block ANOVA are shown. Dotted lines represent the mean viability of HT-22 neuronal cells incubated in supernatants from unstimulated BV-2 microglial cells. 56 3.1.4 Effects of Inflammasome Inhibitors on the Secretion of IL-1 and TNF by LPS-Stimulated BV-2 Murine Microglia MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to BV-2 murine microglia cell cultures 30 min before stimulation with LPS (100 ng/ml). Following 24 h incubation, cell-free supernatant was collected and concentrations of IL-1 or TNF were determined using an ELISA. Fig. 11A-C demonstrate IL-1 concentrations in the supernatant from LPS-stimulated BV-2 murine microglia cells, and Fig. 12A-C illustrate TNF concentrations in the supernatant from LPS-stimulated BV-2 murine microglia cells. Since the supernatant was collected during the transfer experiments described in Section 3.1.3, the corresponding BV-2 cell viability data can be viewed in Fig. 8B, D, F for MCC950, shikonin, and oridonin, respectively. Unstimulated cells secreted detectable levels of IL-1 but not of TNF (see dotted lines on Fig. 11A-C, 12A-C). Treatment of BV-2 cells with inhibitors or their vehicle solution did not upregulate IL-1 or TNF secretion in the absence of LPS stimulation (data not shown). LPS stimulation of BV-2 cells induced IL-1 and TNF secretion. When added to BV-2 murine microglial cells before their stimulation with LPS, neither of the three inhibitors significantly affected the concentration of IL-1 (see Fig. 11A-C). MCC950, shikonin, and oridonin also did not have significant effects on LPS-induced TNF secretion by BV-2 cells (Fig. 12A-C). Interestingly, oridonin exhibited a trend towards downregulation of both IL-1 or TNF secretion by LPS-stimulated BV-2 cells (Fig. 11C, 12C). 57 Figure 11. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS-stimulated BV-2 murine microglia. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before they were stimulated with LPS (100 ng/ml). After 24 h incubation, concentrations of IL-1 in cell-free supernatant were determined using an ELISA. The viability of BV-2 microglial cells in the corresponding samples is presented as Fig. 8B, D, F in the previous section. Data from four independent experiments performed on separate days are presented as means  SEM. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of IL-1 by unstimulated BV-2 murine microglia. 0 2 5 20 50050100150200250IL-1b (pg/ml)MCC950 (mM)LPS + + + + +N = 4F = 0.6P = 0.6A0 40 100 200 400050100150200250IL-1b (pg/ml)N = 4F = 0.7P = 0.5Shikonin (nM)LPS + + + + + B0 1 2 4050100150200250IL-1b (pg/ml)N = 4F = 2.9P = 0.1Oridonin (mM)LPS + + + + C 58 Figure 12. Effects of inflammasome inhibitors on the secretion of TNF by LPS-stimulated BV-2 murine microglia. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 24 h incubation, concentrations of TNF in cell-free supernatant were determined using an ELISA. The viability of BV-2 microglial cells in the corresponding samples is presented as Fig. 8B, D, F in the previous section. Data from four independent experiments performed on separate days are presented as means  SEM. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of TNF by unstimulated BV-2 murine microglia. 0 2 5 20 50050100150200250TNF (pg/ml)N = 4F = 0.6P = 0.6MCC950 (mM)LPS + + + + +A0 1 2 4050100150200250TNF (pg/ml)N = 4F = 1.9P = 0.2Oridonin (mM)LPS + + + +C0 40 100 200 400050100150200250TNF (pg/ml)N = 4 F = 0.6P = 0.6Shikonin (nM)LPS + + + + +B 59 3.1.5 Effects of Inflammasome Inhibitors on the Secretion of Glutamate by LPS-Stimulated BV-2 Murine Microglia Concentrations of glutamate were measured in BV-2 microglial supernatants. Fig. 13A-C demonstrate above 2 mM glutamate concentrations in LPS-stimulated BV-2 murine microglial supernatants. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to BV-2 cell cultures for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. Unstimulated BV-2 microglial cells secreted detectable levels of glutamate (see dashed lines on Fig. 13A-C). As noted above (see Fig. 8B, D, F), at the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS-stimulated BV-2 microglial cells according to the MTT assay and thus the effects seen by MCC950 and shikonin on the secretion of glutamate by BV-2 microglia were not due to their toxicity towards microglial cells. In the absence of stimuli, the inflammasome inhibitors had no effect on the secretion of glutamate (data not shown). In the experiments shown in Fig. 13C, LPS-stimulation of BV-2 murine microglia did not upregulate the secretion of glutamate; however, glutamate secretion was upregulated in the experiments shown in Fig. 13A, B. MCC950 and shikonin reduced the secretion of glutamate by LPS-stimulated BV-2 murine microglia (Fig. 13A, B). MCC950 reduced the secretion of glutamate at two highest concentrations (5 M and 50 M), while shikonin reduced the secretion of glutamate at only the highest concentration tested (400 nM). Both MCC950 and shikonin only slightly reduced glutamate secretion, by about 15-20%; oridonin did not have a significant effect on glutamate secretion. However, these data may not be reliable, and the experiments shown in Fig. 13C should be repeated since the BV-2 cells did not respond to LPS-stimulation. 60 Figure 13. Effects of inflammasome inhibitors on the secretion of glutamate by LPS-stimulated BV-2 murine microglia. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 24 h incubation, concentrations of glutamate in cell-free supernatant were determined. The viability of BV-2 microglial cells in the corresponding samples is presented as Fig. 8B, D, F in the previous section. Data from four to six independent experiments performed on separate days are presented as means  SEM. * P < 0.05, ** P < 0.01 according to the Dunnett’s post-hoc test. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed line represents the detection limit of the glutamate assay, and the dotted lines represent the secretion of glutamate by unstimulated BV-2 murine microglia. 61 3.1.6 Effects of Inflammasome Inhibitors on the Phagocytosis of Latex Beads by Unstimulated and LPS-Stimulated BV-2 Murine Microglia In addition to the experiments determining the secretion of toxins and cytokines by BV-2 murine microglia, I studied phagocytosis (engulfment) of latex beads by BV-2 murine microglia. Unstimulated and LPS-stimulated BV-2 murine microglia were used to study the phagocytosis of latex beads. MCC950 (50 M), shikonin (400 nM), oridonin (4 M), or their vehicle solution (DMSO) was added to BV-2 cell cultures for 30 min prior to stimulation with LPS (400 ng/ml). Following 24 h incubation, fluorescent latex beads were added to cell cultures. After the plate was incubated for one h, fluorescence intensity of internalized latex beads was measured in viable cells identified by their intact nuclei. The phagocytosis of latex beads by LPS-stimulated BV-2 microglial cells was upregulated compared to unstimulated BV-2 microglial cells, and LPS also upregulated phagocytosis in the presence of each of the three inflammasome inhibitors (Fig. 14A-C). In the absence of LPS stimulation, shikonin and oridonin upregulated BV-2 microglial phagocytic activity by almost two-fold (Fig. 14B, C), while MCC950 displayed a trend towards significant upregulation of phagocytosis of the fluorescent latex beads (Fig. 14A). Similarly, shikonin and oridonin significantly upregulated LPS-stimulated BV-2 microglial phagocytic activity by approximately 25% (Fig. 14B, C), while MCC950 displayed a trend towards significant upregulation of LPS-stimulated BV-2 microglia phagocytosis of fluorescent latex beads (Fig. 14A). 62 Figure 14. Effects of inflammasome inhibitors on the phagocytosis of latex beads by unstimulated and LPS-stimulated BV-2 murine microglia. MCC950 (50 M) (A), shikonin (400 nM) (B), oridonin (4 M) (C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures, which were left unstimulated or stimulated 30 min later with LPS (400 ng/ml). Following 24 h incubation, fluorescent latex beads were added to cell cultures at a 10:1 bead to cell ratio. One h later, fluorescence intensity of beads in live cells was measured using a widefield microscope. Data from four to five independent experiments (N = 154 - 197 cells total for each experimental condition) are presented as means  SEM. * P < 0.05, ** P < 0.01 according to the Tukey’s post-hoc test. P and F values displayed on the graphs were calculated using the ordinary one-way ANOVA. 63 3.1.7 Effects of Oridonin in the Presence of PKD and JNK Inhibitors on the Secretion of Nitric Oxide by LPS-Stimulated BV-2 Murine Microglia Since oridonin affected the cytotoxic activity, the secretion of NO, and phagocytosis of latex beads by BV-2 murine microglia, I studied effects of oridonin in the presence of PKD/JNK inhibitors on the secretion of NO by BV-2 cells (Fig. 15A, B). Figure 15C illustrates the effects of inhibitors on the viability of LPS-stimulated BV-2 cells. CID755673 (50 M), a PKD inhibitor, and SP600125 (20 M), a JNK inhibitor, or their vehicle solution (DMSO) was added to BV-2 microglial cells for 30 min prior to the addition of oridonin (4 M) or its vehicle solution (DMSO). Following 30 min incubation, the cells were stimulated with LPS (100 ng/ml) for 24 h. Fig. 15C illustrates that the viability of BV-2 microglial cells in the absence of drugs was reduced by LPS stimulation; however, at the concentrations tested, oridonin, CID755673, and SP600125 did not further affect the viability of LPS-stimulated BV-2 microglial cells according to the MTT assay, and thus the effects of the inhibitors on the secretion of NO were not due to their toxicity toward BV-2 cells. Unstimulated BV-2 microglial cells did not secrete detectable levels of NO, while LPS stimulation induced the secretion. In the absence of stimuli, combinations of oridonin with CID755673 or SP600125 had no effect on NO secretion (data not shown). Oridonin plus CID755673 (Fig. 15A) and oridonin plus SP600125 (Fig. 15B) reduced the secretion of NO by LPS-stimulated BV-2 murine microglia. The combination of oridonin with a PKD or JNK inhibitor was more effective in reducing the secretion of NO by LPS-stimulated BV-2 murine microglia when compared to oridonin on its own. The combination of oridonin plus CID755673 illustrates an additive effect in the reduction of the secretion of NO when compared to oridonin or CID755673 on their own. An additive effect is when the combination of drugs is more efficient than each drug on its own. Oridonin plus CID755673 reduced the secretion of NO by 64 LPS-stimulated BV-2 microglia by nearly 75% when compared to oridonin or CID755673 on their own. The combination of oridonin plus SP600125 did not exhibit an additive effect in the reduction of the secretion of NO. Oridonin plus SP600125 significantly reduced the secretion of NO by LPS-stimulated BV-2 microglia by about 40% compared to SP600125 alone, and insignificantly reduced NO secretion by BV-2 microglia by about 30% compared to oridonin on its own. 65 Figure 15. Effects of oridonin in the presence of PKD and JNK inhibitors on the secretion of nitric oxide by LPS-stimulated BV-2 murine microglia. CID755673 (50 M) (A, C), SP600125 (20 M) (B, C), or their vehicle solution (DMSO) was added to BV-2 microglial cell cultures 30 min before the addition of oridonin (4 M) or its vehicle solution (DMSO). Following 30 min incubation, the cells were stimulated with LPS (100 ng/ml). After 24 h incubation, concentrations of nitrite were measured using the Griess assay (A, B) and the viability of BV-2 microglial cells was measured by the MTT assay (C). Data from five independent experiments performed on separate days are presented as means  SEM. * P < 0.05, ** P < 0.01, according to the Tukey’s post-hoc test. P and F values shown were calculated by the one-way randomized block ANOVA. Dashed lines represent the detection limit of the Griess assay (A, B), and the dotted line represents the viability of cells incubated in the cell culture medium only (C). 66 3.2 Human Microglia-Like Cells 3.2.1 Effects of Inflammasome Inhibitors on the Cytotoxicity of LPS plus IFN--Stimulated THP-1 Human Microglia-Like Cells towards SH-SY5Y Human Neuronal Cells When SH-SY5Y human neuronal cells are exposed to toxins secreted by THP-1 human microglia-like cells, the neuronal viability decreases; therefore, inflammasome inhibitors that increase the neuronal viability, when added to microglia-like cells, exhibit neuroprotective effects. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to THP-1 microglia-like cells for 30 min before stimulation with LPS (100 ng/ml) plus IFN- (150 U/ml). A combination of two stimulants was used in this case since the cytotoxic response of THP-1 cells cannot be induced by LPS alone, which is different from BV-2 murine microglia. Following 48 h incubation, THP-1 microglia-like cell supernatants were transferred onto SH-SY5Y neuronal cells and THP-1 microglia-like cell viability was measured using the MTT assay (Fig. 16B, D, F). After 72 h incubation with supernatant from THP-1 microglia-like cell cultures, viability of SH-SY5Y neuronal cells were determined using the MTT assay (Fig. 16A, C, E). At the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS plus IFN--stimulated THP-1 microglia-like cells according to the MTT assay (Fig. 16B, D, F) and thus the effect of oridonin on THP-1 microglia-like cells was not due to microglial cell death. Unstimulated THP-1 microglia-like cells did not influence the viability of SH-SY5Y neuronal cells significantly (see dotted lines on Fig. 16A, C, E), while supernatants from LPS plus IFN--stimulated THP-1 microglia-like cells reduced the viability of SH-SY5Y neuronal cells. Treatment of THP-1 cells with inflammasome inhibitors or their vehicle solution in the absence of stimulation did not induce the cytotoxic response of THP-1 microglia-like cells towards SH-SY5Y neuronal cells (data not shown). 67 MCC950 and shikonin had no significant effect on LPS plus IFN--stimulated THP-1 microglia-like cells toxicity towards SH-SY5Y neuronal cells (Fig. 16A, C), while oridonin, at the highest concentration used (4 M), significantly reduced LPS plus IFN--stimulated THP-1 microglia-like cell toxicity (Fig. 16E) indicating small but significant reduction in secretion of toxins by LPS plus IFN--stimulated THP-1 microglia-like cell. 68 Figure 16. Effects of inflammasome inhibitors on the cytotoxicity of LPS plus IFN--stimulated THP-1 human microglia-like cells towards SH-SY5Y human neuronal cells. Increasing concentrations of MCC950 (2-50 M) (A, B), shikonin (40-400 nM) (C, D), oridonin (1-4 M) (E, F), or their vehicle solution (DMSO) was added to THP-1 microglia-like cell cultures 30 min before their stimulation with LPS (100 ng/ml) plus IFN- (150 U/ml). Following 48 h incubation, THP-1 microglia-like cell supernatants were transferred onto SH-SY5Y neuronal cell cultures, and the viability of THP-1 microglia-like cells was measured by the MTT assay (B, D, F). After 72 h incubation with supernatants from THP-1 microglia-like cells, the viability of SH-SY5Y neuronal cells was measured by the MTT assay (A, C, E). Data (means ± 69 SEM) from four independent experiments performed on separate days are presented as a percent compared to values obtained from SH-SY5Y neuronal cells not exposed to supernatants from THP-1 microglia-like cells (A, C, E) or as a percent compared to THP-1 microglia-like cells incubated in cell culture medium only (B, D, F). * P < 0.05, according to the Dunnett’s post-hoc test. P and F values calculated by the one-way randomized block ANOVA are shown. Dotted lines represent the mean viability of SH-SY5Y neuronal cells incubated in supernatants from unstimulated THP-1 microglia-like cells (A, C, E) or the mean viability of unstimulated THP-1 microglia-like cells (B, D, F). 70 3.2.2 Effects of Inflammasome Inhibitors on the Secretion of IL-1 and TNF by LPS plus IFN--Stimulated THP-1 Human Microglia-Like Cells Fig. 17A-C demonstrate the secretion of IL-1 by LPS plus IFN-γ-stimulated THP-1 microglia-like cell supernatants, and Fig. 18A-C illustrate the secretion of TNF by LPS plus IFN-γ-stimulated THP-1 microglia-like cell supernatants. