Science, Faculty of
Non UBC
DSpace
International Journal of Molecular Sciences 26 (9): 4019 (2025)
Chao, Howard Wen-Haur
Chao, Windsor Wen-Jin
Chao, Hsiao-Ming
Retinal ischemic disorders present significant threats to vision, characterized by inadequate blood supply oxygen–glucose deprivation (OGD), oxidative stress, and cellular injury, often resulting in irreversible injury. Catalpol, an iridoid glycoside derived from Rehmannia glutinosa, has demonstrated antioxidative and neuroprotective effects. This study aimed at investigating the protective effects and mechanisms of catalpol against oxidative stress or OGD in vitro and retinal ischemia in vivo, focusing on the modulation of key biomarkers of retinal ischemia, including HIF-1α, vascular endothelial growth factor (VEGF), angiopoietin-2, MCP-1, and the Wnt/β-catenin pathway. Cellular viability was assessed using retinal ganglion cell-5 (RGC-5) cells cultured in DMEM; a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. H2O2 (1 mM)/OGD was utilized. Vehicle or different catalpol concentrations were administered 15 min before the ischemic-like insults. The Wistar rat eyes’ intraocular pressure was increased to 120 mmHg for 60 min to induce retinal ischemia. Intravitreous injections of catalpol (0.5 or 0.25 mM), Wnt inhibitor DKK1 (1 μg/4 μL), anti-VEGF Lucentis (40 μg/4 μL), or anti-VEGF Eylea (160 μg/4 μL) were administered to the rats’ eyes 15 min before or after retinal ischemia. Electroretinogram (ERG), fluorogold retrograde labeling RGC, Western blotting, ELISA, RT-PCR, and TUNEL were utilized. In vitro, both H2O2 and OGD models significantly (p < 0.001/p < 0.001; H2O2 and OGD) induced oxidative stress/ischemic-like insults, decreasing RGC-5 cell viability (from 100% to 55.14 ± 2.19%/60.84 ± 4.57%). These injuries were insignificantly (53.85 ± 1.28% at 0.25 mM)/(63.46 ± 3.30% at 0.25 mM) and significantly (p = 0.003/p = 0.012; 64.15 ± 2.41%/77.63 ± 8.59% at 0.5 mM) altered by the pre-administration of catalpol, indicating a possible antioxidative and anti-ischemic effect of 0.5 mM catalpol. In vivo, catalpol had less effect at 0.25 mM for ERG amplitude ratio (median [Q1, Q3] 14.75% [12.64%, 20.48%]) and RGC viability (mean ± SE 63.74 ± 5.13%), whereas (p < 0.05 and p < 0.05) at 0.5 mM ERG’s ratio (35.43% [24.35%, 43.08%]) and RGC’s density (74.34 ± 5.10%) blunted the ischemia-associated significant (p < 0.05 and p < 0.01) reduction in ERG b-wave amplitude (6.89% [4.24%, 10.40%]) and RGC cell viability (45.64 ± 3.02%). Catalpol 0.5 mM also significantly protected against retinal ischemia supported by the increased amplitude ratio of ERG a-wave and oscillatory potential, along with recovering a delayed a-/b-wave response time ratio. When contrasted with DKK1 or Lucentis, catalpol exhibited similar protective effects against retinal ischemia via significantly (p < 0.05) blunting the ischemia-induced overexpression of β-catenin, VEGF, or angiopoietin-2. Moreover, ischemia-associated significant increases in apoptotic cells in the inner retina, inflammatory biomarker MCP-1, and ischemic indicator HIF-1α were significantly nullified by catalpol. Catalpol demonstrated antiapoptotic, anti-inflammatory, anti-ischemic (in vivo retinal ischemia or in vitro OGD), and antioxidative (in vitro) properties, counteracting retinal ischemia via suppressing upstream Wnt/β-catenin and inhibiting downstream HIF-1α, VEGF, and angiopoietin-2, together with its decreasing TUNEL apoptotic cell number and inflammatory MCP-1 concentration.
