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Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases Lee, Moonhee; McGeer, Edith G; McGeer, Patrick L Feb 8, 2016

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RESEARCH Open AccessSodium thiosulfate attenuates glial-mediated neuroinflammation indegenerative neurological diseasesMoonhee Lee, Edith G. McGeer and Patrick L. McGeer*AbstractBackground: Sodium thiosulfate (STS) is an industrial chemical which has also been approved for the treatment ofcertain rare medical conditions. These include cyanide poisoning and calciphylaxis in hemodialysis patients withend-stage kidney disease. Here, we investigated the anti-inflammatory activity of STS in our glial-mediatedneuroinflammatory model.Methods: Firstly, we measured glutathione (GSH) and hydrogen sulfide (H2S, SH−) levels in glial cells after treatmentwith sodium hydrosulfide (NaSH) or STS. We also measured released levels of tumor necrosis factor-α (TNFα) andinterleukin-6 (IL-6) from them. We used two cell viability assays, MTT and lactate dehydrogenase (LDH) release assays,to investigate glial-mediated neurotoxicity and anti-inflammatory effects of NaSH or STS. We also employed Westernblot to examine activation of intracellular inflammatory pathways.Results: We found that STS increases H2S and GSH expression in human microglia and astrocytes. When humanmicroglia and astrocytes are activated by lipopolysaccharide (LPS)/interferon-γ (IFNγ) or IFNγ, they release materials thatare toxic to differentiated SH-SY5Y cells. When the glial cells were treated with NaSH or STS, there was a significantenhancement of neuroprotection. The effect was concentration-dependent and incubation time-dependent. Suchtreatment reduced the release of TNFα and IL-6 and also attenuated activation of P38 MAPK and NFκB proteins. Thecompounds tested were not harmful when applied directly to all the cell types.Conclusions: Although NaSH was somewhat more powerful than STS in these in vitro assays, STS has already beenapproved as an orally available treatment. STS may therefore be a candidate for treating neurodegenerative disordersthat have a prominent neuroinflammatory component.Keywords: NaSH, Neurotoxicity, TNFα, IL-6, Alzheimer’s disease, Parkinson’s diseaseBackgroundSodium thiosulphate (Na2S2O3, STS) is an industrial com-pound which is typically available as the pentahydrate,Na2S2O3 · 5H2O. It also has medical uses in the treatmentof some rare medical conditions. These include calciphy-laxis in hemodialysis patients with end-stage kidney dis-ease [1] as well as cyanide poisoning [2]. It also hasfunctions as a preservative in table salt (less than 0.1 %)and alcoholic beverages (less than 0.0005 %). GMP prod-ucts are widely available. While these amounts are verysmall, they indicate that the general population isconsuming STS on a regular basis and increasing the dosemay have important therapeutic applications.Recently, STS was demonstrated to function as an anti-inflammatory agent [3]. For example, in acute liver failureinduced in mice by lipopolysaccharide (LPS) or LPS/D-galactosamine, the survival rate was improved by hydro-gen sulfide (H2S) and STS [4, 5]. STS is also reported toprotect neuronal cells from ischemia [6].This at least is partially due to the antioxidative functionof these two agents; STS reacts with GSSG (oxidizedglutathione) to produce reduced glutathione in the pres-ence of hydroxyl radicals or peroxides. In addition, STShas a potential to produce hydrogen sulfide (H2S) by reac-tion with trans-sulfuration enzymes [7–10].* Correspondence: mcgeerpl@mail.ubc.caKinsmen Laboratory of Neurological Research, University of British Columbia,2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada© 2016 Lee et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Lee et al. Journal of Neuroinflammation  (2016) 13:32 DOI 10.1186/s12974-016-0488-8Previously, we reported that depletion of glutathione(GSH) in glial cells induces neuroinflammation resulting inneuronal death [11]. Neuroinflammation is, at least in part,characterized by the microglial release of proinflammatoryfactors such as cytokines and free radicals. Its purpose is toremove the source of harm so healing can take place. Butwhen the inflammation is prolonged, it may cause neuronaldysfunction and death [12]. Chronic neuroinflammation isclosely associated with the pathogenesis of several neurode-generative diseases, including Alzheimer’s disease (AD) andParkinson’s disease (PD).In earlier studies, we demonstrated that H2S or H2S-re-leasing moieties, exhibited antioxidative, anti-inflammatory,and neuroprotective properties [13–15]. In this investiga-tion, we studied the effects of sodium hydrosulfide (NaSH)or STS on SH-SY5Y neuronal cell death induced by super-natants from LPS and interferon-γ (IFNγ)-activated glialcells. These included cultured human astroglial and micro-glial cells and human THP-1 and U373 cells.We found that NaSH and STS generated H2S and GSHin THP-1 and U373 cells, reduced release of proinflamma-tory cytokines such as tumor necrosis factor-α (TNFα) andinterleukin-6 (IL-6) from LPS/IFNγ-stimulated microgliaand THP-1 cells, as well as IFNγ-stimulated astrocytes andU373 cells. The toxicity was attenuated in a concentration-dependent and incubation time-dependent manner. Thiswas due to reduced activation of intracellular inflammatorypathways such as P38 MAPK and NFκB proteins.MethodsMaterialsAll reagents were purchased from Sigma (St. Louis, MO)unless stated otherwise. The following substances wereapplied to the cell cultures: bacterial LPS (Escherichia coli055:B5) and human recombinant IFNγ (Bachem Califor-nia, Torrance, CA). Sodium thiosulfate anhydrous (STS)was purchased from Sciencelab.com Inc. (Houston, TX).Cell culture and experimental protocolsThe human monocyte THP-1 and astrocytoma U373 celllines were obtained from the American Type CultureCollection (Manassas, VA). The human neuroblastoma SH-SY5Y cell line was a gift from Dr R. Ross, Fordham