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to THP-1 microglia-like cells for 30 min before stimulation with LPS (100 ng/ml) plus IFN- (150 U/ml). Following 48 h incubation, cell-free supernatant was collected and concentrations of IL-1 or TNF were determined using an ELISA. Since the supernatant was collected during the transfer experiments described in Section 2.5.4, the corresponding THP-1 microglia-like cell viability data can be viewed in Fig. 17B, D, F for MCC950, shikonin, and oridonin, respectively. At the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS plus IFN--stimulated THP-1 microglia-like cells, according to the MTT assay (Fig. 16B, D, F) and thus the effect of MCC950 on the secretion of IL-1 by THP-1 microglia-like cells was not due to microglial cell death. Unstimulated THP-1 cells did not secrete detectable IL-1 or TNF levels (see dotted lines in Fig. 17A-C, 18A-C). Treatment of THP-1 microglia-like cells with inhibitors or their vehicle solution did not upregulate IL-1β or TNF secretion in the absence of LPS stimulation (data not shown). LPS stimulation of THP-1 cells induced IL-1β and TNF secretion. MCC950 (2 M, 20 M, and 50 M) significantly reduced the secretion of IL-1β by LPS plus IFN-γ-stimulated THP-1 microglia-like cells by approximately 60% (Fig. 17A), while shikonin and oridonin had no effect (Fig. 17B, C). MCC950, shikonin, and oridonin did not have significant effects on LPS plus IFN-γ-induced TNF secretion by THP-1 microglia-like cells (Fig. 18A-C). 71 Figure 17. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS plus IFN--stimulated THP-1 human microglia-like cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to THP-1 microglia-like cell cultures 30 min before their stimulation with LPS (100 ng/ml) plus IFN- (150 U/ml). After 48 h incubation, concentrations of IL-1 in cell-free supernatant were determined using an ELISA. The viability of THP-1 microglia-like cells in the corresponding samples is presented as Fig. 16B, D, F in the previous section. Data from three to four independent experiments performed on separate days are presented as means  SEM. * P < 0.05 according to the Dunnett’s post-hoc test. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of IL-1 by unstimulated THP-1 human microglia-like cells. 72 Figure 18. Effects of inflammasome inhibitors on the secretion of TNF by LPS plus IFN--stimulated THP-1 human microglia-like cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to THP-1 microglia-like cell cultures 30 min before their stimulation with LPS (100 ng/ml) plus IFN- (150 U/ml). After 48 h incubation, concentrations of TNF in cell-free supernatant were determined using an ELISA. The viability of THP-1 microglia-like cells in the corresponding samples is presented as Fig. 16B, D, F in the previous section. Data from three independent experiments performed on separate days are presented as means  SEM. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of TNF by unstimulated THP-1 human microglia-like cells. 0 2 5 20 500100200300400500600TNF (pg/ml)N = 3F = 1.3P = 0.3MCC950 (mM)LPS + + + + + IFN-g + + + + + A0 1 2 40100200300400500600TNF (pg/ml)N = 3F = 1.7P = 0.2Oridonin (mM)LPS + + + + IFN-g + + + + C0 40 100 200 4000100200300400500600TNF (pg/ml)N = 3F = 0.1P = 0.8Shikonin (nM)LPS + + + + + IFN-g + + + + + B 73 3.2.3 Effects of Inflammasome Inhibitors on the Secretion of ROS by DMSO-Differentiated HL-60 Cells Primed with LPS and Stimulated with fMLP The respiratory burst assay was used to determine the release of ROS by immune-stimulated microglia-like cells. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to DMSO-differentiated HL-60 human microglia-like cells for 30 min before being primed with LPS (500 ng/ml). Following 24 h incubation, cells were stimulated with fMLP (1.2 M) to induce the release of ROS, which react with luminol to produce CHL. The resulting CHL was measured over a 25 min period. The viability of DMSO-differentiated HL-60 microglia-like cells in the absence of the inflammasome inhibitors was reduced by LPS priming. Shikonin at 200 nM, but not MCC950 or oridonin, reduced the viability of DMSO-differentiated HL-60 microglia-like cells (Fig. 19D). Even though shikonin decreased cell viability at 200 nM (Fig. 19D), decreased ROS was observed at the lowest concentration (40 nM) and thus this effect was not due to the toxicity of shikonin toward HL-60 cells. MCC950 and oridonin also did not affect the viability of LPS-primed HL-60 microglia-like cells, according to the MTT assay (Fig. 19B, F); therefore, the inhibitory effect of MCC950 observed on the secretion of ROS was not due to toxicity. Unprimed DMSO-differentiated HL-60 microglia-like cells did not secrete high levels of ROS (see dotted-dashed line in Fig. 19A, C, E), while LPS-priming enhanced the secretion of ROS in response to fMLP stimulation as determined by increase in CHL signal. In the absence of priming, the inflammasome inhibitors had no effect on ROS secretion (data not shown). MCC950 and shikonin significantly reduced the secretion of ROS by LPS-primed and fMLP-stimulated DMSO-differentiated HL-60 microglia-like cells (Fig. 19A, C), while oridonin did not have a significant effect (Fig. 19E). MCC950 significantly reduced the secretion of ROS by 74 approximately 20% at the highest concentration tested (50 M), while shikonin reduced the secretion of ROS by over 35% at the lowest concentration tested (40 nM). Figure 19. Effects of inflammasome inhibitors on the secretion of ROS by DMSO-differentiated HL-60 cells primed with LPS and stimulated with fMLP. Increasing concentrations of MCC950 (2-50 M) (A, B), shikonin (40-400 nM) (C, D), oridonin (1-4 M) (E, F), or their vehicle solution (DMSO) was added to DMSO-differentiated HL-60 microglia-like cell cultures 30 min before their priming with LPS (500 ng/ml) for 24 h. Cells were stimulated with fMLP (1.2 M) and CHL was measured over a 25 min period (A, C, E).The viability of HL-60 microglia-like cells was measured by the MTT assay (B, D, F). Data from six to eight independent experiments performed on separate days are presented as means ± SEM. * P < 0.05 according to the Dunnett’s post-hoc test. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dotted lines represent the viability 75 of cells incubated in the cell culture medium only, and the dotted-dashed lines represent the secretion of ROS by unprimed HL-60 microglia-like cells. 3.3 U-118 MG Human Astrocyte-like cells 3.3.1 Effects of Inflammasome Inhibitors on LPS-Stimulated U-118 MG Human Astrocytic Cell-Mediated Cytotoxicity and IL-1 plus IFN--Stimulated U-118 MG Human Astrocytic Cell-Mediated Cytotoxicity towards SH-SY5Y Human Neuronal Cells When SH-SY5Y human neuronal cells are exposed to toxins secreted by U-118 MG human astrocytic cells, the neuronal viability decreases; therefore, inflammasome inhibitors which suppress the release of toxins by U-118 MG astrocytic cells may provide neuroprotection resulting in increased neuronal viability. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cells for 30 min before stimulation with IL-1 (150 U/ml) plus IFN- (150 U/ml). Following 48 h incubation, U-118 MG astrocytic cell supernatants were transferred onto SH-SY5Y neuronal cells and U-118 MG astrocytic cell viability was measured. At the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS-stimulated or IL-1 plus IFN--stimulated U-118 MG astrocytic cells, according to the MTT assay (Fig. 20B, D, F and Fig. 21B, D, F) and thus, the effect of shikonin on viability of SH-SY5Y neuronal cells was not due to astrocytic cell death. After 72 h incubation with supernatant from U-118 MG astrocytic cell cultures, the viability of SH-SY5Y neuronal cells were determined using the MTT assay (Fig. 20A, C, E and Fig. 21A, C, E). Unstimulated U-118 MG astrocytic cells did not significantly influence the viability of SH-SY5Y neuronal cells (see dotted lines on Fig. 20A, C, E and Fig. 21A, C, E), while LPS-stimulated U-118 MG astrocytic cells minimally reduced the viability of SH-SY5Y neuronal cells. Treatment of U-118 MG cells with inflammasome inhibitors or their vehicle solution 76 (DMSO) in the absence of stimulation did not induce the cytotoxic response of U-118 MG astrocytic cells towards SH-SY5Y neuronal cells (data not shown). The inflammasome inhibitors had no effect on LPS-stimulated or IL-1 plus IFN--stimulated U-118 MG astrocytic cell viability (Fig. 20B, D, F and 21B, D, F). The inflammasome inhibitors had no further significant effect on LPS-stimulated U-118 MG astrocytic cell toxicity towards SH-SY5Y neuronal cells (Fig. 20A, C, E). MCC950 and oridonin had no effect on IL-1 plus IFN--stimulated U-118 MG astrocytic cell toxicity towards SH-SY5Y neuronal cells (Fig. 21A, E). At the highest concentration tested, shikonin (400 nM) slightly reduced IL-1 plus IFN--stimulated U-118 MG astrocytic cell toxicity (Fig. 21C) indicating small but significant reduction in secretion of toxins. 77 Figure 20. Effects of inflammasome inhibitors on the cytotoxicity of LPS-stimulated U-118 MG human astrocytic cells towards SH-SY5Y human neuronal cells. Increasing concentrations of MCC950 (2-50 M) (A, B), shikonin (40-400 nM) (C, D), oridonin (1-4 M) (E, F), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures 30 min before their stimulation with LPS (100 ng/ml). Following 48 h incubation, U-118 MG astrocytic supernatants were transferred onto SH-SY5Y neuronal cell cultures, and the viability of U-118 MG astrocytic cells was measured by the MTT assay (B, D, F). After 72 h incubation with supernatants from U-118 MG astrocytic cells, the viability of SH-SY5Y neuronal cells was measured by the MTT assay (A, C, E). Data (means ± SEM) from three independent experiments performed on separate days are presented as a percent compared to values obtained from SH-SY5Y neuronal cells not exposed to supernatants from U-118 MG astrocytic cells (A, C, E) or as a percent compared to U-118 MG astrocytic cells incubated in cell culture medium only (B, D, F). Dotted lines represent the mean viability of SH-SY5Y neuronal cells incubated in supernatants from unstimulated U-118 MG astrocytic cells (A, C, E) or the mean viability of unstimulated U-118 MG astrocytic cells (B, D, F). 0 2 5 20 50020406080100%SH-SY5YCell ViabilityMCC950 (mM)N = 3F = 1.2P = 0.3LPS + + + + + A0 40 100 200 400020406080100%SH-SY5Y Cell ViabilityShikonin (nM)N = 3 F = 1.0P = 0.4LPS + + + + + C0 1 2 4020406080100%SH-SY5Y Cell ViabilityOridonin (mM)LPS + + + + N = 3F = 1.4P = 0.3E0 2 5 20 50020406080100%U-118 MG Cell ViabilityMCC950 (mM)N = 3F = 3.1P = 0.2LPS + + + + + B0 40 100 200 400020406080100%U-118 MG Cell ViabilityShikonin (nM)N = 3F = 0.3P = 0.7LPS + + + + + D0 1 2 4020406080100%U-118 MG Cell ViabilityOridonin (mM)N = 3F = 1.6P = 0.2LPS + + + + F 78 Figure 21. Effects of inflammasome inhibitors on the cytotoxicity of IL-1 plus IFN--stimulated U-118 MG human astrocytic cells towards SH-SY5Y human neuronal cells. Increasing concentrations of MCC950 (2-50 M) (A, B), shikonin (40-400 nM) (C, D), oridonin (1-4 M) (E, F), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures 30 min before their stimulation with IL-1 (150 U/ml) plus IFN- (150 U/ml). Following 48 h incubation, U-118 MG astrocytic supernatants were transferred onto SH-SY5Y neuronal cell cultures, and the viability of U-118 MG astrocytic cells was measured by the MTT assay (B, D, F). After 72 h incubation with supernatants from U-118 MG astrocytic cells, the viability of SH-SY5Y neuronal cells was measured by the MTT assay (A, C, E). Data (means ± SEM) from four to five independent experiments performed on separate days are presented as a percent compared to values obtained from SH-SY5Y neuronal cells not exposed to supernatants from U-118 MG astrocytic cells (A, C, E) or as a percent compared to U-118 MG astrocytic cells incubated in cell culture medium only (B, D, F). * P < 0.05 according to the Dunnett’s post-hoc test. P and F values calculated by the one-way randomized block ANOVA are shown. Dotted 79 lines represent the mean viability of SH-SY5Y neuronal cells incubated in supernatants from unstimulated U-118 MG astrocytic cells (A, C, E) or the mean viability of unstimulated U-118 MG astrocytic cells (B, D, F). 3.3.2 Effects of Inflammasome Inhibitors on the Secretion of IL-1 and TNF by LPS-Stimulated U-118 MG Human Astrocytic Cells Fig. 22A-C demonstrate the secretion of IL-1 by LPS-stimulated U-118 MG astrocytic cells, and Fig. 23A-C illustrate the secretion of TNF by LPS-stimulated U-118 MG astrocytic cells. MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures for 30 min before stimulation with LPS (100 ng/ml). Following 48 h incubation, cell-free supernatant was collected and concentrations of IL-1 or TNF were determined using an ELISA. Since the supernatant was collected during the transfer experiments described in Section 3.3.1, the corresponding U-118 MG astrocytic cell viability data can be viewed in Fig. 21B, D, F for MCC950, shikonin, and oridonin, respectively. As noted above (see Fig. 21B, D, F), at the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS-stimulated U-118 MG astrocytic cells according to the MTT assay and thus, the effects of the inflammasome inhibitors on the secretion of IL-1 by U-118 MG astrocytes were not due to astrocytic cell death. Unstimulated cells did not secrete detectable IL-1 or TNF levels (see dotted lines in Fig. 22A-C, Fig. 23A-C). Treatment of U-118 MG astrocytic cells with inhibitors or their vehicle solution did not upregulate IL-1 or TNF secretion in the absence of LPS stimulation (data not shown). LPS stimulation of U-118 MG astrocytic cells induced IL-1 and TNF secretion. All inflammasome inhibitors significantly reduced the secretion of IL-1 by LPS-stimulated U-118 MG astrocytic cells (See Fig. 22A-C). In U-118 MG astrocytic cells stimulated with LPS, MCC950 (2 M) and shikonin (40 and 400 nM) reduced the secretion of IL-1 by about half, 80 while oridonin (1 M and 4 M) reduced the secretion of IL-1 by approximately 75%. Neither of the three inhibitors affected the secretion of TNF by LPS-stimulated U-118 MG astrocytic cells (Fig. 23A-C). Figure 22. Effects of inflammasome inhibitors on the secretion of IL-1 by LPS-stimulated U-118 MG human astrocytic cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 48 h incubation, concentrations of IL-1 in cell-free supernatant were determined using an ELISA. The viability of U-118 MG astrocytic cells in the corresponding samples is presented as Fig. 21B, D, F in the previous section. Data from five to six independent experiments performed on separate days are presented as means  SEM. * P < 0.05 according to the Dunnett’s post-hoc test. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of IL-1 by unstimulated U-118 MG astrocytic cells. 81 Figure 23. Effects of inflammasome inhibitors on the secretion of TNF by LPS-stimulated U-118 MG human astrocytic cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 48 h incubation, concentrations of TNF in cell-free supernatant were determined using an ELISA. The viability of U-118 MG astrocytic cells in the corresponding samples is presented as Fig. 21B, D, F in the previous section. Data from five to six independent experiments performed on separate days are presented as means  SEM. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed lines represent the detection limit of the ELISA, and the dotted lines represent the secretion of TNF by unstimulated U-118 MG astrocytic cells. 0 2 5 20 50050100150200 TNF (pg/ml)MCC950 (mM)LPS + + + + + N = 6 F = 1.5P = 0.2A0 1 2 4050100150200TNF (pg/ml)Oridonin (mM)LPS + + + + N = 6 F = 1.0P = 0.3C0 40 200 400050100150200TNF (pg/ml)Shikonin (nM)LPS + + + + N = 5F = 3.3P = 0.05B 82 3.3.3 Effects of Inflammasome Inhibitors on the Secretion of Glutamate by LPS-Stimulated U-118 MG Human Astrocytic Cells Concentrations of glutamate in LPS-stimulated U-118 MG astrocytic cell supernatants were determined (Fig. 24A-C). MCC950 (2-50 M), shikonin (40-400 nM), oridonin (1-4 M), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures for 30 min prior to stimulation with LPS (100 ng/ml) for 48 h. As noted above (see Fig. 20B, D, F), at the concentrations tested, MCC950, shikonin, and oridonin did not affect the viability of LPS-stimulated U-118 MG astrocytic cells according to the MTT assay. Thus, the increase in glutamate levels in the presence of oridonin was not due to astrocytic cell death since cell death is usually linked with decreased secretion of toxins. Unstimulated U-118 MG astrocytic cells secreted detectable levels of glutamate (see dotted line, Fig. 24A-C), while LPS stimulation upregulated the secretion of glutamate. In the absence of stimuli, the inflammasome inhibitors had no effect on the secretion of glutamate (data not shown). MCC950 and shikonin did not affect the secretion of glutamate by LPS-stimulated U-118 MG astrocytes (Fig. 24A, B); however, oridonin (4 M) increased the secretion of glutamate by approximately 25% at the highest concentration tested (Fig. 24C). 