Univer-sity, NY. These cells were grown in DMEM/F12 mediumcontaining 10 % fetal bovine serum (FBS) and 100 IU/mLpenicillin and 100 μg/mL streptomycin (Invitrogen, Carls-bad, CA) under humidified 5 % CO2 and 95 % air.Human astroglial and microglial cells were isolated fromsurgically resected temporal lobe tissue as described previ-ously [15]. Briefly, tissues were rinsed with a phosphate-buffered saline (PBS) solution and chopped into small(<2 mm3) pieces with a sterile scalpel. They were incu-bated in 10 mL of a 0.25 % trypsin solution at 37 °C for20 min. Subsequently, DNase I (from bovine pancreas,Pharmacia Biotech, Baie d’Urfé, PQ, Canada) was addedto reach a final concentration of 50 μg/mL. Tissues wereincubated for an additional 10 min at 37 °C. After centrifu-gation at 275g for 10 min, the cell pellet was resuspendedin the serum-containing medium and passed through a100-μm nylon cell strainer (Becton Dickinson, FranklinLakes, NJ). The cell suspension was then centrifuged (275gfor 10 min), resuspended into 10 mL of Dulbecco’s modi-fied Eagle medium (DMEM)-F12 with 10 % FBS containinggentamicin (50 μg/mL), and plated onto tissue cultureplates (Becton Dickinson) in a humidified 5 % CO2, 95 %air atmosphere at 37 °C for 2 h. This achieved adherence ofmicroglial cells. The non-adherent astrocytes along withmyelin debris were transferred into new culture plates. As-trocytes adhered slowly and were allowed to grow by re-placing the medium once a week. New passages of cellswere generated by harvesting confluent astrocyte culturesusing a trypsin–EDTA solution (0.25 % trypsin with EDTA,Invitrogen, Carlsbad, CA). Human astrocytes from up tothe fifth passage from four surgical cases were used in thestudy.For estimating the purity of astrocytic and microglial cellcultures, aliquots of the cultures were placed on glass slidesat 37 °C for 48 h. The attached cells were then fixed with4 % paraformaldehyde for 1 h at 4 °C and permeablizedwith 0.1 % Triton X-100 for 1 h at room temperature. Afterwashing twice with PBS, the astrocytic culture slides weretreated with a monoclonal anti-GFAP antibody (1/4,000,DAKO) and the microglial slides with the polyclonal anti-Iba-1 antibody (1/500, Wako Chemicals, Richmond, VA)for 3 h at room temperature. The slides were then incu-bated with Alexa Fluor 488-conjugated goat anti-mouseIgG antibody (Invitrogen, 1:500) and Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody (Invitrogen,1:500) in the dark for 3 h at room temperature to yieldred fluorescence for Iba-1 positive cells and green fluores-cence for GFAP positive cells. To visualize all cells, theslides were washed twice with PBS. Images were acquiredusing an Olympus BX51 microscope and a digital camera(Olympus DP71). Fluorescent images were colocalizedwith ImagePro software (Improvision Inc., Waltham,MA). We randomly chose 30 microscopic fields. Eachfield contained a total of about 500 cells. The numbers ofastrocytes which appeared in microglial culture fields av-eraged 3.11 ± 0.12 cells per field. The numbers of micro-glia appearing in astrocytic culture fields was 2.05 ± 0.34cells per field. The purity of microglia and astrocytic cul-tures was more than 99 % (Fig. 1).To achieve SH-SY5Y differentiation, the undifferentiatedcells were treated for 4 days with a high concentration ofretinoic acid (RA, 5 μM) in DMEM/F12 medium contain-ing 5 % FBS, 100 IU/mL penicillin, and 100 μg/mL strepto-mycin [16]. The RA-including medium was changed every2 days. Differentiated SH-SY5Y cells demonstrated neuriteLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 2 of 14extension, indicative of their differentiation [17], and actlike a fully-differentiated human neuron-like cells [18].Experimental protocolsProtocol 1Human astrocytes, U373 astrocytoma cells and THP-1cells (5 × 105 cells), and human microglial cells (5 × 104cells) were seeded into 24-well plates in 1 mL of DMEM/F12 medium containing 5 % FBS. NaSH or STS was thenintroduced at concentrations of 1 to 500 μM (the stocksolutions were prepared just before use with sterilized de-ionized water). Incubation of the mixtures was carried outfor 2, 4, 8, or 12 h. Cells were washed with PBS twice andreplated in 800 μL DMEM/F12 medium containing 5 %FBS. One set of cells was then incubated at 37 °C for 2 daysin the presence of inflammatory stimulants. For microgliaand THP-1 cells, the stimulants were LPS at 1 μg/mL andIFNγ at 333 U/mL. For astrocytes and U373 cells, thestimulant was IFNγ alone at 150 U/mL. A comparable setof cells was incubated in media without inflammatorystimulants. After incubation, the supernatants (400 μL)were transferred to differentiated human neuroblastomaSH-SY5Y cells (2 × 105 cells per well). The cells were incu-bated for a further 72 h and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were per-formed as described below.Protocol 2Since it is perceived that both NaSH and STS could be use-ful as pharmaceutical agents, they must be shown to benontoxic to humans. To determine whether they were dir-ectly affecting SH-SY5Y cell viability in the presence ofLPS/IFNγ-stimulated THP-1 conditioned medium (CM) orIFNγ-stimulated U373 CM, NaSH or STS was added to aglial cell supernatant (400 μL) just before the supernatantswere added to the SH-SY5Y cells. The glial cell superna-tants were from THP-1 cells or U373 cells that had beenactivated for 2 days with the inflammatory stimulants previ-ously described. The subsequent procedures were the sameas in protocol 1.SH-SY5Y cell viability assaysThe viability of SH-SY5Y cells following incubation withglial cell supernatants was evaluated by MTT assays aspreviously described [19]. Briefly, the viability was deter-mined by adding MTT to the SH-SY5Y cell cultures toreach a final concentration of 1 mg/mL. Following a 1 hincubation at 37 °C, the dark crystals formed were dis-solved by adding a SDS/DMF extraction buffer (300 μL,20 % sodium dodecyl sulfate, 50 % N,N-dimethylforma-mide, pH 4.7). Subsequently, plates were incubated over-night at 37 °C, and optical densities at 570 nm weremeasured by transferring 100 μL aliquots to 96-well platesand using a plate reader with a corresponding filter. Dataare presented as a percentage of the values obtained fromcells incubated in fresh medium only.The viability of differentiated SH-SY5Y cells was investi-gated by the lactate dehydrogenase (LDH) release assay[19]. Briefly, cell culture supernatants (100 μl) were pipet-ted into the wells of 96-well plates, followed by theaddition of 15 μl lactate solution (36 mg/ml in PBS) and15 μl p-iodonitrotetrazolium violet (INT) solution (2 mg/ml in PBS). The enzymatic reaction was started byaddition of 15 μl of NAD+/diaphorase solution (3 mg/mlNAD+; 2.3 mg solid/ml diaphorase). After 1 h, opticaldensities were measured with a Model 450 microplatereader (Bio-Rad Laboratories, Richmond, CA) using a490-nm filter. The amount of LDH released was expressedFig. 1 Immunofluorescence staining of Iba-1 (a, d red) in microglia and GFAP (b, e green) and merged images (c, f) in astrocytes in the microglial(a–c) and astrocytic cultures (d–f). The results suggested that more than 99 % of cells prepared from human brains are microglia and astrocytes,respectively. The calibration bar in F: 50 μmLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 3 of 14as a percentage of the value obtained in comparative wellswhere cells were 100 % lysed by 1 % Triton X-100.Measurement of TNFα and IL-6 releaseCytokine levels were measured in cell-free supernatantsfollowing 48 h incubation of THP-1 cells, U373 cells,microglial cells, and astrocytes. The cell stimulation proto-cols in these experiments were the same as that used inprotocol 1. Quantitation was performed with ELISA de-tection kits (Peprotech, NJ) following protocols describedby the manufacturer.For determining TNFα and IL-6 depletion, protocolspublished in earlier studies were followed [17, 20]. Briefly,microglia were exposed to LPS/IFNγ, and astrocytes toIFNγ, for 2 days. Their supernatants were transferred to24-well plates which had been coated with anti-TNFα oranti-IL-6 antibodies (10 mg/ml). After 3-h incubation, thesupernatants were transferred to SH-SY5Y cells. After in-cubation at 37 °C for 3 days, MTT assays were performedon the SH-SY5Y cells.Activation of P38 MAPK and NFκB protein by WesternBlottingWestern blotting on cell lysates was performed as de-scribed previously [19]. Briefly, microglia and astrocyteswere exposed to NaSH and STS at 100 μM for 8 h andwere subsequently exposed to stimulants for 2 h. Humanmicroglia and astrocytes were treated with a lysis buffer(150 mm NaCl, 12 mm deoxycholic acid, 0.1 % NonidetP-40, 0.1 % Triton X-100, and 5 mm Tris-EDTA, pH 7.4).The protein concentration of the cell lysates was then de-termined using a BCA protein assay reagent kit (Pierce,Rockford, IL). Proteins in each sample were loaded ontogels and separated by 10 % sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) (150 V, 1.5 h).The loading quantities of lysate proteins were 100 μg. Fol-lowing SDS-PAGE, proteins were transferred to a PVDFmembrane (Bio-Rad, CA) at 30 mA for 2 h. The mem-branes were blocked with 5 % milk in PBS-T (80 mmNa2HPO4, 20 mm NaH2PO4, 100 mm NaCl, 0.1 % Tween20, pH 7.4) for 1 h and incubated overnight at 4 °C with apolyclonal anti-phospho-P38 MAP kinase antibody (9211,Cell Signaling, Beverly, MA, 1/2,000) or anti-phospho-P65NFκB antibody (3031, Cell Signaling, 1/1,000). The mem-branes were then treated with a horseradish peroxidase-conjugated anti-IgG (P0448, DAKO, Mississauga, Ontario,CA, 1:2,000) or the secondary antibody anti-mouse IgG(A3682, Sigma, 1/3,000) for 3 h at room temperature, andthe bands were visualized with an enhanced chemilumin-escence system and exposure to photographic film(Hyperfilm ECL™, Amersham Biosciences). Equalization ofprotein loading was assessed independently using α-tubulin as the housekeeping protein. The primary anti-body was anti-α-tubulin (T6074, Sigma, 1/2,000) and thesecondary antibody anti-mouse IgG (A3682, Sigma, 1/3,000). Primary antibody incubation was overnight at 4 °C,and the secondary antibody incubation was for 3 h atroom temperature.Measurement of H2S LevelsH2S (HS−) levels were measured using a previously de-scribed method [15]. To suppress endogenous productionof H2S by CBS, all experiments were done with 0.1 mM ofthe specific CBS inhibitor hydroxylamine added to the so-lutions. Two sets of experiments were conducted: one inwhich the THP-1 cells and U373 cells were unstimulatedand a second where they were stimulated with inflamma-tory mediators for 48 h. For THP-1 cells, the stimulationwas LPS at 1 μg/ml and IFNγ at 333 U/ml, and for U373cells, it was IFNγ at 150 U/ml. The cells in each case weretreated with hydroxylamine plus NaSH or hydroxylamineplus STS (100 μM each) for 2, 4, 8, and 12 h. Followingtreatment, they were homogenized in 250 μl of ice-cold100 mM potassium phosphate buffer (pH 7.4) containingtrichloroacetic acid (10 % w/v). Zinc acetate (1 % w/v,250 μl) was injected to trap the generated H2S. A solutionof N,N-dimethyl-p-phenylenediamine sulfate (20 μM;133 μl) in 7.2 m HCl and FeCl3 (30 μM; 133 μl) in 1.2 MHCl was added. Absorbance at 670 nm of the resultingmixture (300 μl) was determined after 10 min using a 96-well microplate reader (Bio-Rad). The H2S concentrationof each sample was calculated against a calibration curveof NaSH (1–500 μmol/ml). The protein concentration wasmeasured with a BCA protein assay reagent kit (Pierce,Rockford, IL). The concentrations were expressed as mi-cromole per gram protein.Glutathione levelThe GSH level was assessed by the method of Hissin andHilf [21] and Lee et al. [11]. This assay detects reducedGSH by its reaction with o-phthalaldehyde (OPT) at pH8.0. Cells (106) in 1.5-ml tubes were washed twice withPBS, and 200 μl of 6.5 % trichloroacetic acid (TCA) wasadded. The mixture was incubated on ice for 10 min andcentrifuged (13,000 rpm, 1 min). The supernatant was dis-carded, and the pellets were resuspended in 200 μl of ice-cold 6.5 % TCA and