83 Figure 24. Effects of inflammasome inhibitors on the secretion of glutamate by LPS-stimulated U-118 MG human astrocytic cells. Increasing concentrations of MCC950 (2-50 M) (A), shikonin (40-400 nM) (B), oridonin (1-4 M) (C), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures 30 min before their stimulation with LPS (100 ng/ml). After 48 h incubation, concentrations of glutamate in cell-free supernatant were determined. The viability of U-118 MG astrocytic cells in the corresponding samples is presented as Fig. 20B, D, F in the previous section. Data from four to five independent experiments performed on separate days are presented as means  SEM. * P < 0.05 according to the Dunnett’s post-hoc test. P and F values displayed on the graphs were calculated using the one-way randomized block ANOVA. The dashed line represents the detection limit of the glutamate assay, and the dotted lines represent the secretion of glutamate by unstimulated U-118 MG astrocytic cells. 84 3.3.4 Effects of Inflammasome Inhibitors on the Phagocytosis of Latex Beads by Unstimulated and LPS-Stimulated U-118 MG Human Astrocytic Cells Unstimulated and LPS-stimulated U-118 MG astrocytic cells were used to study the phagocytosis of latex beads. MCC950 (50 M), shikonin (400 nM), oridonin (4 M), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures for 30 min prior to stimulation with LPS (400 ng/ml). Following 48 h incubation, fluorescent latex beads were added to cell cultures. After the cells were incubated for one h, fluorescence intensity of internalized latex beads was measured in viable cells identified by their intact nuclei. The phagocytosis of latex beads by U-118 MG astrocytic cells was upregulated, or showed a trend towards upregulation, in the presence of LPS stimulation when compared to unstimulated U-118 MG astrocytic cells. LPS also upregulated phagocytosis in the presence of shikonin and oridonin, but not MCC950 (Fig. 25A-C). In the absence of LPS stimulation, MCC950 significantly upregulated phagocytic activity (Fig. 25A), while shikonin and oridonin induced trends towards upregulated phagocytosis (Fig. 25B, C). MCC950 and shikonin induced trends towards upregulated LPS-stimulated U-118 MG astrocytic phagocytosis of latex beads, while oridonin significantly upregulated phagocytic activity of LPS-stimulated U-118 MG cells. 85 Figure 25. Effects of inflammasome inhibitors on the phagocytosis of latex beads by unstimulated and LPS-stimulated U-118 MG human astrocytic cells. MCC950 (50 M) (A), shikonin (400 nM) (B), oridonin (4 M) (C), or their vehicle solution (DMSO) was added to U-118 MG astrocytic cell cultures, which were left unstimulated or stimulated 30 min later with LPS (400 ng/ml). Following 48 h incubation, fluorescent latex beads were added to cell cultures at a 10:1 bead to cell ratio. One h later, fluorescence intensity of beads in live cells was measured using a widefield microscope. Data from three to four independent experiments (N = 112-151 cells total for each experimental condition) are presented as means  SEM. * P < 0.05, ** P < 0.01 according to the Tukey’s post-hoc test. P and F values displayed on the graphs were calculated using the ordinary one-way ANOVA. 86 3.4 Summary of Results Tables 2-4 summarize the results of the study on the effects of MCC950, shikonin, and oridonin on BV-2 murine microglia, THP-1 and HL-60 human microglia-like cells, and U-118 MG human astrocytic-like cells. Arrows ( and ) indicate an increase or decrease in the effect of the inflammasome inhibitors. The star (*) symbol represents significance, and “X” indicates no effect was observed. Table 2. The effects of MCC950, shikonin, and oridonin on BV-2 murine microglia. MCC950 Shikonin Oridonin NO secretion * * * BV-2 mediated toxicity towards NSC-34 neuronal cells X X * BV-2 mediated toxicity towards HT-22 neuronal cells X * * IL-1 secretion X X  TNF secretion X X  Glutamate secretion * * X Phagocytosis  * * 87 Table 3. The effects of MCC950, shikonin, and oridonin on human microglia-like cells. MCC950 Shikonin Oridonin THP-1 mediated toxicity towards SH-SY5Y neuronal cells X X * IL-1 secretion by THP-1 microglia-like cells * X X TNF secretion by THP-1 microglia-like cells X X X ROS secretion by HL-60 microglia-like cells * *  Table 4. The effects of MCC950, shikonin, and oridonin on U-118 MG human astrocytic-like cells. MCC950 Shikonin Oridonin U-118 MG mediated toxicity towards SH-SY5Y neuronal cells X * X IL-1 secretion * * * TNF secretion X X X Glutamate secretion X X * Phagocytosis   * 88 Chapter 4: Discussion 4.1 Cells Reactive glial cells release inflammatory cytokines and neurotoxins, thus contributing to the progression of neurodegeneration. Inflammasome assembly and structure in glial cells have been described in a recent review by Mata-Martínez et al. (2022) and in section 1.3.2 of this thesis. It is known that the activity of inflammasomes can influence inflammation in the brain and alter microglial function (Chiarini et al., 2020; Heneka et al., 2013; Liu et al., 2020; Nizami et al., 2019). The effects of the inflammasome inhibitors MCC950, shikonin, and oridonin on cytokine secretion, cytotoxicity, and phagocytic activity of microglia have been described (Dempsey et al., 2017; Li et al., 2022; Nam et al., 2008; Nie et al., 2021; Xu et al., 2009). However, the effects of these inflammasome inhibitors on astrocyte phagocytosis have not been well characterized. Previous data investigating the roles of these inhibitors on the secretion of pro-inflammatory cytokines, such as TNF, are conflicting and requires further investigation. Hence, I hypothesized that the three structurally different inflammasome inhibitors, MCC950, shikonin, and oridonin, regulate a broad range of immune responses of microglial and astrocytic cells. Species specificity of inflammasomes is an important consideration and several studies have established that a given stimulus can have differential effects across several species. It has been reported that murine monocytes employ an all-or-nothing response while human monocytes adopt a gradual response to inflammasome activation (Gaidt et al., 2016); accordingly, the pro-inflammatory responses observed in murine cells are stronger than in human cells. LPS triggered the secretion of IL-1 by human monocytes but not murine monocytes (Gaidt et al., 2016; Sha et al., 2014), and thus did not activate the NLRP3 inflammasome in murine cells given that IL-1 is 89 a product of inflammasome activation. In THP-1 human monocytes and HEK293T human embryonic kidney cells, but not murine macrophages isolated from bone marrow, the NLRP3 inflammasome could be activated by the bacterium Francisella tularensis (Atianand et al., 2011), which further provides evidence that NLRP3 activation is distinct between human and murine cells. In another study, it was determined that a single signal, such as LPS, can be used to prime human THP-1 cells for inflammasome activation, but murine cells may need an additional secondary signal, such as ATP (Wang et al., 2013); this suggests that human microglia may have increased sensitization for NLRP3 activation compared to murine microglia. Both microglial and astrocytic cell lines were studied in this thesis work; therefore, the similarities and differences between these two glial cell types and their inflammasome activation must also be considered. A and -synuclein have previously been established to induce NLRP3 activation in BV-2 murine microglial cells and primary human microglia (Liu et al., 2020; Pike et al., 2021). Interestingly, it has been suggested that increased secretion of IL-1 by C57BL/6 mouse brain microglia indicates the expression level of the NLRP3 (Gustin et al., 2015). Human astrocytes have been shown to express the NLRP2 inflammasome (Minkiewicz et al., 2013) and the NLRP3 inflammasome (Lu et al., 2014). A more recent study reported that stimulated murine and human primary astrocytes express the NLRP3 inflammasome to a stronger extent than microglia (Freeman et al., 2017). The aforementioned differences in inflammasome activation between the two glial cell types, astrocytes and microglia, are seen throughout the experiments described in this thesis. 90 4.2 Toxins 4.2.1. Nitric Oxide LPS stimulation of murine microglia induces production of NO, which can lead to tissue injury when secreted at high levels (Corradin et al., 1993; Han et al., 2021; Lo et al., 2022). The relevance of NO secretion by murine microglia to human pathologies is not well understood since mice have differently regulated enzymes that synthesize NO compared to human cells (Schoedon et al., 1993; Vodovotz et al., 1994). Nevertheless, previous studies have determined that MCC950, shikonin derivatives, and oridonin inhibit the release of NO by primary rat microglia (Danielski et al., 2020; Nam et al., 2008; Xu et al., 2009). For example, Danielski et al. (2020) determined that MCC950 reduced NO secretion in sepsis-induced primary rat microglia. Nam et al. (2008) found that shikonin derivatives, isobutytylshikonin and isovalerylshikonin reduced the secretion of NO by LPS-stimulated primary rat microglia, and Xu et al. (2009) reported that oridonin reduced the secretion of NO by LPS-stimulated primary rat microglia. My experiments confirmed these findings by demonstrating that MCC950, shikonin, and oridonin significantly reduced NO secretion by LPS-stimulated BV-2 murine microglia. To the best of my knowledge, this is the first demonstration that MCC950, shikonin, and oridonin downregulate the secretion of NO by immortalized murine microglia. 4.2.2. Glutamate To date, no studies have established the effects of the inflammasome inhibitors on glutamate secretion by microglia; however, LPS-induced BV-2 microglial glutamate release has been studied (Lee et al., 2018). An increase in extracellular glutamate can trigger the release of ATP from glial cells, which then activates the NLRP3 inflammasome (Yuan et al., 2022). The 91 data presented in this thesis demonstrated that MCC950 and shikonin, but not oridonin, significantly reduced the secretion of glutamate by LPS-stimulated BV-2 murine microglia. In LPS-stimulated U-118 MG astrocytic cells, neither MCC950 nor shikonin had a significant effect on glutamate secretion, while oridonin significantly increased the secretion of glutamate. Glutamate processing is known to occur in astrocytes, and metabolism and uptake of glutamate is an important mechanism in astrocytes, which is not considered a main function of microglia (Mahmoud et al., 2019). The use of U-118 MG cells as astrocyte models in this thesis work is supported by findings from Tabatabaee & Menard (2020) who showed that these cells respond to extracellular glutamate in the same manner as primary astrocytes. Likewise, McNaught & Jenner (2000) determined that the secretion of glutamate can be induced by LPS stimulation of cultured primary astrocytes from Wistar rats. To the best of my knowledge, this is the first study to determine the effects of inflammasome inhibitors on glutamate secretion by U-118 MG cells. Excess extracellular glutamate induces neuronal excitotoxicity (Malarkey & Parpura, 2008). Yao et al. (2022) obtained results similar to my observations by using primary rat neurons treated with glutamate in vitro. They showed that shikonin protected neurons against glutamate-induced toxicity by regulating endoplasmic reticulum stress (Yao et al., 2022). Interestingly, Lin et al. (2019) used kainic acid to upregulate glutamate secretion by PC-12 rat adrenal medulla cells and demonstrated that oridonin (0.5 – 2 M) abrogated the kainic acid-mediated increase in glutamate secretion. Although previous studies demonstrated that shikonin and oridonin reduced glutamate secretion, these results were obtained using neuronal cells and cannot be directly compared to the data derived from the experiments with glial cells. 92 4.2.3 Reactive Oxygen Species DMSO-differentiated human HL-60 cells were used to model the respiratory burst response of microglia. The high intracellular generation of ROS can be beneficial by causing the apoptosis of tumor or cancer cells (Duan et al., 2014); however, in neurodegenerative diseases, high levels of ROS are harmful to both glia and neurons. Previous studies determined that LPS stimulation induced the activation of the inflammasome in microglia and downstream generation of ROS (Arioz et al., 2019; Kim et al., 2019; Ślusarczyk et al., 2018). The data presented in this thesis show that MCC950 and shikonin significantly reduced the secretion of ROS by immune primed and activated HL-60 human microglia-like cells, while oridonin insignificantly decreased the secretion of ROS by these cells. Recently, Li et al. (2022) determined that MCC950 decreased the secretion of ROS by oxygen-glucose deprived/reperfused THP-1 microglia-like cells. THP-1 cells were not utilized to study ROS secretion in this thesis since they have a delayed secretion of ROS in response to immune stimulation. For example, ROS secretion by THP-1 microglia-like cells could be detected one to ten h after treatment with phorbol-12-myristate-13-acetate (PMA) (Kasai et al., 2005), which is a slow response compared to the immediate ROS release (in about 30 s) by HL-60 microglia-like cells stimulated with fMLP. A previous study demonstrated that shikonin increased the secretion of ROS by HL-60 cells when used at a highly toxic concentration of 5 M (Duan et al., 2014), which is over 12 times the non-toxic concentration determined in this thesis work. Although toxic concentrations are valuable in cancer studies, non-toxic concentrations are desirable in neurodegenerative disease studies to ensure that treatment does not reduce the number of living glial cells in the brain and prevent normal functioning. Studies have also determined that oridonin, at non-toxic concentrations, attenuated ROS accumulation by LPS-stimulated human kidney (HK)-2 cells and LPS plus IFN--stimulated monocyte-derived Kupffer cells (Huang et al., 2020; Zhu et al., 2021). Oridonin in 93 my hands exhibited a trend toward decreasing the secretion of ROS by LPS-primed and fMLP-stimulated HL-60 human microglia-like cells. MCC950 and shikonin significantly reduced the secretion of ROS by LPS-primed and fMLP-stimulated HL-60 human microglia-like cells. 4.2.4 Toxins Summary The results presented in this thesis confirm previous research concerning the actions of MCC950, shikonin, and oridonin on the secretion of NO by murine microglial cells. To date, no studies have determined the effects of the inflammasome inhibitors on the secretion of glutamate by murine and human microglia or astrocytes. MCC950 was previously shown to decrease the production of ROS by LPS-stimulated THP-1 cells and oridonin by LPS-stimulated kidney cells. Additionally, shikonin has been previously studied using stimulated HL-60 cells at highly toxic concentrations. Therefore, to the best of my knowledge, the work in this thesis is the first to determine the effects of the inflammasome inhibitors on DMSO-differentiated LPS-primed and fMLP-stimulated HL-60 microglia-like cells at non-toxic concentrations. 4.3 Pro-inflammatory Cytokines In LPS-stimulated BV-2 murine microglia, oridonin exhibited a trend toward decreasing the secretion of IL-1 while MCC950 and shikonin did not have a significant effect. As presented in this thesis, none of the inflammasome inhibitors had a significant effect on LPS-stimulated BV-2 murine microglial secretion of TNF. Previous research on stimulated murine microglia determined the effects of inflammasome inhibitors on pro-inflammatory cytokine secretion and/or expression and concluded that MCC950, shikonin, and oridonin might have an effect on the secretion of IL-1, TNF, IL-6, and IL-10 (Danielski et al., 2020; Dempsey et al., 2017; Nam et al., 2008; Xu et al., 2009). Specifically, Danielski et al. (2020) concluded that MCC950 decreased the secretion of IL-1, TNF, IL-6, and IL-10 by sepsis-induced primary rat 94 microglial cells. On the contrary, Dempsey et al. (2017) determined that MCC950 decreased the secretion of IL-1, but not TNF or IL-6, by LPS plus A-stimulated primary murine microglia. Nam et al. (2008) found that shikonin derivatives reduced the secretion of TNF and IL-1, as well as the expression of COX-2, a key enzyme of inflammatory pathways, by LPS-stimulated primary rat microglia and reduced NF-kB DNA binding activity. Xu et al. (2009) concluded that oridonin inhibited the secretion of TNF, IL-1, and IL-6 by LPS-stimulated primary rat microglia by blocking NF-kB DNA binding activity and thus exhibiting immunoregulatory effects. In BV-2 microglia, it has been shown that shikonin and oridonin at potentially toxic concentrations may reduce the LPS-induced secretion of pro-inflammatory cytokines (Liang et al., 2022; Prasad et al., 2015). For example, Prasad et al. (2015) determined that shikonin (800 nM) effectively reduced the secretion of TNF and expression of COX-2 by LPS-stimulated BV-2 murine microglia. These effects were seen at a concentration of shikonin twice what I determined to be the highest non-toxic concentration to BV-2 murine microglia cells. Liang et al. (2022) found that oridonin (10 - 20 M) effectively reduced the secretion of IL-1 and IL-18 by LPS-stimulated BV-2 murine microglia, but they did not observe any effects of oridonin at concentrations of 5 M or lower. The highest non-toxic concentration of oridonin toward BV-2 murine microglia cells presented in this study was 4 M. While Liang et al. (2022) results showed that these higher concentrations (10-20 M) were not toxic to BV-2 murine microglia cells, my studies determined that such high concentrations are toxic and therefore should not be used in experiments determining specific inhibition of cytokine secretion by this cell type. In LPS plus IFN--stimulated THP-1 microglia-like cells, MCC950 significantly reduced the secretion of IL-1 while shikonin and oridonin did not have an effect. To the best of my 95 knowledge, the effects of oridonin on the secretion of TNF and IL-1 have not been studied before in human microglia or microglia-like cells. In contrast to my results in THP-1 microglia-like cells, a recent study determined that MCC950 did not have an effect on the secretion of IL-1 by oxygen-glucose deprivation/reperfusion-stimulated human microglial clone-3 cells (Arik et al., 2022). However, in a study using THP-1 microglia-like cells treated with low-density lipoprotein, MCC950 reduced IL-1 and IL-18 protein levels but mRNA transcription levels of the NLRP3 inflammasome proteins were not affected (Zeng et al., 2021); therefore, MCC950 had no effect on the priming of the NLRP3 inflammasome, but inhibited its assembly and activation. Further studies regarding the effects of MCC950 on human microglial IL-1 secretion is warranted. In this thesis work, none of the inflammasome inhibitors had a significant effect on stimulated human microglia-like cell secretion of TNF; however, MCC950 exhibited a trend toward decreasing the secretion of TNF by stimulated THP-1 microglia-like cells. Zeng et al. (2021) discovered that MCC950 did not influence TNF transcription or secretion by THP-1 cells that were treated with low-density lipoprotein, which is consistent with the data presented in this thesis showing no effect of MCC950 on TNF secretion by THP-1 cells. Inconsistent with my results, a recent study using THP-1 microglia-like cells that had been stimulated with LPS plus nigericin, a microbial toxin that triggers NLRP3 activation, demonstrated that shikonin (2-10 M) decreased the secretion of IL-1, in addition to TNF, IL-6, and IP-10 (Cardoso et al., 2022). I determined that the highest non-toxic concentration of shikonin toward microglia-like cells was 400 nM, and therefore the concentrations presented by Cardoso et al. (2022) should not be used when confirming specific inhibition of pro-inflammatory molecules. 96 Notably, in LPS-stimulated U-118 MG human astrocytic cells, MCC950, shikonin, and oridonin significantly reduced the secretion of IL-1. In the same cells, MCC950 and shikonin exhibited an insignificant trend toward decreasing TNF secretion while oridonin did not have an effect. A previous study found that MCC950 reduced IL-1 and TNF secretion by sepsis-induced primary rat microglia, but not primary rat astrocytes (Danielski et al., 2020). More recent results indicated that MCC950 inhibited NLRP3 protein expression and protein levels of cleaved caspase-1 in primary murine astrocytes from M83 -synuclein transgenic mice (Ren et al., 2022). The same study showed that MCC950 had no effect on the secretion of IL-1. Consistent with the results in this thesis, shikonin has recently been shown to reduce the secretion of TNF, IL-1, and IL-6 by astrocytes from a PD model C57BL/6 mice (Guo et al., 2022). Lastly, in LPS-stimulated primary astrocytes from C57BL/6 mice, oridonin significantly attenuated IL-1 levels (Li et al., 2022). This study, in addition to my own, indicates that oridonin is a strong candidate for reducing IL-1 secretion by astrocytic cells. While the inflammasome inhibitors I studied do not have a significant effect on IL-1 secretion by LPS-stimulated BV-2 murine microglia, they significantly decrease the secretion of IL-1 by LPS-stimulated U-118 MG human astrocytic cells. The differing results may be due to species or cell-type specificities. MCC950 significantly reduced the secretion of IL-1 by LPS plus IFN--stimulated THP-1 microglia-like cells, but shikonin and oridonin did not have an effect. MCC950, shikonin, and oridonin exhibit no significant effect on the secretion of TNF by stimulated BV-2 murine microglia, THP-1 microglia-like cells, or U-118 MG astrocytic cells. Although the results presented in this thesis indicate that shikonin may induce a trend toward decreased secretion of TNF by U-118 MG cells, these data conflict with previous studies and therefore more research on the effects of the inflammasome inhibitors on the secretion of TNF 97 by human cells is necessary. It is important to consider that the secretion of IL-1 is mediated by both inflammasome-dependent and inflammasome-independent pathways; therefore, the lack of effect of MCC950, shikonin, and oridonin on the secretion of IL-1 by microglia-like cells could be due to the inflammasome-independent pathways being responsible for the release of IL-1 in these cells. 4.4 Cytotoxicity Previous studies have reported the neurotoxic effects of LPS-stimulated BV-2 murine microglia cells toward NSC-34 and HT-22 neuronal cells and have demonstrated that LPS-stimulated murine primary and immortal microglia-like cells reduced the viability of neuronal cells (Chang et al., 2021; Lee et al., 2013; Li et al., 2007; Wenzel et al., 2021). The results presented in this thesis indicated that MCC950 did not influence the cytotoxicity of the immortal BV-2 microglial cells toward immortal neuronal cells. Shikonin significantly reduced the cytotoxicity of LPS-stimulated BV-2 murine microglia toward HT-22 murine neuronal cells, but not NSC-34 murine neuronal cells, which may be a result of HT-22 cells being more sensitive to the secreted ROS and other toxins that shikonin inhibited. Lastly, oridonin significantly reduced the cytotoxicity of LPS-stimulated BV-2 murine microglia cells toward both HT-22 and NSC-34 murine neuronal cells. A-stimulated BV-2 murine microglia have been shown to decrease HT-22 neuronal cell viability (Liu et al., 2020). The same study indicated that blocking the TLR4/NLRP3 pathway, with the TLR4 inhibitor CLI-095, in BV-2 murine microglia significantly increased HT-22 cell viability. BV-2 cells have also been shown to be neurotoxic to HT-22 neuronal cells when stimulated with LPS (Ogunrinade et al., 2021). Another study confirmed the involvement of the NLRP3 inflammasome in LPS-stimulated BV-2 neurotoxicity toward HT-22 and NSC-34 98 neuronal cells in a cell co-culture system (Ma et al., 2022). In BV-2 murine microglia, MCC950 reduced the cytotoxicity of acrylamide, which is known to have toxic effects on the nervous system (Sui et al., 2020); however, the study did not determine the protective effect of MCC950 on neurons. To the best of my knowledge, my study is the first to demonstrate the protective effects of MCC950 and oridonin against microglia-mediated toxicity. Other studies have determined the neurotoxicity of LPS plus IFN--stimulated THP-1 microglia-like cells towards SH-SY5Y human neuronal cells (Hashioka et al., 2009; McKenzie et al., 2019), but no studies have reported effects of MCC950, shikonin, or oridonin on neurotoxicity of these human microglia-like cells towards human neuronal cells. According to my results, oridonin, but not MCC950 or shikonin, significantly reduced the cytotoxicity of stimulated THP-1 microglia-like cells towards SH-SY5Y human neuronal cells. Prior research has demonstrated the neurotoxicity of stimulated U-118 MG human astrocytic cells towards SH-SY5Y neuronal cells (Bahniwal et al., 2017). My results indicated that MCC950 and oridonin did not significantly reduce the cytotoxicity of LPS-stimulated U-118 MG cells towards SH-SY5Y human neuronal cells. Meanwhile, shikonin significantly reduced the cytotoxicity of LPS-stimulated U-118 MG cells towards SH-SY5Y cells. To the best of my knowledge, my study is the first to determine the effects of MCC950, shikonin, and oridonin on astrocyte-mediated toxicity towards neuronal cells. Pro-inflammatory cytokine production is important to consider when discussing the neurotoxic effect of glial cells. For example, in this thesis I provide evidence that MCC950 and oridonin are decreasing the secretion of IL-1 by LPS-stimulated U-118 MG astrocytic cells but are not able to decrease the secretion of this cytokine enough to protect neuronal cells from death. However, future studies on the involvement of other pro-inflammatory cytokines such as 99 IL-18, IL-6, and IP-10 would be warranted to determine if the inflammasome inhibitors act on the secretion of other cytokines. Furthermore, the significant increase in glutamate secretion by LPS-stimulated U-118 MG astrocytic cells in the presence of oridonin could explain the lack of inhibition this drug exhibited on the cytotoxicity of stimulated U-118 MG cells towards SH-SY5Y neuronal cells. The release of potentially cytotoxic molecules, including IL-1, TNF, and glutamate, by U-118 MG cells decreases SH-SY5Y neuronal cell viability, so while oridonin reduced the secretion of IL-1 by U-118 MG cells, oridonin increased the secretion of glutamate, which could explain the observed lack of its effect on the overall cytotoxicity. 4.5 Phagocytosis The effects of the inflammasome inhibitors on phagocytosis, a protective function of microglia to remove extracellular debris and clear A, were also determined in this thesis work. My results indicate that MCC950, shikonin, and oridonin significantly increased LPS-stimulated BV-2 murine microglial phagocytosis of fluorescent latex beads. In microglia, A binds to TLR4 and activates their phagocytosis and cytokine production (reviewed by Yang et al., 2020). Murine microglial A phagocytosis has been shown to increase in cells that are deficient in NLRP3 or caspase-1 (Yang et al., 2020). Dempsey et al. (2017) concluded that IL-1 inhibited phagocytic activity of primary murine microglia; therefore, the study concluded that increased phagocytosis in the presence of MCC950 is expected. The same study also found that MCC950 decreased the accumulation of A in APP/PS1 mice due to increased phagocytosis (Dempsey et al., 2017). Additionally, in a cerebral hemorrhage model, shikonin upregulated primary murine microglial phagocytosis of red blood cells (RBC) (Nie et al., 2021). Moreover, oridonin was shown to increase phagocytosis by macrophages, such as UV-irradiated U937 and RAW264.7 100 cells (Liu et al., 2005; Zang et al., 2012, 2013, 2019), but the effect of oridonin on microglial phagocytosis has not been investigated until now. Astrocytes have the ability to act as phagocytes in the brain and can compensate for dysfunctional microglial phagocytosis (Konishi et al., 2020). Activated astrocytes phagocytose extracellular debris and their phagocytic activity can be stimulated by LPS (Kalmár et al., 2001); therefore, I determined the effects of the inflammasome inhibitors on the phagocytosis of fluorescent latex beads by unstimulated and LPS-stimulated astrocytic cells. In LPS-stimulated U-118 MG human astrocytic cells, adding MCC950 and shikonin led to a trend towards increasing phagocytosis of latex beads while oridonin significantly increased phagocytosis of latex beads. To the best of my knowledge, this is the first study demonstrating the effects of the inflammasome inhibitors, MCC950, shikonin, and oridonin, on astrocytic phagocytosis. 4.7 PKD and JNK NLRP3 phosphorylation is triggered by PKD and JNK, among other enzymes, which can regulate the inflammasome (Sandall & MacDonald, 2019; Zhang et al., 2017). Previous studies have shown that oridonin attenuates JNK signaling in LPS-stimulated HK-2 human kidney cells and pancreatic cancer cells (Huang et al., 2020; Wang et al., 2019). Other studies show that oridonin activates JNK pathway as seen in unstimulated HCT-15 cancer cells (Zhang et al., 2019). Importantly, the effects of oridonin on microglial JNK signaling have not been studied until now. Because of this, I used the JNK inhibitor, SP600125, in combination with oridonin to determine the effects of this combination on the secretion of NO by LPS-stimulated BV-2 murine microglia. My results indicate that oridonin may act on the JNK pathway since there is not a significant additive effect when oridonin and SP600125 are combined. 101 PKD activity is a requirement for effective inflammasome activation, particularly in the recruitment of ASC to the NLRP3 protein (Zhang et al., 2017). There are no current studies that have determined the effects of oridonin on PKD signaling. My results indicate that oridonin does not influence PKD signaling since there is an additive effect when oridonin and CID755673, a PKD inhibitor, are combined. These results demonstrate an important point that a combination of drugs could be necessary to fully prevent the activation of the NLRP3 inflammasome. 4.8 Summary AD is the most prevalent form of neurodegeneration that plagues our elderly. Current research is focused on the inhibition of overactivated glial cells because of their role in the release of pro-inflammatory cytokines. Discovering novel molecules that inhibit the production of toxins by astrocytes and microglia, but enhance their phagocytic activity, is important in the search for a cure to AD. The data presented in this thesis suggest that shikonin and oridonin are effective at inhibiting the NLRP3 inflammasome in LPS-stimulated murine microglia. Oridonin proved to be the most efficacious inhibitor of the NLRP3 inflammasome in human astrocytic cells when compared to MCC950 and shikonin. Oridonin strongly inhibited the secretion of NO by BV-2 cells, reduced BV-2 cytotoxicity towards NSC-34 and HT-22 neuronal cells, reduced THP-1 toxicity towards SH-SY5Y neuronal cells and inhibited the secretion of IL-1 by U-118 MG astrocytic cells. The aforementioned results suggest that oridonin, rather than MCC950 or shikonin, might be the most successful in treating neurodegenerative diseases. MCC950, shikonin, and oridonin inhibited the secretion of IL-1 in U-118 MG cells and hence these inflammasome inhibitors could be considered efficacious candidates in preventing NLRP3 activation and downstream neurodegeneration. Furthermore, MCC950, shikonin, and oridonin upregulated both microglial and astrocytic phagocytosis, which is important in regulation and 102 elimination of dying cells and cell debris, thus displaying activity that is potentially beneficial in neurodegenerative diseases. The concept of inflammasomes in glia is a relatively new development, but evidence suggests that inflammasome inhibitors could be used as efficacious drugs for the treatment of neurodegenerative diseases. Taken together, the findings presented in this thesis demonstrate that inflammasome inhibitors can modify the secretion of some pro-inflammatory mediators, such as NO, ROS and glutamate. Future studies are necessary to determine the effects of MCC950, shikonin, and oridonin on the release of other pro-inflammatory cytokines, such as IL-6 and IL-18, by human microglial and astrocytic cells. Confirmation of the anti-neuroinflammatory activity of inflammasome inhibitors in animal models of neurodegenerative disease is also a necessary next step in the preclinical studies of these drugs. 