centrifuged again (13,000 rpm,2 min). Supernatants (7.5 μl) were transferred to 96-wellplates containing 277.5 μl phosphate-EDTA buffer (pH8.0) in 1 M NaOH solution. Then, 15 μl OPT (1 mg/ml inmethanol) was added. The reaction mixture was incubatedin the dark at room temperature for 25 min. The fluores-cence at 350 nm excitation/420 nm emission was mea-sured in a multiwell plate reader. The concentration wascalculated from a standard curve using serial dilutions ofreduced GSH. The concentration was expressed as micro-mole per gram protein.Lee et al. Journal of Neuroinflammation  (2016) 13:32 Page 4 of 14Data analysisThe significance of differences between data sets was an-alyzed by one-way or two-way ANOVA tests. Multiplegroup comparisons were followed by a post hoc Bonfer-roni test. P values are given in the figure legends.ResultsIn these experiments, we compared NaSH with sodiumthiosulfate (STS). We firstly measured H2S (SH−) andGSH levels in THP-1 and U373 cells after treatment withSTS or NaSH. These cells are regarded as surrogate cellsof microglia and astrocytes. Intracellular H2S (SH−) andGSH concentrations were measured after treatment withLPS/IFNγ for THP-1 cells and IFNγ for U373 cells(100 μM each). STS and NaSH were exposed to the cellsfor 0, 2, 4, 8, and 12 h. The results are shown in Fig. 2. Inboth cell types, the H2S (SH−) and GSH content increasedin an incubation time-dependent manner. However, thelevels were dramatically decreased when THP-1 cells wereexposed to LPS/IFNγ, and U373 cells to IFNγ, for 2 days.For the NaSH groups, LPS/IFNγ treatment reduced bothH2S and GSH content in THP-1 cells by 75 % after 12 h.IFNγ treatment reduced H2S and GSH by 80 and 85 %, re-spectively, in U373 cells after 12 h. For STS groups, LPS/IFNγ treatment reduced H2S and GSH by 75 and 90 %, re-spectively, in THP-1 cells after in 12 h, and IFNγ treat-ment reduced by 80 % in H2S and by 90 % in GSH inU373 cells after 12 h. NaSH generated somewhat moreH2S (SH−) and GSH than STS in both unstimulated andstimulated cells. The data indicate that 12 h after NaSH orSTS treatment, substantial amounts of H2S (SH−) andGSH were still found in both types of cells after 2 days.Therefore, we chose 2, 4, 8, and 12 h incubation time pe-riods for further experiments.Release of inflammatory cytokinesInflammatory stimulation of microglia or THP-1 cellscauses them to release the inflammatory cytokines TNFαand IL-6 [22, 23]. Figure 3 shows the effect on TNFα re-lease by treatment of glial cells with NaSH and STS (1–500 μM for 8 h pre-incubation, protocol 1). THP-1 releaseof TNFα (Fig. 3a) and IL-6 (Fig. 3b) and human microglialrelease of TNFα (Fig. 3c) and IL-6 (Fig. 3d) are illustrated.The release of TNFα (Fig. 3a) and IL-6 (Fig. 3b) wasFig. 2 Intracellular H2S (SH−) and GSH concentrations produced from NaSH and STS in THP-1 cells (a, c) and U373 cells (b, d) with or withoutstimulation for 2 days (for THP-1 cells: LPS/IFNγ and for U373 cells: IFNγ). NaSH and STS (100 μM each) were separately added to THP-1 cells. See“Methods” section for details. Values are mean ± SEM, n = 4. Two-way ANOVA was carried out to test significance. Multiple comparisons werefollowed with post hoc Bonferroni tests. a–d **P < 0.01 for STS-treated groups compared with NaSH groups in the same condition (without stimulants)between 2 and 12 h, ##P < 0.01 for LPS/IFNγ- or IFNγ-activated groups compared with groups without stimulants between 2 and 12 h, ++P < 0.01 forSTS-treated groups compared with NaSH groups in the presence of stimulants between 2 and 12 h. Activation of both cells with stimulants dramaticallyreduced H2S and GSH. NaSH was a somewhat more powerful agent than STS, but there was no qualitative difference between the two agentsLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 5 of 14reduced by NaSH and STS exposure in a concentration-dependent manner (Fig. 3, NaSH: P < 0.01 for 1 μM orhigher and for STS: P < 0.01 for 10 μM or higher). The in-hibitory effects of NaSH were more powerful than STS (P< 0.01 for 1 μM or higher and for TNFα and P < 0.01 for 3μM or higher for IL-6). The IC50 values for NaSH and STSshown in Table 1 (A, B) were confirmatory. The patternwas similar in microglia (Fig. 3c, d). LPS/IFNγ stimulationcaused a 9.5-fold increase of TNFα and an 11-fold increaseof IL-6. Treatment with NaSH and STS reduced this release(NaSH: 75 % and STS: 50–60 % at 500 μM, P < 0.01).For astrocytes, IL-6 is the main inflammatory mediatorthat is generated [24]. Figure 4 shows comparable data forIL-6 release from U373 cells (Fig. 4a) and cultured astro-cytes (Fig. 4b). Cells were activated with IFNγ accordingto experimental protocol 1 and were then treated similarlyto the THP-1 and microglial cells shown in Fig. 3. The re-lease of IL-6 was more than fourfold higher in primarycultured astrocytes compared with U373 cells. However,both compounds reduced the release of IL-6 from IFNγ-activated U373 cells or astrocytes in a concentration-dependent manner (P < 0.01 NaSH: P < 0.01 from 1 μMand for STS: P < 0.01 from 10 μM). Again, NaSH isstronger in reducing IL-6 release in both cell types (P <0.01 from 1 μM). The IC50 values for NaSH and STS areshown in Table 1 (C). NaSH is more potent than STS inattenuating release of these cytokines.We investigated, as described in methods, the direct ef-fects of TNFα and IL-6 treatment, alone and in combin-ation, on the viability of SH-SY5Y cells. MTT assays wereperformed after 3 days incubation at 37 °C. There were nochanges in viability as shown in Fig. 5. However, the ef-fects were different when the cells were exposed tocytokine-depleted CM from microglia and astrocytes. Fordepleted microglial conditioned medium (CM), the SH-SY5Y viability was increased by 10.6 % for TNFα, 8.7 %for IL-6, and 21 % for TNFα + IL-6 (Fig. 5a). For astro-cytes, the corresponding increase in viability for IL-6 de-pletion was 22 % (Fig. 5b).Neuroprotective effect of NaSH and STS againstmicroglial, astrocytic, THP-1, and U373 cell toxicityWe investigated the effects of pre-treatment with NaSHand STS on the toxicity of CM toward SH-SY5Y cells. TheCM was obtained by 2 days treatment of LPS/IFNγ-Fig. 3 Effect of pre-treatment with NaSH or STS on released levels of TNFα (a, c) or IL-6 (b, d) from LPS/IFNγ-activated THP-1 cells (a, b) or LPS/IFNγ-activated microglia (c, d). 8 h pre-incubation with NaSH or STS was performed before LPS/IFNγ was exposed to the cells for 2 days. Valuesare mean ± SEM, n = 4. a, b Two-way ANOVA was carried out to test significance. Multiple comparisons were followed with post hoc Bonferronitests. +P < 0.01 for LPS/IFNγ-activated groups compared with control (CON) groups in each condition, *P < 0.01 for NaSH- or STS-treated groupscompared with LPS/IFNγ-activated groups, #P< 0.01 for STS-treated groups compared with NaSH groups in each condition. Note that both compoundsattenuated released levels of TNFα and IL-6 from both cell types in a concentration-dependent mannerLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 6 of 14activated THP-1 cells and IFNγ-activated U373 cells. Ex-perimental protocol 1 was followed.It was found that NaSH and STS each attenuated SH-SY5Y cell viability loss by THP-1 CM (Fig. 6). The effectwas concentration-dependent in the 1–500 μM range andwas time-dependent up to 12 h (Fig. 6a: 2 h pre-incubation, 6b: 4 h pre-incubation, 6c: 8 h pre-incubationand 6d: 12 h pre-incubation). At the shortest time intervalof 2 h and at the lowest concentration of 1 μM, the pro-tective effect of NaSH was minimal. But the toxicity wasreduced to about half at a concentration of 500 μM (P <0.01 from 1 μM). By 12 h the protective effect had in-creased to the point where the lowest concentration re-duced the toxicity by about one third while the highestconcentration reduced it by about five- to sixfold. Pre-treatment with STS also attenuated THP-1 toxicity towardSH-SY5Y cells at 2 h pre-incubation (P < 0.01 from50 μM) but was less effective than NaSH (P < 0.01 from3 μM); protective levels were 75 % of those obtained withNaSH.Figure 7 shows the effects of exposing SH-SY5Y cells tothe neurotoxic CM from U373 cells that had been stimu-lated with 150 U of IFNγ according to experimental proto-col 1. The figure shows highly similar data to thoseobtained from stimulated THP-1 cells (Fig. 7a: 2 h pre-incubation, 7b: 4 h pre-incubation, 7c: 8 h pre-incubationand 7d: 12 h pre-incubation) were obtained. Again, the pro-tective effect of the both agents was both concentration-dependent and incubation time-dependent. NaSH wasmore neuroprotective than STS (MTT test data: P < 0.01Table 1 IC50 (μM) based on stimulated glial cell-released TNFα and IL-6 data of NaSH and STS at 8 h pre-incubation times(A) TNFα: THP-1 cells and microgliaTHP-1 cells MicrogliaPre-incubation time NaSH STS NaSH STS8 h 31.15 ± 3.66 424.53 ± 18.25* 26.71 ± 4.32 579.53 ± 27.75*(B) IL-6: THP-1 cells and microgliaTHP-1 cells MicrogliaPre-incubation time NaSH STS NaSH STS8 h 63.84 ± 7.95 621.53 ± 24.25* 33.36 ± 5.32 87.28 ± 6.79*(C) IL-6: U373 cells and astrocytesU373 cells AstrocytesPre-incubation time NaSH STS NaSH STS8 h 432.26 ± 54.14 1356.53 ± 224.25* 7.44 ± 0.87 102.79 ± 11.25*The tables summarized the IC50 results of the studies in Fig. 3 (Table 1 (A, B)) and Fig. 4 (Table 1 (C)). Values are mean ± SEM, n = 4. One-way ANOVA was carriedout to test significance. Note that there was a significant reduction in IC50 values of NaSH compared with those of STS in all groups*P < 0.01Fig. 4 Effect of pre-treatment with NaSH or STS on released levels of IL-6 from IFNγ-activated U373 cells (a) or IFNγ-activated astrocytes (b). 8 hpre-incubation with NaSH or STS was performed before IFNγ was exposed to the cells for 2 days. Values are mean ± SEM, n = 4. a, b Two-wayANOVA was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. +P < 0.01 for LPS/IFNγ-activatedgroups compared with CON groups for each condition, *P < 0.01 for NaSH- or STS-treated groups compared with LPS/IFNγ-activated groups,#P < 0.01 for STS-treated groups compared with NaSH groups in each conditions. Note that both compounds attenuated released levels of IL-6from both cell types in a concentration-dependent mannerLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 7 of 14from 50 μM at 2 h, P < 0.01 from 30 μM at 4 h, P < 0.01from 10 μM at 8 h, P < 0.01 from 3 μM at 12 h).Table 2 summarized the IC50 results of these studies. Asshown in Figs. 6 and 7, the concentrations of NaSH re-quired to reach the level of IC50, for both THP-1 and U373cells, are approximately threefold lower than those requiredfor STS at all the incubation times in both THP-1 andU373 cells. The data indicate that IC50s of NaSH and STSare reduced with a longer pre-incubation time. The IC50s ofNaSH were at least three times lower than those of STS ineach pre-incubation time in both cell types.We also investigated the effect of supernatants fromLPS/IFNγ-stimulated microglia and IFNγ-stimulated as-trocytes on SH-SY5Y cell viability after treatment withall the agents. Due to the limited availability of microgliaand astrocytes, they were pre-exposed to the compoundsat only one time period (8 h) prior to treatment withstimulatory agents (experimental protocol 1). For meas-uring SH-SY5Y cell viability, the MTT and LDH releaseassays were utilized (upper panel: MTT assays and lowerpanel: LDH release assays). It was observed that bothNaSH and STS reduced the toxicity of both LPS/IFNγ-stimulated microglia (Fig. 8a) and IFNγ-stimulated astro-cytes (Fig. 8b) toward SH-SY5Y cells (MTT assay data:for NaSH: P < 0.01 from 1 μM and for STS: P < 0.01from 10 μM). Again, NaSH had a greater neuroprotec-tive effect than STS (MTT assay data: P < 0.01 from1 μM) (Table 3). These data demonstrate that the effectsobserved with cultured microglia and astrocytes arecomparable to those observed with the THP-1 and U373cell lines.However, pre-treatment with NaSH or STS of THP-1cells and U373 cells for 12 h and of microglia and astro-cytes for 8 h with both compounds did not change the via-bility of any glial