103 Chapter 5: Conclusion 5.1 Hypotheses Addressed Hypothesis one is partially correct; selective inhibition of inflammasomes decreases the secretion of multiple murine microglia-derived toxins and increases phagocytic activity of murine microglia, thereby demonstrating a neuroprotective effect. MCC950, shikonin, and oridonin enhance phagocytic activity, and inhibit the secretion of two toxins, NO and glutamate, by LPS-stimulated BV-2 murine microglia. MCC950 and shikonin lower the secretion of NO and glutamate by BV-2 murine microglia but do not decrease these toxins at a sufficient level to reduce overall cytotoxicity of BV-2 microglia towards NSC-34 murine neuronal cells. Furthermore, oridonin was found to possibly interact with the JNK pathway due to its lack of significant additive effect with the JNK inhibitor, which is known to prevent inflammasome activation. MCC950, shikonin, and oridonin are considered inflammasome inhibitors because they inhibit the assembly of the inflammasome complex, even though they do not directly affect the secretion of IL-1β in LPS-stimulated BV-2 murine microglia. Hypothesis two is partially correct; selective inhibition of inflammasomes decreases the secretion of human microglia-derived toxins. MCC950, but not shikonin or oridonin, decreases the secretion of IL-1β by THP-1 human microglia-like cells demonstrating its effect on the NLRP3 inflammasome. MCC950 and shikonin decrease the secretion of ROS but do not lower this toxin at a sufficient level to reduce overall cytotoxicity of THP-1 microglia-like cells towards SH-SY5Y neuronal cells. Oridonin does not decrease select toxins, such as ROS, but protects neurons from cell death and therefore must act on other THP-1 human microglia-like derived toxins, such as IL-6 or IL-18. 104 Hypothesis three is correct; selective inhibition of inflammasomes decreases the secretion of astrocyte-derived toxins and increases phagocytic activity of astrocytes. MCC950, shikonin and oridonin decrease the secretion of the astrocytic toxins, IL-1β and TNF, and increase phagocytic activity. MCC950, shikonin, and oridonin are NLRP3 inflammasome inhibitors in U-118 MG human astrocytic cells as shown by significantly decreased secretion of IL-1β. 5.2 Limitations of the Study In this study, I use immortalized cell lines exclusively. While the use of immortalized cell lines can provide us with insight into the morphology and functions of the target cells, their gene expression differs drastically from primary cells. To elaborate further, primary cells are considered a better option for cell-based studies; however, they also have disadvantages, such as morphological changes and limited growth potential once they leave their native environment. The results in this thesis allow us to gain an understanding of the fundamental actions of these drugs on monolayer culture, but our research is limited to this 2D structure. Current technology facilitates the use of organoids derived from inducible pluripotent stem cells. This technique has previously been used to study neurodegenerative diseases and inflammasome activation. Advantages of organoids over monoculture include composition involving multiple cell types, and opportunities to analyze cellular interactions (Kim et al., 2020; Pollen et al., 2019). Furthermore, the research in this thesis utilizes a pharmacological approach to finding a treatment for AD. However, pharmaceutical approaches may not effectively prevent AD on their own and thus should be used in combination with other treatments. For example, targeting protein-protein interactions is proven to be a disease-modifying strategy, such as in the case of Aducanumab which is a United States Food and Drug Administration (FDA) approved monoclonal antibody for the treatment of AD (Alzheimer’s Association, 2023). 105 In this thesis, I determine the effects of MCC950, shikonin, and oridonin on both murine and human cell lines. A further understanding on species specific actions of inflammasome inhibitors could provide more insight as to why there is reduction in certain cytokines in human cells but not in murine. Furthermore, the relevance of NO secretion from BV-2 murine microglia is not well understood. While NO is a toxin secreted by murine microglial cells, it is not known if this applies to human cells. To resolve this, human cell lines should be used to confirm the effects I saw in BV-2 murine microglia. 5.3 Future Directions In this thesis, I provide insight into the effects of MCC950, shikonin, and oridonin on activated glial cells. Further mechanistic experiments, such as co-immunoprecipitation of PI3K and NLRP3 inflammasome in the presence of these inhibitors, are necessary to understand if these inhibitors act on pathways beyond the inflammasome. My results indicate a lack of inflammasome inhibition in microglial cells; therefore, studies to determine if these inhibitors decrease other pro-inflammatory cytokines, such as IL-18, could provide more feedback as to whether or not these inhibitors are in fact acting on the inflammasome pathway. It is imperative to establish the extent to which MCC950, shikonin, and oridonin inhibit pathways outside of the inflammasome before moving forward with clinical trials. For example, it would be interesting to determine the effects of these inflammasome inhibitors after the addition of a PI3K inhibitor in human cells. Although this thesis provides valuable insights into the effects of the inflammasome inhibitors on glial cells, further research is required to fully understand the molecular mechanisms underlying the development and progression of AD. Given the limitations of our cell model, the study of human primary cells and organoids using these inflammasome inhibitors is warranted. Mouse and human organoids could be useful 106 in studying the effects of MCC950, shikonin, and oridonin on the secretion of pro-inflammatory cytokines. The combination of a pharmacological approach, such as an inflammasome inhibitor, and gene therapy may also prove to be an effective advancement in the search for a treatment of AD. Lastly, experiments detailing the effects of MCC950, shikonin, and oridonin on astrocytes are sparce. This thesis provides novel information on astrocyte phagocytic activity and the release of pro-inflammatory cytokines in the presence of these inflammasome inhibitors. Further studies on the mechanism of action of the inhibitors in astrocytes is imperative. 5.4 Research Significance This thesis demonstrates the effects of MCC950, shikonin, and oridonin on immortalized cell line models of murine microglia, human microglia, and human astrocytes. I determine that: (1) MCC950, shikonin, and oridonin modulate murine microglial and human astrocytic phagocytosis, (2) oridonin may act on intracellular pathways other than those linked to the NLRP3 inflammasome, (3) MCC950 and oridonin modulate the secretion of toxins by human microglia-like cells, (4) all inhibitors decrease the secretion of NO by BV-2 murine microglia and ROS by DMSO-differentiated HL-60 microglia-like cells, (5) MCC950, shikonin, and oridonin inhibit the NLRP3 inflammasome in human astrocytic cells. These data provide insight into the possible effects of MCC950, shikonin, and oridonin on neuroinflammatory processes indicating their potential as novel therapeutic agents for preventing neurodegeneration. Neurodegenerative diseases, such as AD, PD, and MS, affect millions of people worldwide: however, there are no effective treatments for these disorders. As the number of patients affected increases, finding viable treatments is more crucial than ever. This thesis contributes to the knowledge of cellular targets of inflammasome inhibitors and the future they 107 may hold in the treatment of neurodegeneration, which is partially characterized by inflammation and activated glia. 108 References Acosta, C., Anderson, H. D., & Anderson, C. M. (2017). Astrocyte dysfunction in Alzheimer disease. Journal of Neuroscience Research, 95(12), 2430–2447. https://doi.org/https://doi.org/10.1002/jnr.24075 Alzheimer’s Association. (2023). Aducanumab Approved for Treatment of Alzheimer’s Disease. https://www.alz.org/alzheimers-dementia/treatments/aducanumab#:~:text=Aducanumab (AduhelmTM) has received,and Drug Administration (FDA). Alzheimer’s disease facts and figures. (2022). Alzheimer’s & Dementia, 18(4), 700–789. https://doi.org/https://doi.org/10.1002/alz.12638 Araki, T., Ikegaya, Y., & Koyama, R. (2021). The effects of microglia- and astrocyte-derived factors on neurogenesis in health and disease. The European Journal of Neuroscience, 54(5), 5880–5901. https://doi.org/10.1111/ejn.14969 Arik, E., Heinisch, O., Bienert, M., Gubeljak, L., Slowik, A., Reich, A., Schulz, J. B., Wilhelm, T., Huber, M., & Habib, P. (2022). Erythropoietin enhances post-ischemic migration and phagocytosis and alleviates the activation of inflammasomes in human microglial cells. Frontiers in Cellular Neuroscience, 16, 915348. https://doi.org/10.3389/fncel.2022.915348 Arioz, B. I., Tastan, B., Tarakcioglu, E., Tufekci, K. U., Olcum, M., Ersoy, N., Bagriyanik, A., Genc, K., & Genc, S. (2019). Melatonin attenuates LPS-induced acute depressive-like behaviors and microglial NLRP3 inflammasome activation through the SIRT1/Nrf2 pathway. In Frontiers in Immunology (Vol. 10). Atianand, M. K., Duffy, E. B., Shah, A., Kar, S., Malik, M., & Harton, J. A. (2011). Francisella tularensis reveals a disparity between human and mouse NLRP3 inflammasome activation. Journal of Biological Chemistry, 286(45), 39033–39042. https://doi.org/10.1074/jbc.M111.244079 Bahniwal, M., Little, J. P., & Klegeris, A. (2017). High glucose enhances neurotoxicity and inflammatory cytokine secretion by stimulated human astrocytes. Current Alzheimer Research, 14(7), 731–741. https://doi.org/10.2174/1567205014666170117104053 Bi, Y., Zhu, Y., Zhang, M., Zhang, K., Hua, X., Fang, Z., Zhou, J., Dai, W., Cui, Y., Li, J., & You, T. (2017). Effect of shikonin on spinal cord injury in rats via regulation of HMGB1/TLR4/NF-kB signaling pathway. Cellular Physiology and Biochemistry, 43(2), 481–491. https://doi.org/10.1159/000480474 BioRender. (2023). No Title. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., & Bistoni, F. (1990). Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus. Journal of Neuroimmunology, 27(2), 229–237. https://doi.org/https://doi.org/10.1016/0165-5728(90)90073-V Brown, G. C. (2019). The endotoxin hypothesis of neurodegeneration. Journal of Neuroinflammation, 16(1), 180. https://doi.org/10.1186/s12974-019-1564-7 Bryan, N. S., & Grisham, M. B. (2007). Methods to detect nitric oxide and its metabolites in biological samples. Free Radical Biology & Medicine, 43(5), 645–657. https://doi.org/10.1016/j.freeradbiomed.2007.04.026 109 Burguillos, M. A., Deierborg, T., Kavanagh, E., Persson, A., Hajji, N., Garcia-Quintanilla, A., Cano, J., Brundin, P., Englund, E., Venero, J. L., & Joseph, B. (2011). Caspase signalling controls microglia activation and neurotoxicity. Nature, 472(7343), 319–324. https://doi.org/10.1038/nature09788 Cajal, S. R. y. (1913). Contribución al conocimiento de la neuroglia del cerebro humano. Cardoso, M., Gaspar, V. M., Ferreira, C., Silvestre, R., Duarte, I. F., & Mano, J. F. (2022). Macrophage-targeted shikonin-loaded nanogels for modulation of inflammasome activation. Nanomedicine: Nanotechnology, Biology and Medicine, 42, 102548. https://doi.org/https://doi.org/10.1016/j.nano.2022.102548 Chang, X., Fang, Y., Zhang, Y., Liu, Y., Fan, L., Nan, L., Xu, W., Lin, Y., Chu, K., & Yan, G. (2021). Gualou guizhi granule suppresses LPS-induced inflammatory response of microglia and protects against microglia-mediated neurotoxicity in HT-22 via Akt/NF-κB signaling pathways. Evidence-Based Complementary and Alternative Medicine, 2021, 9957459. https://doi.org/10.1155/2021/9957459 Chang, Y., Zhu, J., Wang, D., Li, H., He, Y., Liu, K., Wang, X., Peng, Y., Pan, S., & Huang, K. (2020). NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury. Journal of Neuroinflammation, 17(1), 219. https://doi.org/10.1186/s12974-020-01879-1 Chao, C. C., Hu, S., Molitor, T. W., Shaskan, E. G., & Peterson, P. K. (1992). Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. The Journal of Immunology, 149(8), 2736 LP – 2741. Chen, L., Huang, C.-F., Li, Y.-C., Deng, W.-W., Mao, L., Wu, L., Zhang, W.-F., Zhang, L., & Sun, Z.-J. (2018). Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma. Cellular and Molecular Life Sciences, 75(11), 2045–2058. https://doi.org/10.1007/s00018-017-2720-9 Chen, X., Liu, G., Yuan, Y., Wu, G., Wang, S., & Yuan, L. (2019). NEK7 interacts with NLRP3 to modulate the pyroptosis in inflammatory bowel disease via NF-κB signaling. Cell Death & Disease, 10(12), 906. https://doi.org/10.1038/s41419-019-2157-1 Chen, Y., Meng, J., Bi, F., Li, H., Chang, C., Ji, C., & Liu, W. (2019). NEK7 Regulates NLRP3 Inflammasome Activation and Neuroinflammation Post-traumatic Brain Injury. In Frontiers in Molecular Neuroscience (Vol. 12). Cheng, J., Liao, Y., Dong, Y., Hu, H., Yang, N., Kong, X., Li, S., Li, X., Guo, J., Qin, L., Yu, J., Ma, C., Li, J., Li, M., Tang, B., & Yuan, Z. (2020). Microglial autophagy defect causes parkinson disease-like symptoms by accelerating inflammasome activation in mice. Autophagy, 16(12), 2193–2205. https://doi.org/10.1080/15548627.2020.1719723 Chiarini, A., Armato, U., Hu, P., & Dal Prà, I. (2020). Danger-sensing/patten recognition receptors and neuroinflammation in Alzheimer’s disease. International Journal of Molecular Sciences, 21(23), 9036. https://doi.org/10.3390/ijms21239036 Coll, R. C., Hill, J. R., Day, C. J., Zamoshnikova, A., Boucher, D., Massey, N. L., Chitty, J. L., Fraser, J. A., Jennings, M. P., Robertson, A. A. B., & Schroder, K. (2019). MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nature Chemical Biology, 15(6), 556–559. https://doi.org/10.1038/s41589-019-0277-7 110 Coll, R. C., Robertson, A. A. B., Chae, J. J., Higgins, S. C., Muñoz-Planillo, R., Inserra, M. C., Vetter, I., Dungan, L. S., Monks, B. G., Stutz, A., Croker, D. E., Butler, M. S., Haneklaus, M., Sutton, C. E., Núñez, G., Latz, E., Kastner, D. L., Mills, K. H. G., Masters, S. L., … O’Neill, L. A. J. (2015). A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nature Medicine, 21(3), 248–255. https://doi.org/10.1038/nm.3806 Collins, S. J. (1987). The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood, 70(5), 1233–1244. https://doi.org/https://doi.org/10.1182/blood.V70.5.1233.1233 Condello, C., Yuan, P., Schain, A., & Grutzendler, J. (2015). Microglia constitute a barrier that prevents neurotoxic protofibrillar Aβ42 hotspots around plaques. Nature Communications, 6, 6176. https://doi.org/10.1038/ncomms7176 Corradin, S. B., Mauël, J., Donini, S. D., Quattrocchi, E., & Ricciardi-Castagnoli, P. (1993). Inducible nitric oxide synthase activity of cloned murine microglial cells. Glia, 7(3), 255–262. https://doi.org/https://doi.org/10.1002/glia.440070309 Dahlgren, C., & Karlsson, A. (1999). Respiratory burst in human neutrophils. Journal of Immunological Methods, 232(1), 3–14. https://doi.org/https://doi.org/10.1016/S0022-1759(99)00146-5 Danielski, L. G., Giustina, A. Della, Bonfante, S., de Souza Goldim, M. P., Joaquim, L., Metzker, K. L., Biehl, E. B., Vieira, T., de Medeiros, F. D., da Rosa, N., Generoso, J., Simoes, L., Farias, H. R., da Silva Lemos, I., Giridharan, V., Rezin, G. T., Fortunato, J. J., Bitencourt, R. M., Streck, E. L., … Petronilho, F. (2020). NLRP3 activation contributes to acute brain damage leading to memory impairment in sepsis-surviving rats. Molecular Neurobiology, 57(12), 5247–5262. https://doi.org/10.1007/s12035-020-02089-9 Danot, O., Marquenet, E., Vidal-Ingigliardi, D., & Richet, E. (2009). Wheel of life, wheel of death: a mechanistic insight into signaling by STAND proteins. Structure, 17(2), 172–182. https://doi.org/https://doi.org/10.1016/j.str.2009.01.001 Dempsey, C., Rubio Araiz, A., Bryson, K. J., Finucane, O., Larkin, C., Mills, E. L., Robertson, A. A. B., Cooper, M. A., O’Neill, L. A. J., & Lynch, M. A. (2017). Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-β and cognitive function in APP/PS1 mice. Brain, Behavior, and Immunity, 61, 306–316. https://doi.org/https://doi.org/10.1016/j.bbi.2016.12.014 Dinarello, C. A. (2000). Proinflammatory cytokines. Chest, 118(2), 503–508. https://doi.org/https://doi.org/10.1378/chest.118.2.503 Ding, A. H., Nathan, C. F., & Stuehr, D. J. (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. The Journal of Immunology, 141(7), 2407 LP – 2412. Dong, J., Wang, X., Xu, C., Gao, M., Wang, S., Zhang, J., Tong, H., Wang, L., Han, Y., Cheng, N., & Han, Y. (2021). Inhibiting NLRP3 inflammasome activation prevents copper-induced neuropathology in a murine model of Wilson’s disease. Cell Death & Disease, 12(1), 87. https://doi.org/10.1038/s41419-021-03397-1 111 Duan, D., Zhang, B., Yao, J., Liu, Y., & Fang, J. (2014). Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Free Radical Biology and Medicine, 70, 182–193. https://doi.org/https://doi.org/10.1016/j.freeradbiomed.2014.02.016 Duncan, J., Bergstralh, D., Yanhong, W., Willingham, S., Zhengmao, Y., G., Z. A., & Pan-Yun, T. J. (2007). Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling. Proceedings of the National Academy of Sciences, 104(19), 8041–8046. https://doi.org/10.1073/pnas.0611496104 Esmaeilzadeh, E., Gardaneh, M., Gharib, E., & Sabouni, F. (2013). Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis. Neurochemical Research, 38(8), 1590–1604. https://doi.org/10.1007/s11064-013-1061-9 Fauzia, E., Barbhuyan, T. K., Shrivastava, A. K., Kumar, M., Garg, P., Khan, M. A., Robertson, A. A. B., & Raza, S. S. (2018). Chick embryo: a preclinical model for understanding ischemia-reperfusion mechanism. In Frontiers in Pharmacology (Vol. 9). Fernández-Tomé, P., Brera, B., Arévalo, M.-A., & de Ceballos, M. L. (2004). β-Amyloid25-35 inhibits glutamate uptake in cultured neurons and astrocytes: modulation of uptake as a survival mechanism. Neurobiology of Disease, 15(3), 580–589. https://doi.org/https://doi.org/10.1016/j.nbd.2003.12.006 Freeman, L., Guo, H., David, C. N., Brickey, W. J., Jha, S., & Ting, J. P.-Y. (2017). NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes. The Journal of Experimental Medicine, 214(5), 1351–1370. https://doi.org/10.1084/jem.20150237 Fu, H., Liu, B., Frost, J. L., Hong, S., Jin, M., Ostaszewski, B., Shankar, G. M., Costantino, I. M., Carroll, M. C., Mayadas, T. N., & Lemere, C. A. (2012). Complement component C3 and complement receptor type 3 contribute to the phagocytosis and clearance of fibrillar Aβ by microglia. Glia, 60(6), 993–1003. https://doi.org/https://doi.org/10.1002/glia.22331 Fu, R., Shen, Q., Xu, P., Luo, J. J., & Tang, Y. (2014). Phagocytosis of microglia in the central nervous system diseases. Molecular Neurobiology, 49(3), 1422–1434. https://doi.org/10.1007/s12035-013-8620-6 Gaidt, M. M., Ebert, T. S., Chauhan, D., Schmidt, T., Schmid-Burgk, J. L., Rapino, F., Robertson, A. A. B., Cooper, M. A., Graf, T., & Hornung, V. (2016). Human monocytes engage an alternative inflammasome pathway. Immunity, 44(4), 833–846. https://doi.org/https://doi.org/10.1016/j.immuni.2016.01.012 Gantner, B. N., LaFond, K. M., & Bonini, M. G. (2020). Nitric oxide in cellular adaptation and disease. Redox Biology, 34, 101550. https://doi.org/10.1016/j.redox.2020.101550 Garwood, C. J., Pooler, A. M., Atherton, J., Hanger, D. P., & Noble, W. (2011). Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture. Cell Death & Disease, 2(6), e167–e167. https://doi.org/10.1038/cddis.2011.50 Gates, E. J. (2020). Signaling Between Gut Microbiota and Brain Immune Cells. The University of British Columbia, Okanagan. 112 Giacoppo, S., Bramanti, P., & Mazzon, E. (2017). Triggering of inflammasome by impaired autophagy in response to acute experimental Parkinson’s disease: involvement of the PI3K/Akt/mTOR pathway. Neuroreport, 28(15), 996–1007. https://doi.org/10.1097/WNR.0000000000000871 Grewer, C., Gameiro, A., Zhang, Z., Tao, Z., Braams, S., & Rauen, T. (2008). Glutamate forward and reverse transport: from molecular mechanism to transporter-mediated release after ischemia. IUBMB Life, 60(9), 609–619. https://doi.org/10.1002/iub.98 Guo, H., Callaway, J. B., & Ting, J. P.-Y. (2015). Inflammasomes: mechanism of action, role in disease, and therapeutics. Nature Medicine, 21(7), 677–687. https://doi.org/10.1038/nm.3893 Guo, L., Li, Y., Li, W., Qiu, J., Du, J., Wang, L., & Zhang, T. (2022). Shikonin ameliorates oxidative stress and neuroinflammation via the Akt/ERK/JNK/NF-κB signalling pathways in a model of Parkinson’s disease. Clinical and Experimental Pharmacology and Physiology, n/a(n/a). https://doi.org/https://doi.org/10.1111/1440-1681.13709 Gustin, A., Kirchmeyer, M., Koncina, E., Felten, P., Losciuto, S., Heurtaux, T., Tardivel, A., Heuschling, P., & Dostert, C. (2015). NLRP3 inflammasome Is expressed and functional in mouse brain microglia but not in astrocytes. PLOS ONE, 10(6), e0130624. Halle, A., Hornung, V., Petzold, G. C., Stewart, C. R., Monks, B. G., Reinheckel, T., Fitzgerald, K. A., Latz, E., Moore, K. J., & Golenbock, D. T. (2008). The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nature Immunology, 9(8), 857–865. https://doi.org/10.1038/ni.1636 Han, H., Sun, W., Feng, L., Wen, Z., Yang, M., Ma, Y., Fu, J., Ma, X., Xu, X., Wang, Z., Yin, T., Wang, X.-M., Lu, G.-H., Qi, J.-L., Lin, H., & Yang, Y. (2021). Differential relieving effects of shikonin and its derivatives on inflammation and mucosal barrier damage caused by ulcerative colitis. PeerJ, 9, e10675–e10675. https://doi.org/10.7717/peerj.10675 Han, Y., Lu, Y., Li, X., Niu, X., Chang, A. K., Yang, Z., Li, X., He, X., & Bi, X. (2021). Novel organoselenides (NSAIDs-Se derivatives) protect against LPS-induced inflammation in microglia by targeting the NOX2/NLRP3 signaling pathway. International Immunopharmacology, 101, 108377. https://doi.org/https://doi.org/10.1016/j.intimp.2021.108377 Hanisch, U.-K., & Kettenmann, H. (2007). Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nature Neuroscience, 10(11), 1387–1394. https://doi.org/10.1038/nn1997 Harris, M. E., Wang, Y., Pedigo Jr., N. W., Hensley, K., Butterfield, D. A., & Carney, J. M. (1996). Amyloid β peptide (25–35) inhibits Na+-dependent glutamate uptake in rat hippocampal astrocyte cultures. Journal of Neurochemistry, 67(1), 277–286. https://doi.org/https://doi.org/10.1046/j.1471-4159.1996.67010277.x Harry, G. J., & Kraft, A. D. (2008). Neuroinflammation and microglia: considerations and approaches for neurotoxicity assessment. Expert Opinion on Drug Metabolism & Toxicology, 4(10), 1265–1277. https://doi.org/10.1517/17425255.4.10.1265 Hashioka, S., Klegeris, A., & McGeer, P. L. (2009). Proton pump inhibitors exert anti-inflammatory effects and decrease human microglial and monocytic THP-1 cell 113 neurotoxicity. Experimental Neurology, 217(1), 177–183. https://doi.org/https://doi.org/10.1016/j.expneurol.2009.02.002 Hashioka, S., Klegeris, A., Schwab, C., & McGeer, P. L. (2009). Interferon-γ-dependent cytotoxic activation of human astrocytes and astrocytoma cells. Neurobiology of Aging, 30(12), 1924–1935. https://doi.org/https://doi.org/10.1016/j.neurobiolaging.2008.02.019 He, H., Jiang, H., Chen, Y., Ye, J., Wang, A., Wang, C., Liu, Q., Liang, G., Deng, X., Jiang, W., & Zhou, R. (2018). Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity. Nature Communications, 9(1), 2550. https://doi.org/10.1038/s41467-018-04947-6 He, M., Liu, J., Cheng, S., Xing, Y., & Suo, W. Z. (2013). Differentiation renders susceptibility to excitotoxicity in HT22 neurons. Neural Regeneration Research, 8(14), 1297–1306. https://doi.org/10.3969/j.issn.1673-5374.2013.14.006 He, Y., Zeng, M. Y., Yang, D., Motro, B., & Núñez, G. (2016). NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature, 530(7590), 354–357. https://doi.org/10.1038/nature16959 Heneka, M. T., Kummer, M. P., Stutz, A., Delekate, A., Schwartz, S., Vieira-Saecker, A., Griep, A., Axt, D., Remus, A., Tzeng, T.-C., Gelpi, E., Halle, A., Korte, M., Latz, E., & Golenbock, D. T. (2013). NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature, 493(7434), 674–678. https://doi.org/10.1038/nature11729 Henn, A., Lund, S., Hedtjärn, M., Schrattenholz, A., Pörzgen, P. and Leist, M. (2009). The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX - Alternatives To, 83–94. https://doi.org/10.14573 Heusinkveld, H. J., & Westerink, R. H. S. (2017). Comparison of different in vitro cell models for the assessment of pesticide-induced dopaminergic neurotoxicity. Toxicology in Vitro, 45, 81–88. https://doi.org/https://doi.org/10.1016/j.tiv.2017.07.030 Hong-rong, X. I. E., Lin-sen, H. U., & Guo-yi, L. I. (2010). SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson’s disease. Chinese Medical Journal, 123(08), 1086–1092. https://doi.org/10.3760/cma.j.issn.0366-6999.2010.08.021 Hounoum, B. M., Vourc’h, P., Felix, R., Corcia, P., Patin, F., Guéguinou, M., Potier-Cartereau, M., Vandier, C., Raoul, C., Andres, C. R., Mavel, S., & Blasco, H. (2016). NSC-34 Motor Neuron-Like Cells Are Unsuitable as Experimental Model for Glutamate-Mediated Excitotoxicity. Frontiers in Cellular Neuroscience, 10, 118. https://doi.org/10.3389/fncel.2016.00118 Hu, J., Akama, K. T., Krafft, G. A., Chromy, B. A., & Van Eldik, L. J. (1998). Amyloid-β peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release. Brain Research, 785(2), 195–206. https://doi.org/https://doi.org/10.1016/S0006-8993(97)01318-8 Huang, J.-H., Lan, C.-C., Hsu, Y.-T., Tsai, C.-L., Tzeng, I.-S., Wang, P., Kuo, C.-Y., & Hsieh, P.-C. (2020). Oridonin attenuates lipopolysaccharide-induced ROS accumulation and inflammation in HK-2 cells. Evidence-Based Complementary and Alternative Medicine : ECAM, 2020, 9724520. https://doi.org/10.1155/2020/9724520 114 Huang, W.-R., Zhang, Y., & Tang, X. (2014). Shikonin inhibits the proliferation of human lens epithelial cells by inducing apoptosis through ROS and caspase-dependent pathway. Molecules (Basel, Switzerland), 19(6), 7785–7797. https://doi.org/10.3390/molecules19067785 Huang, W., Huang, M., Ouyang, H., Peng, J., & Liang, J. (2018). Oridonin inhibits vascular inflammation by blocking NF-κB and MAPK activation. European Journal of Pharmacology, 826, 133–139. https://doi.org/https://doi.org/10.1016/j.ejphar.2018.02.044 Hutton, H. L., Ooi, J. D., Holdsworth, S. R., & Kitching, A. R. (2016). The NLRP3 inflammasome in kidney disease and autoimmunity. Nephrology, 21(9), 736–744. https://doi.org/https://doi.org/10.1111/nep.12785 Iliff, J., Minghuan, W., Yonghong, L., A., P. B., Weiguo, P., A., G. G., Helene, B., Edward, V. G., Rashid, D., A., G. S., A., N. E., & Maiken, N. (2012). A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid β. Science Translational Medicine, 4(147), 147ra111-147ra111. https://doi.org/10.1126/scitranslmed.3003748 Inagaki, Y., Yamagishi, S., Amano, S., Okamoto, T., Koga, K., & Makita, Z. (2002). Interferon-γ-induced apoptosis and activation of THP-1 macrophages. Life Sciences, 71(21), 2499–2508. https://doi.org/https://doi.org/10.1016/S0024-3205(02)02042-8 Iravani, M. M., Sadeghian, M., Leung, C. C. M., Jenner, P., & Rose, S. (2012). Lipopolysaccharide-induced nigral inflammation leads to increased IL-1β tissue content and expression of astrocytic glial cell line-derived neurotrophic factor. Neuroscience Letters, 510(2), 138–142. https://doi.org/https://doi.org/10.1016/j.neulet.2012.01.022 Ising, C., Venegas, C., Zhang, S., Scheiblich, H., Schmidt, S. V, Vieira-Saecker, A., Schwartz, S., Albasset, S., McManus, R. M., Tejera, D., Griep, A., Santarelli, F., Brosseron, F., Opitz, S., Stunden, J., Merten, M., Kayed, R., Golenbock, D. T., Blum, D., … Heneka, M. T. (2019). NLRP3 inflammasome activation drives tau pathology. Nature, 575(7784), 669–673. https://doi.org/10.1038/s41586-019-1769-z Jia, T., Cai, M., Ma, X., Li, M., Qiao, J., & Chen, T. (2019). Oridonin inhibits IL-1β-induced inflammation in human osteoarthritis chondrocytes by activating PPAR-γ. International Immunopharmacology, 69, 382–388. https://doi.org/https://doi.org/10.1016/j.intimp.2019.01.049 Jia, Y., Tong, Y., Min, L., Li, Y., & Cheng, Y. (2021). Protective effects of oridonin against cerebral ischemia/reperfusion injury by inhibiting the NLRP3 inflammasome activation. Tissue and Cell, 71, 101514. https://doi.org/https://doi.org/10.1016/j.tice.2021.101514 Jones, R. S., Minogue, A. M., Connor, T. J., & Lynch, M. A. (2013). Amyloid-β-induced astrocytic phagocytosis is mediated by CD36, CD47 and RAGE. Journal of Neuroimmune Pharmacology, 8(1), 301–311. https://doi.org/10.1007/s11481-012-9427-3 Jung, Y.-J., & Chung, W.-S. (2018). Phagocytic Roles of Glial Cells in Healthy and Diseased Brains. Biomolecules & Therapeutics, 26(4), 350–357. https://doi.org/10.4062/biomolther.2017.133 Kalmár, B., Kittel, Á., Lemmens, R., Környei, Z., & Madarász, E. (2001). Cultured astrocytes react to LPS with increased cyclooxygenase activity and phagocytosis. Neurochemistry 115 International, 38(5), 453–461. https://doi.org/https://doi.org/10.1016/S0197-0186(00)00090-5 Kasai, S., Shiku, H., Torisawa, Y., Noda, H., Yoshitake, J., Shiraishi, T., Yasukawa, T., Watanabe, T., Matsue, T., & Yoshimura, T. (2005). Real-time monitoring of reactive oxygen species production during differentiation of human monocytic cell lines (THP-1). Analytica Chimica Acta, 549(1), 14–19. https://doi.org/https://doi.org/10.1016/j.aca.2005.06.034 Kaur, G., & Dufour, J. M. (2012). Cell lines: Valuable tools or useless artifacts. Spermatogenesis, 2(1), 1–5. https://doi.org/10.4161/spmg.19885 Kim, J.-J., & Jo, E.-K. (2013). NLRP3 inflammasome and host protection against bacterial infection. Journal of Korean Medical Science, 28(10), 1415–1423. https://doi.org/10.3346/jkms.2013.28.10.1415 Kim, J., Koo, B.-K., & Knoblich, J. A. (2020). Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology, 21(10), 571–584. https://doi.org/10.1038/s41580-020-0259-3 Kim, S. M., Ha, J. S., Han, A. R., Cho, S.-W., & Yang, S.-J. (2019). Effects of α-lipoic acid on LPS-induced neuroinflammation and NLRP3 inflammasome activation through the regulation of BV-2 microglial cells activation. BMB Reports, 52(10), 613–618. https://doi.org/10.5483/BMBRep.2019.52.10.026 Klegeris, A., Bissonnette, C. J., & McGeer, P. L. (2005). Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system. Neurobiology of Aging, 26(5), 673–682. https://doi.org/https://doi.org/10.1016/j.neurobiolaging.2004.06.012 Klegeris, A., & McGeer, P. L. (2003). Toxicity of human monocytic THP-1 cells and microglia toward SH-SY5Y neuroblastoma cells is reduced by inhibitors of 5-lipoxygenase and its activating protein FLAP. Journal of Leukocyte Biology, 73(3), 369–378. https://doi.org/https://doi.org/10.1189/jlb.1002482 Konishi, H., Okamoto, T., Hara, Y., Komine, O., Tamada, H., Maeda, M., Osako, F., Kobayashi, M., Nishiyama, A., Kataoka, Y., Takai, T., Udagawa, N., Jung, S., Ozato, K., Tamura, T., Tsuda, M., Yamanaka, K., Ogi, T., Sato, K., & Kiyama, H. (2020). Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction. The EMBO Journal, 39(22), e104464. https://doi.org/https://doi.org/10.15252/embj.2020104464 Lancaster, C. E., Fountain, A., Dayam, R. M., Somerville, E., Sheth, J., Jacobelli, V., Somerville, A., Terebiznik, M. R., & Botelho, R. J. (2021). Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes. The Journal of Cell Biology, 220(9), e202005072. https://doi.org/10.1083/jcb.202005072 Lau, A., & Tymianski, M. (2010). Glutamate receptors, neurotoxicity and neurodegeneration. Pflügers Archiv - European Journal of Physiology, 460(2), 525–542. https://doi.org/10.1007/s00424-010-0809-1 Lee, S., Kim, Y. K., Shin, T.-Y., & Kim, S.-H. (2013). Neurotoxic effects of bisphenol AF on calcium-induced ROS and MAPKs. Neurotoxicity Research, 23(3), 249–259. https://doi.org/10.1007/s12640-012-9353-4 116 Lee, W. J., Ham, S. A., Yoo, H., Hwang, J. S., Yoo, T., Paek, K. S., Lim, D.-S., Han, S. G., Lee, C.-H., Hong, K., & Seo, H. G. (2018). Activation of PPARδ attenuates neurotoxicity by inhibiting lipopolysaccharide-triggered glutamate release in BV-2 microglial cells. Journal of Cellular Biochemistry, 119(7), 5609–5619. https://doi.org/https://doi.org/10.1002/jcb.26732 Levy, R., Rotrosen, D., Nagauker, O., Leto, T. L., & Malech, H. L. (1990). Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression. The Journal of Immunology, 145(8), 2595 LP – 2601. Li, C.-Y., Wang, Q., Shen, S., Wei, X.-L., & Li, G.