cells by stimulants (LPS/IFNγ for THP-1cells and microglia: Additional file 1: Figure S1A and S1C,and IFNγ for U373 cells and strocytes: Additional file 1:Figure S1B and S1D).It was also found that NaSH and STS were not directlyprotective of SH-SY5Y cells (Additional file 1: Figure S2).When they were added to the CM after stimulation hadtaken place (protocol 2, A: THP-1 cells and B: U373 cells),they had no effect. As the figure shows, there was no dif-ference between the agents and there was no effect of con-centration. This establishes that the agents were workingby inhibiting the glial inflammatory response.In summary, NaSH and STS were not toxic to the glialcells in the presence of inflammatory stimuli. The viabilityof SH-SY5Y cells treated with the CM from LPS/IFNγ-stimulated THP-1 and IFNγ-stimulated U373 cells wasunchanged.As further control experiments, we tested the effects ofNaSH and STS on the growth and survival of humanmicroglia and astrocytes under normal and inflammatoryconditions. Human microglia and astrocytes were treatedwith NaSH or STS for 3 days. MTT assays were then per-formed on the cultures. Microglia and astrocytes werenext treated with stimulants plus NaSH or STS for 2 days.The stimulants were LPS/IFNγ for microglia and IFNγ forastrocytes. Comparative MTT assays were then performedon the cultures. The results are shown in Additional file 1:Figure S3. It was found that STS and NaSH did notFig. 5 a Contribution of TNFα and IL-6 released from LPS/IFNγ-stimulated microglia on SH-5Y5Y cell viability. Direct treatment of RA-differentiatedSH-SY5Y cells with TNFα (800 pg/ml) and/or IL-6 (600 pg/ml) was not changed SH-SY5Y cell viability (TNFa, IL-6, and TNFa + IL-6 in x-axis). Effectsof removing TNFα and/or IL-6 using their specific antibody (ELISA methods) from neurotoxic secretions from microglia stimulated with LPS/IFNγ(TNFa Ab, IL-6 Ab, and TNFa Ab + IL-6 Ab in x-axis). CON and ST mean unstimulation and stimulation, respectively. See “Methods” section for details.One-way ANOVA was carried out to test significance. *P < 0.01 vs CON group, **P < 0.01 vs ST group, and ***P < 0.01 vs TNFa Ab and IL-6 Ab groups.b Contribution of IL-6 released from IFNγ-stimulated astrocytes on SH-5Y5Y cell viability. For details, see “Methods” section. Direct treatment of RA-differentiated SH-SY5Y cells with IL-6 (120 ng/ml) was not changed SH-SY5Y cell viability (IL-6 in x-axis). Effects of removing IL-6 using their specificantibody (ELISA methods) from neurotoxic secretions from astrocytes stimulated with IFNγ (IL-6 Ab in x-axis). CON and ST mean unstimulation andstimulation, respectively. See “Methods” section for details. One-way ANOVA was carried out to test significance. *P < 0.01 vs CON group and **P < 0.01vs ST groupLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 8 of 14Fig. 6 Effect of treatment with NaSH or STS on SH-SY5Y cell viability changes induced by LPS/IFNγ-activated THP-1 cell conditioned medium(CM) as followed by MTT and lactate LDH release assays (protocol 1). a 2 h pre-incubation, b 4 h pre-incubation, c 8 h pre-incubation, and d 12 hpre-incubation. Upper panel: MTT assay data and lower panel: LDH release assay data. Values are mean ± SEM, n = 4. Two-way ANOVA was carriedout to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. +P < 0.01 for LPS/IFNγ-activated groups comparedwith CON groups in the same concentrations, *P < 0.01 for NaSH- or STS-treated groups compared with LPS/IFNγ-activated groups, #P < 0.01 forSTS-treated groups compared with NaSH roups in the same concentrations (a–d). Note that both compounds attenuated a reduction of SH-SY5Ycell viability induced by LPS/IFNγ-activated THP-1 cell CM in a concentration-dependent and pre-incubation time-dependent mannerLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 9 of 14Fig. 7 Effect of treatment with NaSH or STS on SH-SY5Y cell viability changes induced by IFNγ-activated U373 cell CM as followed by MTT andLDH release assays (protocol 1). a 2 h pre-incubation, b 4 h pre-incubation, c 8 h pre-incubation, and d 12 h pre-incubation. Upper panel: MTTassay data and lower panel: LDH release assay data. Values are mean ± SEM, n = 4. Two-way ANOVA was carried out to test significance. Multiplecomparisons were followed with post hoc Bonferroni tests. +P < 0.01 for LPS/IFNγ-activated groups compared with CON groups in the sameconcentrations, *P < 0.01 for NaSH- or STS-treated groups compared with LPS/IFNγ-activated groups, #P < 0.01 for STS-treated groups comparedwith NaSH groups at the same concentrations (a–d). Note that both compounds attenuated the reduction of SH-SY5Y cell viability induced byIFNγ-activated U373 CM in a concentration-dependent and pre-incubation time-dependent mannerLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 10 of 14change the viability of microglia and astrocytes under anyof the experimental conditions.Activation of intracellular inflammatory pathway inmicroglia and astrocytesIn the final set of experiments, we investigated the ef-fects of pre-treatment with NaSH and STS at 100 μMon phospho-P38 MAPK and phospho-NFκB proteins.These are indicative of intracellular inflammatory activa-tion. The data are shown in Fig. 9. Upon exposure to thestimulants, both microglia and astrocytes showed an in-crease in these proteins. For microglia, P38 MAPK wasincreased sevenfold and NFκB eightfold. For astrocytes,P38 MAPK and NFκB were increased sevenfold. Bothcompounds attenuated these increases, and NaSH wasagain more powerful than STS (NaSH: 80 % reductionand STS: 60–65 % reduction).DiscussionBrain damage is known to cause neuroinflammation byactivating microglia and astrocytes to release proinflam-matory factors such as cytokines, toxic free radicals, andproteases. Many previous publications indicate that oxi-dative stress in neuronal cells plays an important role intheir death [25]. Indeed, we first reported that depletionof GSH in both microglia and astrocytes induces neuro-inflammation and results in neurotoxicity. It is