-X. (2018). Oridonin inhibits migration, invasion, adhesion and TGF-β1-induced epithelial-mesenchymal transition of melanoma cells by inhibiting the activity of PI3K/Akt/GSK-3β signaling pathway. Oncology Letters, 15(1), 1362–1372. https://doi.org/10.3892/ol.2017.7421 Li, C., Zhu, Y., Wu, Y., Fu, M., Wu, Y., Wu, Y., Qiu, Y., Zhang, H., & Ding, M. (2022). Oridonin alleviates LPS-induced depression by inhibiting NLRP3 inflammasome via activation of autophagy. Frontiers in Medicine, 8, 813047. https://doi.org/10.3389/fmed.2021.813047 Li, L., Lu, J., Tay, S. S. W., Moochhala, S. M., & He, B. P. (2007). The function of microglia, either neuroprotection or neurotoxicity, is determined by the equilibrium among factors released from activated microglia in vitro. Brain Research, 1159, 8–17. https://doi.org/https://doi.org/10.1016/j.brainres.2007.04.066 Li, Xiaodong, Wu, X., Wang, Q., Xu, W., Zhao, Q., Xu, N., Hu, X., Ye, Z., Yu, S., Liu, J., He, X., Shi, F., Zhang, Q., & Li, W. (2022). Sanguinarine ameliorates DSS induced ulcerative colitis by inhibiting NLRP3 inflammasome activation and modulating intestinal microbiota in C57BL/6 mice. Phytomedicine, 104, 154321. https://doi.org/https://doi.org/10.1016/j.phymed.2022.154321 Li, Xue-zhong, Bai, L., Yang, Y., Luo, W., Hu, W., Chen, J., Mao, C., & Liu, C. (2009). Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation. Neuroscience Research, 65(3), 252–258. https://doi.org/https://doi.org/10.1016/j.neures.2009.07.007 Li, Y., Liu, L., Barger, S. W., & Griffin, W. S. T. (2003). Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway. The Journal of Neuroscience, 23(5), 1605 LP – 1611. https://doi.org/10.1523/JNEUROSCI.23-05-01605.2003 Lian, H., Roy, E., & Zheng, H. (2016). Microglial Phagocytosis Assay. Bio-Protocol, 6(21), e1988. https://doi.org/10.21769/BioProtoc.1988 Liang, L., Wang, H., Hu, Y., Bian, H., Xiao, L., & Wang, G. (2022). Oridonin relieves depressive-like behaviors by inhibiting neuroinflammation and autophagy impairment in rats subjected to chronic unpredictable mild stress. Phytotherapy Research, 36(8), 3335–3351. https://doi.org/https://doi.org/10.1002/ptr.7518 Liang, L., Zheng, Y., Xie, Y., Xiao, L., & Wang, G. (2021). Oridonin ameliorates insulin resistance partially through inhibition of inflammatory response in rats subjected to chronic unpredictable mild stress. International Immunopharmacology, 91, 107298. 117 https://doi.org/https://doi.org/10.1016/j.intimp.2020.107298 Lin, K.-H., Li, C.-Y., Hsu, Y.-M., Tsai, C.-H., Tsai, F.-J., Tang, C.-H., Yang, J.-S., Wang, Z.-H., & Yin, M.-C. (2019). Oridonin, A natural diterpenoid, protected NGF-differentiated PC12 cells against MPP+- and kainic acid-induced injury. Food and Chemical Toxicology, 133, 110765. https://doi.org/https://doi.org/10.1016/j.fct.2019.110765 Liu, C., He, L., Wang, J., Wang, Q., Sun, C., Li, Y., Jia, K., Wang, J., Xu, T., Ming, R., Wang, Q., & Lin, N. (2020). Anti-angiogenic effect of Shikonin in rheumatoid arthritis by downregulating PI3K/AKT and MAPKs signaling pathways. Journal of Ethnopharmacology, 260, 113039. https://doi.org/https://doi.org/10.1016/j.jep.2020.113039 Liu, J., Li, L., & Suo, W. Z. (2009). HT22 hippocampal neuronal cell line possesses functional cholinergic properties. Life Sciences, 84(9), 267–271. https://doi.org/https://doi.org/10.1016/j.lfs.2008.12.008 Liu, X., Xu, J., Zhou, J., & Shen, Q. (2021). Oridonin and its derivatives for cancer treatment and overcoming therapeutic resistance. Genes & Diseases, 8(4), 448–462. https://doi.org/https://doi.org/10.1016/j.gendis.2020.06.010 Liu, Y.-Q., You, S., Zhang, C.-L., Tashiro, S., Onodera, S., & Ikejima, T. (2005). Oridonin enhances phagocytosis of UV-irradiated apoptotic U937 cells. Biological and Pharmaceutical Bulletin, 28(3), 461–467. https://doi.org/10.1248/bpb.28.461 Liu, Yang, Dai, Y., Li, Q., Chen, C., Chen, H., Song, Y., Hua, F., & Zhang, Z. (2020). Beta-amyloid activates NLRP3 inflammasome via TLR4 in mouse microglia. Neuroscience Letters, 736, 135279. https://doi.org/https://doi.org/10.1016/j.neulet.2020.135279 Liu, Yuanbin, Peterson, D. A., Kimura, H., & Schubert, D. (1997). Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Journal of Neurochemistry, 69(2), 581–593. https://doi.org/https://doi.org/10.1046/j.1471-4159.1997.69020581.x Liu, Z., Li, J., Lin, S., Wu, Y., He, D., & Qu, P. (2021). PI3K regulates the activation of NLRP3 inflammasome in atherosclerosis through part-dependent AKT signaling pathway. Experimental Animals, 70(4), 488–497. https://doi.org/10.1538/expanim.21-0002 Lo, J., Liu, C.-C., Li, Y.-S., Lee, P.-Y., Liu, P.-L., Wu, P.-C., Lin, T.-C., Chen, C.-S., Chiu, C.-C., Lai, Y.-H., Chang, Y.-C., Wu, H.-E., Chen, Y.-R., Huang, Y.-K., Huang, S.-P., Wang, S.-C., & Li, C.-Y. (2022). Punicalagin attenuates LPS-induced inflammation and ROS production in microglia by inhibiting the MAPK/NF-κB signaling pathway and NLRP3 inflammasome activation. Journal of Inflammation Research, 15, 5347–5359. https://doi.org/10.2147/JIR.S372773 Lu, A., Magupalli, V. G., Ruan, J., Yin, Q., Atianand, M. K., Vos, M. R., Schröder, G. F., Fitzgerald, K. A., Wu, H., & Egelman, E. H. (2014). Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell, 156(6), 1193–1206. https://doi.org/10.1016/j.cell.2014.02.008 Lu, C., Chen, C., Chen, A., Wu, Y., Wen, J., Huang, F., & Zeng, Z. (2020). Oridonin attenuates myocardial ischemia/reperfusion injury via downregulating oxidative stress and NLRP3 inflammasome pathway in mice. Evidence-Based Complementary and Alternative Medicine : ECAM, 2020, 7395187. https://doi.org/10.1155/2020/7395187 118 Lu, M., Sun, X.-L., Qiao, C., Liu, Y., Ding, J.-H., & Hu, G. (2014). Uncoupling protein 2 deficiency aggravates astrocytic endoplasmic reticulum stress and nod-like receptor protein 3 inflammasome activation. Neurobiology of Aging, 35(2), 421–430. https://doi.org/https://doi.org/10.1016/j.neurobiolaging.2013.08.015 Lu, Y.-C., Yeh, W.-C., & Ohashi, P. S. (2008). LPS/TLR4 signal transduction pathway. Cytokine, 42(2), 145–151. https://doi.org/https://doi.org/10.1016/j.cyto.2008.01.006 Lučiūnaitė, A., McManus, R. M., Jankunec, M., Rácz, I., Dansokho, C., Dalgėdienė, I., Schwartz, S., Brosseron, F., & Heneka, M. T. (2020). Soluble Aβ oligomers and protofibrils induce NLRP3 inflammasome activation in microglia. Journal of Neurochemistry, 155(6), 650–661. https://doi.org/https://doi.org/10.1111/jnc.14945 Ma, B., Liu, Y., Zhang, X., Zhang, R., Zhang, Z., Zhang, Z., Liu, J., Juan, Z., Sun, X., Sun, L., Huang, J., & Feng, J. (2022). TSPO ligands protect against neuronal damage mediated by LPS-induced BV-2 microglia activation. Oxidative Medicine and Cellular Longevity, 2022, 5896699. https://doi.org/10.1155/2022/5896699 Mahmoud, S., Gharagozloo, M., Simard, C., & Gris, D. (2019). Astrocytes maintain glutamate homeostasis in the CNS by controlling the balance between glutamate uptake and release. Cells, 8(2), 184. https://doi.org/10.3390/cells8020184 Malarkey, E. B., & Parpura, V. (2008). Mechanisms of glutamate release from astrocytes. Neurochemistry International, 52(1), 142–154. https://doi.org/https://doi.org/10.1016/j.neuint.2007.06.005 Marín-Aguilar, F., Castejón-Vega, B., Alcocer-Gómez, E., Lendines-Cordero, D., Cooper, M. A., de la Cruz, P., Andújar-Pulido, E., Pérez-Alegre, M., Muntané, J., Pérez-Pulido, A. J., Ryffel, B., Robertson, A. A. B., Ruiz-Cabello, J., Bullón, P., & Cordero, M. D. (2020). NLRP3 inflammasome inhibition by MCC950 in aged mice improves health via enhanced autophagy and PPARα activity. The Journals of Gerontology: Series A, 75(8), 1457–1464. https://doi.org/10.1093/gerona/glz239 Mata-Martínez, E., Díaz-Muñoz, M., & Vázquez-Cuevas, F. G. (2022). Glial cells and brain diseases: inflammasomes as relevant pathological entities. In Frontiers in Cellular Neuroscience (Vol. 16). McKenzie, J. A., Barghash, R. F., Alsaggaf, A. T., Kulkarni, O., Boudreau, K., Menard, F., Neeland, E. G., & Klegeris, A. (2019). Synthesis and evaluation of novel pyrazole ethandiamide compounds as inhibitors of human THP-1 monocytic cell neurotoxicity. In Cells (Vol. 8, Issue 7). https://doi.org/10.3390/cells8070655 McKenzie, J. A., & Klegeris, A. (2018). Modulation of microglial functions by methyl jasmonate. Neural Regeneration Research, 13(7), 1290–1293. https://doi.org/10.4103/1673-5374.235078 McNaught, K. S. ., & Jenner, P. (2000). Extracellular accumulation of nitric oxide, hydrogen peroxide, and glutamate in astrocytic cultures following glutathione depletion, complex I inhibition, and/or lipopolysaccharide-induced activation. Biochemical Pharmacology, 60(7), 979–988. https://doi.org/https://doi.org/10.1016/S0006-2952(00)00415-9 Minkiewicz, J., de Rivero Vaccari, J. P., & Keane, R. W. (2013). Human astrocytes express a novel NLRP2 inflammasome. Glia, 61(7), 1113–1121. 119 https://doi.org/https://doi.org/10.1002/glia.22499 Miranda, K. M., Espey, M. G., & Wink, D. A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide, 5(1), 62–71. https://doi.org/https://doi.org/10.1006/niox.2000.0319 Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1), 55–63. https://doi.org/https://doi.org/10.1016/0022-1759(83)90303-4 Murray, T. E., Wenzel, T. J., Simtchouk, S., Greuel, B. K., Gibon, J., & Klegeris, A. (2022). Extracellular cardiolipin modulates select immune functions of astrocytes in toll-like receptor (TLR) 4-dependent manner. Mediators of Inflammation, 2022, 9946439. https://doi.org/10.1155/2022/9946439 Nagar, A., Rahman, T., & Harton, J. A. (2021). The ASC speck and NLRP3 inflammasome function are spatially and temporally distinct. In Frontiers in Immunology (Vol. 12). Nakanishi, A., Kaneko, N., Takeda, H., Sawasaki, T., Morikawa, S., Zhou, W., Kurata, M., Yamamoto, T., Akbar, S. M. F., Zako, T., & Masumoto, J. (2018). Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system. Inflammation and Regeneration, 38, 27. https://doi.org/10.1186/s41232-018-0085-6 Nam, K. N., Son, M.-S., Park, J.-H., & Lee, E. H. (2008). Shikonins attenuate microglial inflammatory responses by inhibition of ERK, Akt, and NF-κB: neuroprotective implications. Neuropharmacology, 55(5), 819–825. https://doi.org/https://doi.org/10.1016/j.neuropharm.2008.06.065 Nango, H., Kosuge, Y., Sato, M., Shibukawa, Y., Aono, Y., Saigusa, T., Ito, Y., & Ishige, K. (2020). Highly efficient conversion of motor neuron-like NSC-34 cells into functional motor neurons by Prostaglandin E(2). Cells, 9(7), 1741. https://doi.org/10.3390/cells9071741 Ni, F., Huang, X., Chen, Z., Qian, W., & Tong, X. (2018). Shikonin exerts antitumor activity in Burkitt’s lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR pathway and acts synergistically with doxorubicin. Scientific Reports, 8(1), 3317. https://doi.org/10.1038/s41598-018-21570-z Nie, S., Zhao, J., Huang, Y., Zhou, C., Gong, F., & Zhao, F. (2021). Study on the effect of shikonin on CD36 expression and phagocytic ability of microglia in the isolated cerebral haemorrhage model. Folia Neuropathologica, 59(2), 198–204. https://doi.org/10.5114/fn.2021.107110 Nizami, S., Hall-Roberts, H., Warrier, S., Cowley, S. A., & Di Daniel, E. (2019). Microglial inflammation and phagocytosis in Alzheimer’s disease: Potential therapeutic targets. British Journal of Pharmacology, 176(18), 3515–3532. https://doi.org/10.1111/bph.14618 Ogunrinade, F. A., Guetchueng, S. T., Katola, F. O., Aderogba, M. A., Akande, I. S., Sarker, S. D., & Olajide, O. A. (2021). Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation. Journal of Pharmacy and Pharmacology, 73(1), 118–134. https://doi.org/10.1093/jpp/rgaa019 120 Pellegrini, C., Antonioli, L., Lopez-Castejon, G., Blandizzi, C., & Fornai, M. (2017). Canonical and non-canonical activation of NLRP3 inflammasome at the crossroad between immune tolerance and intestinal inflammation. In Frontiers in Immunology (Vol. 8, p. 36). Perera, A. P., Fernando, R., Shinde, T., Gundamaraju, R., Southam, B., Sohal, S. S., Robertson, A. A. B., Schroder, K., Kunde, D., & Eri, R. (2018). MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice. Scientific Reports, 8(1), 8618. https://doi.org/10.1038/s41598-018-26775-w Pihlaja, R., Koistinaho, J., Malm, T., Sikkilä, H., Vainio, S., & Koistinaho, M. (2008). Transplanted astrocytes internalize deposited β-amyloid peptides in a transgenic mouse model of Alzheimer’s disease. Glia, 56(2), 154–163. https://doi.org/https://doi.org/10.1002/glia.20599 Pike, A. F., Varanita, T., Herrebout, M. A. C., Plug, B. C., Kole, J., Musters, R. J. P., Teunissen, C. E., Hoozemans, J. J. M., Bubacco, L., & Veerhuis, R. (2021). α-Synuclein evokes NLRP3 inflammasome-mediated IL-1β secretion from primary human microglia. Glia, 69(6), 1413–1428. https://doi.org/https://doi.org/10.1002/glia.23970 Pollen, A. A., Bhaduri, A., Andrews, M. G., Nowakowski, T. J., Meyerson, O. S., Mostajo-Radji, M. A., Di Lullo, E., Alvarado, B., Bedolli, M., Dougherty, M. L., Fiddes, I. T., Kronenberg, Z. N., Shuga, J., Leyrat, A. A., West, J. A., Bershteyn, M., Lowe, C. B., Pavlovic, B. J., Salama, S. R., … Kriegstein, A. R. (2019). Establishing cerebral organoids as models of human-specific brain evolution. Cell, 176(4), 743-756.e17. https://doi.org/https://doi.org/10.1016/j.cell.2019.01.017 Prasad, R. G., Choi, Y. H., & Kim, G.-Y. (2015). Shikonin isolated from Lithospermum erythrorhizon downregulates proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglial cells by suppressing crosstalk between reactive oxygen species and NF-κB. Biomolecules & Therapeutics, 23(2), 110–118. https://doi.org/10.4062/biomolther.2015.006 Rauf, A., Badoni, H., Abu-Izneid, T., Olatunde, A., Rahman, M. M., Painuli, S., Semwal, P., Wilairatana, P., & Mubarak, M. S. (2022). Neuroinflammatory Markers: Key Indicators in the Pathology of Neurodegenerative Diseases. In Molecules (Vol. 27, Issue 10). https://doi.org/10.3390/molecules27103194 Reen, D. J. (1994). Enzyme-linked immunosorbent assay (ELISA). BT - Basic Protein and Peptide Protocols, 461–466. https://doi.org/10.1385/0-89603-268-X:461 Ren, P., Wu, D., Appel, R., Zhang, L., Zhang, C., Luo, W., Robertson, A. A. B., Cooper, M. A., Coselli, J. S., Milewicz, D. M., Shen, Y. H., & LeMaire, S. A. (2020). Targeting the NLRP3 inflammasome with inhibitor MCC950 prevents aortic aneurysms and dissections in mice. Journal of the American Heart Association, 9(7), e014044–e014044. https://doi.org/10.1161/JAHA.119.014044 Ren, Y., Wang, Q., Yang, Z., Feng, L., & Zhang, Y. (2022). MCC950 ameliorates the dementia symptom at the early age of line M83 mouse and reduces hippocampal α-synuclein accumulation. Biochemical and Biophysical Research Communications, 611, 23–30. https://doi.org/https://doi.org/10.1016/j.bbrc.2022.04.076 Ribrault, C., Sekimoto, K., & Triller, A. (2011). From the stochasticity of molecular processes to the variability of synaptic transmission. Nature Reviews Neuroscience, 12(7), 375–387. 121 https://doi.org/10.1038/nrn3025 Roemeling, C. A. von, Wang, Y., Qie, Y., Yuan, H., Zhao, H., Liu, X., Yang, Z., Yang, M., Deng, W., Bruno, K. A., Chan, C. K., Lee, A. S., Rosenfeld, S. S., Yun, K., Johnson, A. J., Mitchell, D. A., Jiang, W., & Kim, B. Y. S. (2020). Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity. Nature Communications, 11(1), 1508. https://doi.org/10.1038/s41467-020-15129-8 Rothman, D. L., Behar, K. L., Hyder, F., & Shulman, R. G. (2003). In vivo NMR studies of the glutamate neurotransmitter flux and neuroenergetics: implications for brain function. Annual Review of Physiology, 65(1), 401–427. https://doi.org/10.1146/annurev.physiol.65.092101.142131 Sandall, C. F., & MacDonald, J. A. (2019). Effects of phosphorylation on the NLRP3 inflammasome. Archives of Biochemistry and Biophysics, 670, 43–57. https://doi.org/https://doi.org/10.1016/j.abb.2019.02.020 Saresella, M., La Rosa, F., Piancone, F., Zoppis, M., Marventano, I., Calabrese, E., Rainone, V., Nemni, R., Mancuso, R., & Clerici, M. (2016). The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer’s disease. Molecular Neurodegeneration, 11, 23. https://doi.org/10.1186/s13024-016-0088-1 Schmid-Burgk, J. L., Chauhan, D., Schmidt, T., Ebert, T. S., Reinhardt, J., Endl, E., & Hornung, V. (2016). A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. The Journal of Biological Chemistry, 291(1), 103–109. https://doi.org/10.1074/jbc.C115.700492 Schoedon, G., Schneemann, M., Hofer, S., Guerrero, L., Blau, N., & Sschaffner, A. (1993). Regulation of the L-arginine-dependent and tetrahydrobiopterin-dependent biosynthesis of nitric oxide in murine macrophages. European Journal of Biochemistry, 213(2), 833–839. https://doi.org/https://doi.org/10.1111/j.1432-1033.1993.tb17826.x Serrano-Pozo, A., Mielke, M. L., Gómez-Isla, T., Betensky, R. A., Growdon, J. H., Frosch, M. P., & Hyman, B. T. (2011). Reactive glia not only associates with plaques but also parallels tangles in Alzheimer’s disease. The American Journal of Pathology, 179(3), 1373–1384. https://doi.org/https://doi.org/10.1016/j.ajpath.2011.05.047 Sha, W., Mitoma, H., Hanabuchi, S., Bao, M., Weng, L., Sugimoto, N., Liu, Y., Zhang, Z., Zhong, J., Sun, B., & Liu, Y.