knownthat H2S is a reducing agent which can increase intracel-lular glutathione levels. Glutathione is a major intracel-lular antioxidant [26] and thereby inhibits oxidativestress [11]. H2S activates adenylate cyclase to increase[cAMP]i which has an inhibitory effect on transcriptionof inflammatory cytokine mRNAs [26]. All of thesemechanisms may be contributing to the results reportedhere.In this study, we have shown that STS has the samebroad spectrum inhibition of toxic inflammatory activityas NaSH. Since neuroinflammation has been demon-strated to occur in degenerative neurological diseases suchas Alzheimer’s disease (AD) and Parkinson’s disease (PD)[27, 28], STS is a potential therapeutic agent for these andother neurodegenerative disorders. It has already been ap-proved as a treatment for arsenic poisoning and calciphy-laxis in hemodialysis patients with end-stage kidneydisease. This is at least partly due to an increase in H2Sand GSH in microglia and astrocytes by STS [7–10].In these studies, we observed that the longer pre-incubation time with STS and NaSH, the lower the con-centrations of NaSH or STS to allow 50 % of SH-SY5Y cellsurvival (IC50s) when exposed to their CMs. This corre-lates with an increase in intracellular GSH and SH− levelsin both glial cell types (Fig. 2). Those increases inhibitedthe proinflammatory pathways involving P38 MAP kinaseand NFκB proteins (Fig. 9). In turn, this led to productionand release of the proinflammatory factors TNFα and IL-6(Figs. 3 and 4). The overall result was a decrease in loss ofSH-SY5Y cells in a concentration and pre-incubationtime-dependent manner (Figs. 6, 7, and 8). We found thatNaSH is more potent than STS (Tables 1, 2, and 3). Thisis apparently due to higher levels of intracellular GSH andSH− reached by NaSH compared with STS treatment.In our previous experiments, we found that TNFα andIL-6, alone and in combination, did not change SH-SY5Y cell viability (Fig. 5). However, they enhanced SH-Table 2 IC50 (μM) based on MTT (A-1 and B-1) and LDHR data (A-2 and B-2) of NaSH and STS at different pre-incubation times inTHP-1 cells and U373 cells(A) MTT dataTHP-1 cells U373 cellsPre-incubation time NaSH STS NaSH STS2 h 28.55 ± 2.32 121.53 ± 3.25 28.55 ± 3.32 95.53 ± 3.254 h 23.83 ± 2.34 96.47 ± 2.44 22.43 ± 3.34 49.63 ± 2.448 h 10.35 ± 2.11 53.55 ± 2.46 9.44 ± 2.11 30.27 ± 2.4612 h 5.83 ± 1.47 35.73 ± 1.78 4.47 ± 1.83 19.57 ± 1.78(A) LDHR dataTHP-1 cells U373 cellsPre-incubation time NaSH STS NaSH STS2 h 15.33 ± 0.88 173.53 ± 18.75 32.48 ± 2.32 98.38 ± 7.254 h 3.83 ± 0.56 100.47 ± 12.44 25.18 ± 2.21 71.05 ± 4.448 h 2.11 ± 0.16 61.81 ± 5.46 10.17 ± 1.11 47.91 ± 3.4612 h 1.42 ± 0.22 33.51 ± 2.31 1.84 ± 0.28 32.22 ± 2.78The tables summarized the IC50 results of the studies in Figs. 6 and 7. Values are mean ± SEM, n = 4. Two-way ANOVA was carried out to test significance. Multiplecomparisons were followed with post hoc Bonferroni tests. Note that there was a significant reduction in IC50 values between any pre-incubation time groups andthat there was a significant reduction in IC50 values of NaSH compared with those of STS in the same incubation time groupsLee et al. Journal of Neuroinflammation  (2016) 13:32 Page 11 of 14SY5Y sensitivity to other inflammatory materials re-leased from LPS/IFNγ-stimulated microglia and IFNγ-stimulated astrocytes [17, 20].Murutani et al. [6], in HPLC-linked measurements, re-ported that treatment of SH-SY5Y cells with Na2S in-creased intracellular and extracellular levels of thiosulfate,but not H2S. In the current study, we measured H2S levelsin THP-1 and U373 cells with the zinc acetate-mediatedH2S-trap methods (i.e., methylene blue method). Giventhe known limitation of this method that utilizes highlyacidic condition [29, 30], we were not able to measurethiosulfate levels in the glial cells in this study.It is known that direct inhalation of H2S is toxic eventhough this gas is present in our body for a wide varietyFig. 8 Effect of pre-treatment with NaSH or STS for 8 h on SH-SY5Y cell viability changes induced by a LPS/IFNγ-activated microglial CM andb IFNγ-activated astrocytic CM as followed by MTT and LDH release assays (protocol 1). Upper panel: MTT assay data and lower panel: LDH releaseassay data. Values are mean ± SEM, n = 4. Two-way ANOVA was carried out to test significance. Multiple comparisons were followed with posthoc Bonferroni tests. +P < 0.01 for LPS/IFNγ- (a) or IFNγ-activated (b) groups compared with CON groups in each condition, *P < 0.01 for NaSH-or STS-treated groups compared with LPS/IFNγ- or IFNγ-activated groups, #P < 0.01 for STS groups compared with NaSH groups in each condition.Note that both compounds attenuated a reduction of SH-SY5Y cell viability induced by LPS/IFNγ-activated THP-1 cell CM in aconcentration-dependent mannerTable 3 IC50 (μM) based on MTT (A-1 and B-1) and LDHR data (A-2 and B-2) of NaSH and STS at different pre-incubation times inmicroglia and astrocytesHuman microglia and astrocytes(B) MTT dataMiroglia AstrocytesPre-incubation time NaSH STS NaSH STS8 h 7.62 ± 0.46 68.43 ± 3.25 * 9.01 ± 0.71 77.53 ± 3.48 *(B) LDHR dataMiroglia AstrocytesPre-incubation time NaSH STS NaSH STS8 h 23.12 ± 1.46 51.43 ± 3.25 * 1.32 ± 0.11 53.06 ± 4.27 *The tables summarized the IC50 results of the studies shown in Fig. 8. Values are mean ± SEM, n = 4. One-way ANOVA was carried out to test significance. Note thatthere was a significant reduction in IC50 values of NaSH compared with those of STS*P < 0.01Lee et al. Journal of Neuroinflammation  (2016) 13:32 Page 12 of 14of functions. STS was not itself toxic to any cell types upto 500 μM under our experimental conditions. Further-more, STS and NaSH did not affect the viability of anyglial cell type (Additional file 1: Figure S1–S3). There-fore, both agents appear to be potentially suitable aspharmaceutical drugs in conditions such as Alzheimer’sdisease and Parkinson’s disease.ConclusionsIn this report, we have shown NaSH and STS to be potentanti-inflammatory agents and STS is 20–40 % less activethan NaSH in most assays, but there are no qualitative dif-ferences and this would merely translate into modestlyhigher doses to achieve the same effect. What is importantis that STS is an approved agent while NaSH must gothrough many regulatory steps before it could be ap-proved. For the present, emphasis should be on STS as apotential broad spectrum therapeutic agent.Additional fileAdditional file 1: Figure S1. Effect of NaSH or STS on cell viabilitychanges after treatment for 2 days with LPS/IFNγ-activated THP-1 cells(A), IFNγ-activated U373 cells (B), LPS/IFNγ-activated human microglia (C)and IFNγ-activated human astrocytes (D) as followed by MTT assays. (A)THP-1 cells and (B) U373 cells: 12 h preincubation with NaSH or STS and(C) microglia and (D) astrocytes: 12 h preincubation with NaSH or STS.Values are mean±SEM, n = 4. One-way ANOVA was carried out to testsignificance. Multiple comparisons were followed with post-hoc Bonfer-roni tests where necessary. Note that there was no viability change wheneach compound was exposed to the cells in the presence of LPS/IFNγ orIFNγ. Figure S2. Effect of treatment with NaSH and STS on SH-SY5Y cellviability changes induced by LPS/IFNγ-activated THP-1 cell CM (A) orIFNγ-activated U373 cell CM (B) as followed by MTT assays (Protocol 2). A:THP-1 cells and B: U373 cells. After THP-1 cells and U373 cells were stimu-lated for 2 days with LPS/IFNγ or IFNγ, respectively their supernatantswere transferred to SH-SY5Y cells. Then NaSH or STS was added. MTTtests were performed after 3 days. Values are mean±SEM, n = 4. One-wayANOVA was carried out to test significance. Multiple comparisons werefollowed with post-hoc Bonferroni tests where necessary. Note that thereare no viability changes when all the compounds were exposed to SH-SY5Y cells after LPS/IFNγ-activated THP-1 cell CM (A) or IFNγ-activatedU373 cell CM (B) were transferred. Figure S3. Effects of treatment withNaSH and STS on microglial and astrocytic viability changes in the pres-ence or absence of stimulants. (A) Microglia: no stimulation, (B) Astro-cytes: no stimulation, (C) Microglia: Stimulation and (D) Astrocytes:Stimulation. (A,B) After microglia and astrocytes were treated with STSand NaSH for 3 days MTT assays were performed. (C,D) After microgliaand astrocytes were treated with STS and NaSH for 12 h stimulants wereadded. After 2 day incubation MTT assays were performed. Values aremean±SEM, n = 4. Note that NaSH and STS did not change SH-SY5Y cellviability in the presence or absence of stimulants in any concentrationwe tested. (DOC 99 kb)AbbreviationsCM: conditioned medium; GSH: glutathione; IFNγ: interferon-γ; IL-6: interleukin-6; LDH: lactate dehydrogenase; LPS: lipopolysaccharide;STS: sodium thiosulfate; TNFα: tumor necrosis factor-α.Competing interestsThe authors declare that they have no competing interest.Authors’ contributionsML, EM and PLM designed experiments. ML performed experiments, ML andPLM analyzed data and wrote-up this manuscript. All authors read and ap-proved the final manuscript.AcknowledgementsThe authors are grateful to Dr. Rick Saway (ricksaway1@hotmail.com) for hisadvice and inspiration for these experiments and to John Anderson forgoing over the manuscript. This research was supported by donations fromthe individual British Columbians.Received: 19 October 2015 Accepted: 19 January 2016References1. Hayden MR, Goldsmith DJ. Sodium thiosulfate: new hope for the treatmentof calciphylaxis. Semin Dial. 2010;23(3):258–62.2. Berlin FR, Baseler LJ, Wilson CR, Kritchevsky JE, Taylor SD. Arsenic toxicosis incattle: meta-analysis of 156 cases. J Vet Intern Met. 2013;27(4):977–81.3. Sakaguchi M, Marutani E, Shin HS, Chen W, Hanaoka K, Xian M, et al.Sodium thiosulfate attenuates acute lung injury in mice. Anesthesiology.2014;121(6):1248–57.4. Tokuda K, Kida K, Marutani E, Crimi E, Bougaki M, Khatri A, et al. Inhaledhydrogen sulfide prevents endotoxin-induced systemic inflammation andimproves survival by altering sulfide metabolism in mice. Antioxid RedoxSignal. 2012;17(1):11–21.Fig. 9 Effect of pre-treatment with NaSH or STS for 8 h on levels ofphospho-P38 MAPK and phospho-P65-NFκB in LPS/IFNγ-activatedhuman microglia a (left panel) and IFNγ-activated astrocytes a (rightpanel). Cell extracts were prepared and the proteins separated bySDS-PAGE. Representative blots are shown in a and quantitativeresults in b. To ensure equal loading, the densitometric value ofeach band was normalized to the corresponding band for α-tubulin.Values are mean ± SEM, n = 3. One-way ANOVA was carried out toexamine the significance of differences. +P < 0.01 for LPS/IFNγ- orIFNγ-activated groups compared with CON groups in each condition,*P < 0.01 for NaSH- or STS-treated groups compared with LPS/IFNγ- orIFNγ-activated groups, #P < 0.01 for STS-treated groups compared withNaSH groups in each conditions. 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Sequentialtreatment of SH-SY5Y cells with retinoic acid and brain-derivedneurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem. 2000;75(3):991–1003.19. Lee M, Kang Y, Suk K, Schwab C, Yu S, McGeer PL. Acidic fibroblast growthfactor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIbprotein. J Biol Chem. 2011;286(48):41230–45.20. Lee M, Suk K, Kang Y, McGeer E, McGeer PL. Neurotoxic factors released bystimulated human monocytes and THP-1 cells. Brain Res. 2011;1400:99–111.21. Hissin PJ, Hilf R. A flourometric method for determination of oxidized andreduced glutathione in tissues. Anal Biochem. 1976;74:214–26.22. Klegeris A, Walker DG, McGeer PL. Toxicity of human THP-1 monocytic cellstowards neuron-like cells is reduced by non-steroidal anti-inflammatorydrugs (NSAIDs). Neuropharmacology. 1999;38(7):1017–25.23. 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Biochim Biophys Acta. 2009;1787(7):856–63.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Lee et al. Journal of Neuroinflammation  (2016) 13:32 Page 14 of 14

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