-J. (2014). Human NLRP3 inflammasome senses multiple types of bacterial RNAs. Proceedings of the National Academy of Sciences, 111(45), 16059–16064. https://doi.org/10.1073/pnas.1412487111 Sharif, H., Wang, L., Wang, W. L., Magupalli, V. G., Andreeva, L., Qiao, Q., Hauenstein, A. V, Wu, Z., Núñez, G., Mao, Y., & Wu, H. (2019). Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature, 570(7761), 338–343. https://doi.org/10.1038/s41586-019-1295-z Sharma, J. N., Al-Omran, A., & Parvathy, S. S. (2007). Role of nitric oxide in inflammatory diseases. Inflammopharmacology, 15(6), 252–259. https://doi.org/10.1007/s10787-007-0013-x Shields, H. J., Traa, A., & Van Raamsdonk, J. M. (2021). Beneficial and detrimental effects of 122 reactive oxygen species on lifespan: a comprehensive review of comparative and experimental studies. In Frontiers in Cell and Developmental Biology (Vol. 9). Sierra, A., Encinas, J. M., Deudero, J. J. P., Chancey, J. H., Enikolopov, G., Overstreet-Wadiche, L. S., Tsirka, S. E., & Maletic-Savatic, M. (2010). Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell, 7(4), 483–495. https://doi.org/10.1016/j.stem.2010.08.014 Ślusarczyk, J., Trojan, E., Głombik, K., Piotrowska, A., Budziszewska, B., Kubera, M., Popiołek-Barczyk, K., Lasoń, W., Mika, J., & Basta-Kaim, A. (2018). Targeting the NLRP3 inflammasome-related pathways via tianeptine treatment-suppressed microglia polarization to the M1 phenotype in lipopolysaccharide-stimulated cultures. In International Journal of Molecular Sciences (Vol. 19, Issue 7). https://doi.org/10.3390/ijms19071965 Song, N., Liu, Z.-S., Xue, W., Bai, Z.-F., Wang, Q.-Y., Dai, J., Liu, X., Huang, Y.-J., Cai, H., Zhan, X.-Y., Han, Q.-Y., Wang, H., Chen, Y., Li, H.-Y., Li, A.-L., Zhang, X.-M., Zhou, T., & Li, T. (2017). NLRP3 phosphorylation is an essential priming event for inflammasome activation. Molecular Cell, 68(1), 185-197.e6. https://doi.org/https://doi.org/10.1016/j.molcel.2017.08.017 Spielman, L. J., Gibson, D. L., & Klegeris, A. (2017). Incretin hormones regulate microglia oxidative stress, survival and expression of trophic factors. European Journal of Cell Biology, 96(3), 240–253. https://doi.org/https://doi.org/10.1016/j.ejcb.2017.03.004 Strowig, T., Henao-Mejia, J., Elinav, E., & Flavell, R. (2012). Inflammasomes in health and disease. Nature, 481(7381), 278–286. https://doi.org/10.1038/nature10759 Sui, X., Yang, J., Zhang, G., Yuan, X., Li, W., Long, J., Luo, Y., Li, Y., & Wang, Y. (2020). NLRP3 inflammasome inhibition attenuates subacute neurotoxicity induced by acrylamide in vitro and in vivo. Toxicology, 432, 152392. https://doi.org/https://doi.org/10.1016/j.tox.2020.152392 Tabatabaee, M. S., & Menard, F. (2020). L-type voltage-gated calcium channel modulators inhibit glutamate-induced morphology changes in U118-MG astrocytoma cells. Cellular and Molecular Neurobiology, 40(8), 1429–1437. https://doi.org/10.1007/s10571-020-00828-z Tapia-Abellán, A., Angosto-Bazarra, D., Martínez-Banaclocha, H., de Torre-Minguela, C., Cerón-Carrasco, J. P., Pérez-Sánchez, H., Arostegui, J. I., & Pelegrin, P. (2019). MCC950 closes the active conformation of NLRP3 to an inactive state. Nature Chemical Biology, 15(6), 560–564. https://doi.org/10.1038/s41589-019-0278-6 Tejera, D., Mercan, D., Sanchez-Caro, J. M., Hanan, M., Greenberg, D., Soreq, H., Latz, E., Golenbock, D., & Heneka, M. T. (2019). Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome. The EMBO Journal, 38(17), e101064–e101064. https://doi.org/10.15252/embj.2018101064 Tremblay, M.-E., Cookson, M. R., & Civiero, L. (2019). Glial phagocytic clearance in Parkinson’s disease. Molecular Neurodegeneration, 14(1), 16. https://doi.org/10.1186/s13024-019-0314-8 Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., & Tada, K. (1980). Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). 123 International Journal of Cancer, 26(2), 171–176. https://doi.org/https://doi.org/10.1002/ijc.2910260208 Uesugi, A., Kataoka, A., Tozaki-Saitoh, H., Koga, Y., Tsuda, M., Robaye, B., Boeynaems, J.-M., & Inoue, K. (2012). Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis. Glia, 60(7), 1094–1105. https://doi.org/https://doi.org/10.1002/glia.22337 Vinaik, R., Barayan, D., Auger, C., Abdullahi, A., & Jeschke, M. G. (2020). Regulation of glycolysis and the Warburg effect in wound healing. JCI Insight, 5(17), e138949. https://doi.org/10.1172/jci.insight.138949 Virchow, R. (1856). Gesammelte abhandlungen zur wissenschaftlichen medizin. Meidinger. Vodovotz, Y., Kwon, N. S., Pospischil, M., Manning, J., Paik, J., & Nathan, C. (1994). Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN-gamma and bacterial lipopolysaccharide. The Journal of Immunology, 152(8), 4110–4118. https://doi.org/10.4049/jimmunol.152.8.4110 Voet, S., Srinivasan, S., Lamkanfi, M., & van Loo, G. (2019). Inflammasomes in neuroinflammatory and neurodegenerative diseases. EMBO Molecular Medicine, 11(6), e10248. https://doi.org/10.15252/emmm.201810248 Wang, B., Shen, C., Li, Y., Zhang, T., Huang, H., Ren, J., Hu, Z., Xu, J., & Xu, B. (2019). Oridonin overcomes the gemcitabine resistant PANC-1/Gem cells by regulating GST pi and LRP/1 ERK/JNK signalling. OncoTargets and Therapy, 12, 5751–5765. https://doi.org/10.2147/OTT.S208924 Wang, H., Mao, L., & Meng, G. (2013). The NLRP3 inflammasome activation in human or mouse cells, sensitivity causes puzzle. Protein & Cell, 4(8), 565–568. https://doi.org/10.1007/s13238-013-3905-0 Wang, S., Yu, L., Yang, H., Li, C., Hui, Z., Xu, Y., & Zhu, X. (2016). Oridonin attenuates synaptic loss and cognitive deficits in an Aβ1-42-induced mouse model of Alzheimer’s disease. PloS One, 11(3), e0151397–e0151397. https://doi.org/10.1371/journal.pone.0151397 Wang, Z., Liu, T., Gan, L., Wang, T., Yuan, X., Zhang, B., Chen, H., & Zheng, Q. (2010). Shikonin protects mouse brain against cerebral ischemia/reperfusion injury through its antioxidant activity. European Journal of Pharmacology, 643(2), 211–217. https://doi.org/https://doi.org/10.1016/j.ejphar.2010.06.027 Ward, R., Li, W., Abdul, Y., Jackson, L., Dong, G., Jamil, S., Filosa, J., Fagan, S. C., & Ergul, A. (2019). NLRP3 inflammasome inhibition with MCC950 improves diabetes-mediated cognitive impairment and vasoneuronal remodeling after ischemia. Pharmacological Research, 142, 237–250. https://doi.org/https://doi.org/10.1016/j.phrs.2019.01.035 Weller, J., & Budson, A. (2018). Current understanding of Alzheimer’s disease diagnosis and treatment. F1000Research, 7, F1000 Faculty Rev-1161. https://doi.org/10.12688/f1000research.14506.1 Wenzel, T. J., Gates, E. J., Ranger, A. L., & Klegeris, A. (2020). Short-chain fatty acids (SCFAs) alone or in combination regulate select immune functions of microglia-like cells. Molecular 124 and Cellular Neuroscience, 105, 103493. https://doi.org/https://doi.org/10.1016/j.mcn.2020.103493 Wenzel, T. J., Ranger, A. L., McRae, S. A., & Klegeris, A. (2021). Extracellular cardiolipin modulates microglial phagocytosis and cytokine secretion in a toll-like receptor (TLR) 4-dependent manner. Journal of Neuroimmunology, 353, 577496. https://doi.org/https://doi.org/10.1016/j.jneuroim.2021.577496 Wiench, B., Eichhorn, T., Paulsen, M., & Efferth, T. (2012). Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells. Evidence-Based Complementary and Alternative Medicine : ECAM, 2012, 726025. https://doi.org/10.1155/2012/726025 Xie, M., Yu, Y., Kang, R., Zhu, S., Yang, L., Zeng, L., Sun, X., Yang, M., Billiar, T. R., Wang, H., Cao, L., Jiang, J., & Tang, D. (2016). PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation. Nature Communications, 7, 13280. https://doi.org/10.1038/ncomms13280 Xu, Y., Xue, Y., Wang, Y., Feng, D., Lin, S., & Xu, L. (2009). Multiple-modulation effects of Oridonin on the production of proinflammatory cytokines and neurotrophic factors in LPS-activated microglia. International Immunopharmacology, 9(3), 360–365. https://doi.org/https://doi.org/10.1016/j.intimp.2009.01.002 Yan, C., Yan, H., Mao, J., Liu, Y., Xu, L., Zhao, H., Shen, J., Cao, Y., Gao, Y., Li, K., & Jin, W. (2020). Neuroprotective effect of oridonin on traumatic brain injury via inhibiting NLRP3 inflammasome in experimental mice. Frontiers in Neuroscience, 14, 557170. https://doi.org/10.3389/fnins.2020.557170 Yang, H., Lv, H., Li, H., Ci, X., & Peng, L. (2019). Oridonin protects LPS-induced acute lung injury by modulating Nrf2-mediated oxidative stress and Nrf2-independent NLRP3 and NF-κB pathways. Cell Communication and Signaling : CCS, 17(1), 62. https://doi.org/10.1186/s12964-019-0366-y Yang, Jing, Ren, X., Zhang, L., Li, Y., Cheng, B., & Xia, J. (2018). Oridonin inhibits oral cancer growth and PI3K/Akt signaling pathway. Biomedicine & Pharmacotherapy, 100, 226–232. https://doi.org/https://doi.org/10.1016/j.biopha.2018.02.011 Yang, Junling, Wise, L., & Fukuchi, K. (2020). TLR4 cross-talk with NLRP3 inflammasome and complement signaling pathways in Alzheimer’s disease. In Frontiers in Immunology (Vol. 11, p. 724). Yang, Yang, Wang, H., Kouadir, M., Song, H., & Shi, F. (2019). Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death & Disease, 10(2), 128. https://doi.org/10.1038/s41419-019-1413-8 Yang, Ying, Wang, J., Yang, Q., Wu, S., Yang, Z., Zhu, H., Zheng, M., Liu, W., Wu, W., He, J., & Chen, Z. (2014). Shikonin inhibits the lipopolysaccharide-induced release of HMGB1 in RAW264.7 cells via IFN and NF-κB signaling pathways. International Immunopharmacology, 19(1), 81–87. https://doi.org/https://doi.org/10.1016/j.intimp.2014.01.003 Yao, M., Li, G., Zhou, L., Zheng, Z., Sun, Y., Liu, S., Wang, Y., & Cui, X. (2022). Shikonin inhibits neuronal apoptosis via regulating endoplasmic reticulum stress in the rat model of 125 double-level chronic cervical cord compression. Cell Biology and Toxicology. https://doi.org/10.1007/s10565-021-09648-3 Yu, T., Xie, W., & Sun, Y. (2019). Oridonin inhibits LPS-induced inflammation in human gingival fibroblasts by activating PPARγ. International Immunopharmacology, 72, 301–307. https://doi.org/https://doi.org/10.1016/j.intimp.2019.04.006 Yuan, Y., Zhao, Y., Shen, M., Wang, C., Dong, B., Xie, K., Yu, Y., & Yu, Y. (2022). Spinal NLRP3 inflammasome activation mediates IL-1β release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats. Molecular Pain, 17448069221093016. https://doi.org/10.1177/17448069221093016 Zagorodnyaya, E. B., Oskol’skii, G. I., Basharov, A. Y., Lushnikova, E. L., Nepomnyashchikh, L. M., & Zagorodnii, A. S. (2013). Biopolymeric film containing bioactive naphthoquinone (shikonin) in combined therapy of inflammatory destructive lesions in the Buccal Mucosa. Bulletin of Experimental Biology and Medicine, 156(2), 232–235. https://doi.org/10.1007/s10517-013-2318-7 Zahid, A., Li, B., Kombe, A. J. K., Jin, T., & Tao, J. (2019). Pharmacological inhibitors of the NLRP3 inflammasome. Frontiers in Immunology, 10, 2538. https://doi.org/10.3389/fimmu.2019.02538 Zang, L., He, H., Xu, Q., Yu, Y., Zheng, N., Liu, W., Hayashi, T., Tashiro, S., Onodera, S., & Ikejima, T. (2013). Reactive oxygen species H2O2 and OH, but not O2− promote oridonin-induced phagocytosis of apoptotic cells by human histocytic lymphoma U937 cells. International Immunopharmacology, 15(2), 414–423. https://doi.org/https://doi.org/10.1016/j.intimp.2013.01.004 Zang, L., Wang, J., Ren, Y., Liu, W., Yu, Y., Zhao, S., Otkur, W., Zhao, Y., Hayashi, T., Tashiro, S., Onodera, S., & Ikejima, T. (2019). Activated toll-like receptor 4 is involved in oridonin-induced phagocytosis via promotion of migration and autophagy-lysosome pathway in RAW264.7 macrophages. International Immunopharmacology, 66, 99–108. https://doi.org/https://doi.org/10.1016/j.intimp.2018.11.014 Zang, L., Xu, Q., Ye, Y., Li, X., Liu, Y., Tashiro, S., Onodera, S., & Ikejima, T. (2012). Autophagy enhanced phagocytosis of apoptotic cells by oridonin-treated human histocytic lymphoma U937 cells. Archives of Biochemistry and Biophysics, 518(1), 31–41. https://doi.org/https://doi.org/10.1016/j.abb.2011.11.019 Zeng, W., Wu, D., Sun, Y., Suo, Y., Yu, Q., Zeng, M., Gao, Q., Yu, B., Jiang, X., & Wang, Y. (2021). The selective NLRP3 inhibitor MCC950 hinders atherosclerosis development by attenuating inflammation and pyroptosis in macrophages. Scientific Reports, 11(1), 19305. https://doi.org/10.1038/s41598-021-98437-3 Zhang, D., Zhou, Q., Huang, D., He, L., Zhang, H., Hu, B., Peng, H., & Ren, D. (2019). ROS/JNK/c-Jun axis is involved in oridonin-induced caspase-dependent apoptosis in human colorectal cancer cells. Biochemical and Biophysical Research Communications, 513(3), 594–601. https://doi.org/https://doi.org/10.1016/j.bbrc.2019.04.011 Zhang, F.-Y., Hu, Y., Que, Z.-Y., Wang, P., Liu, Y.-H., Wang, Z.-H., & Xue, Y.-X. (2015). Shikonin inhibits the migration and invasion of human glioblastoma cells by targeting 126 phosphorylated β-catenin and phosphorylated PI3K/Akt: A potential mechanism for the anti-glioma efficacy of a traditional Chinese herbal medicine. International Journal of Molecular Sciences, 16(10), 23823–23848. https://doi.org/10.3390/ijms161023823 Zhang, J., Zhou, J., & Xiao, S. (2020). Shikonin inhibits growth, invasion and glycolysis of nasopharyngeal carcinoma cells through inactivating the phosphatidylinositol 3 kinase/AKT signal pathway. Anti-Cancer Drugs, 31(9). Zhang, Y., Lv, X., Hu, Z., Ye, X., Zheng, X., Ding, Y., Xie, P., & Liu, Q. (2017). Protection of Mcc950 against high-glucose-induced human retinal endothelial cell dysfunction. Cell Death & Disease, 8(7), e2941–e2941. https://doi.org/10.1038/cddis.2017.308 Zhang, Z.-Y., Daniels, R., & Schluesener, H. J. (2013). Oridonin ameliorates neuropathological changes and behavioural deficits in a mouse model of cerebral amyloidosis. Journal of Cellular and Molecular Medicine, 17(12), 1566–1576. https://doi.org/10.1111/jcmm.12124 Zhang, Z., Meszaros, G., He, W.-T., Xu, Y., de Fatima Magliarelli, H., Mailly, L., Mihlan, M., Liu, Y., Puig Gámez, M., Goginashvili, A., Pasquier, A., Bielska, O., Neven, B., Quartier, P., Aebersold, R., Baumert, T. F., Georgel, P., Han, J., & Ricci, R. (2017). Protein kinase D at the Golgi controls NLRP3 inflammasome activation. The Journal of Experimental Medicine, 214(9), 2671–2693. https://doi.org/10.1084/jem.20162040 Zhao, G., Zhang, T., Ma, X., Jiang, K., Wu, H., Qiu, C., Guo, M., & Deng, G. (2017). Oridonin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-induced RAW264.7 cells and acute lung injury. Oncotarget, 8(40), 68153–68164. https://doi.org/10.18632/oncotarget.19249 Zhong, J., Wang, Z., Xie, Q., Li, T., Chen, K., Zhu, T., Tang, Q., Shen, C., & Zhu, J. (2020). Shikonin ameliorates D-galactose-induced oxidative stress and cognitive impairment in mice via the MAPK and nuclear factor-κB signaling pathway. International Immunopharmacology, 83, 106491. https://doi.org/https://doi.org/10.1016/j.intimp.2020.106491 Zhou, X., Fernando, S. M., Pan, A. Y., Laposa, R., Cullen, K. R., Klimes-Dougan, B., & Andreazza, A. C. (2021). Characterizing the NLRP3 inflammasome in mood disorders: overview, technical development, and measures of peripheral activation in adolescent patients. International Journal of Molecular Sciences, 22(22), 12513. https://doi.org/10.3390/ijms222212513 Zhu, H., Jian, Z., Zhong, Y., Ye, Y., Zhang, Y., Hu, X., Pu, B., Gu, L., & Xiong, X. (2021). Janus kinase inhibition ameliorates ischemic stroke injury and neuroinflammation through reducing NLRP3 inflammasome activation via JAK2/STAT3 pathway inhibition. Frontiers in Immunology, 12, 714943. https://doi.org/10.3389/fimmu.2021.714943 Zhu, Y., Ruan, S., Shen, H., Guan, Q., Zhai, L., & Yang, Y. (2021). Oridonin regulates the polarized state of Kupffer cells to alleviate nonalcoholic fatty liver disease through ROS-NF-κB. International Immunopharmacology, 101, 108290. https://doi.org/https://doi.org/10.1016/j.intimp.2021.108290 Zorman, M., Sušjan, P., & Hafner-Bratkovic, I. (2016). Shikonin suppresses NLRP3 and AIM2 inflammasomes by direct inhibition of caspase-1. PloS One, 11, e0159826. https://doi.org/10.1371/journal.pone.0159826
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Klegeris, Andis
Potential chemical inhibitors of inflammasomes modulate microglial and astrocytic functions
Text