Academic Editor: J. FieldingHejtmancikReceived: 20 February 2025Revised: 7 April 2025Accepted: 7 April 2025Published: 24 April 2025Citation: Chao, H.W.-H.; Chao,W.W.-J.; Chao, H.-M. Catalpol ProtectsAgainst Retinal Ischemia ThroughAntioxidation, Anti-Ischemia,Downregulation ofβ-Catenin, VEGF,and Angiopoietin-2: In Vitro and InVivo Studies. Int. J. Mol. Sci. 2025, 26,4019. https://doi.org/10.3390/ijms26094019Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ArticleCatalpol Protects Against Retinal Ischemia ThroughAntioxidation, Anti-Ischemia, Downregulation of β-Catenin,VEGF, and Angiopoietin-2: In Vitro and In Vivo Studies †Howard Wen-Haur Chao 1,2, Windsor Wen-Jin Chao 1,3 and Hsiao-Ming Chao 4,5,6,*1 Department of Medicine, School of Medicine, Aston University, Birmingham B4 7ET, UK2 Department of Medical Education, Leeds University, Leeds LS2 9JT, UK3 Department of Science, University of British Columbia, Vancouver, BC V6T 1Z4, Canada4 Department of Chinese Medicine, School of Chinese Medicine, China Medical University,Taichung 404, Taiwan5 Institute of Pharmacology, Department of Medicine, School of Medicine,National Yang Ming Chiao Tung University, Taipei 11221, Taiwan6 Department of Ophthalmology, Shin Kong Hospital, Taipei 105, Taiwan* Correspondence: hsiaoming.chao@gmail.com† This study was accepted to perform both a Rapid Fire oral talk and a poster presentation at the26th European Association for Vision and Eye Research (EVER) Congress on 26–28 October 2023at Valencia Conference Center, Spain.Abstract: Retinal ischemic disorders present significant threats to vision, characterizedby inadequate blood supply oxygen–glucose deprivation (OGD), oxidative stress, andcellular injury, often resulting in irreversible injury. Catalpol, an iridoid glycoside de-rived from Rehmannia glutinosa, has demonstrated antioxidative and neuroprotective ef-fects. This study aimed at investigating the protective effects and mechanisms of catalpolagainst oxidative stress or OGD in vitro and retinal ischemia in vivo, focusing on themodulation of key biomarkers of retinal ischemia, including HIF-1α, vascular endothelialgrowth factor (VEGF), angiopoietin-2, MCP-1, and the Wnt/β-catenin pathway. Cellu-lar viability was assessed using retinal ganglion cell-5 (RGC-5) cells cultured in DMEM;a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed.H2O2 (1 mM)/OGD was utilized. Vehicle or different catalpol concentrations wereadministered 15 min before the ischemic-like insults. The Wistar rat eyes’ intraocu-lar pressure was increased to 120 mmHg for 60 min to induce retinal ischemia. In-travitreous injections of catalpol (0.5 or 0.25 mM), Wnt inhibitor DKK1 (1 µg/4 µL),anti-VEGF Lucentis (40 µg/4 µL), or anti-VEGF Eylea (160 µg/4 µL) were adminis-tered to the rats’ eyes 15 min before or after retinal ischemia. Electroretinogram (ERG),fluorogold retrograde labeling RGC, Western blotting, ELISA, RT-PCR, and TUNELwere utilized. In vitro, both H2O2 and OGD models significantly (p < 0.001/p < 0.001;H2O2 and OGD) induced oxidative stress/ischemic-like insults, decreasing RGC-5cell viability (from 100% to 55.14 ± 2.19%/60.84 ± 4.57%). These injuries were in-significantly (53.85 ± 1.28% at 0.25 mM)/(63.46 ± 3.30% at 0.25 mM) and signifi-cantly (p = 0.003/p = 0.012; 64.15 ± 2.41%/77.63 ± 8.59% at 0.5 mM) altered by the pre-administration of catalpol, indicating a possible antioxidative and anti-ischemic effect of0.5 mM catalpol. In vivo, catalpol had less effect at 0.25 mM for ERG amplitude ratio(median [Q1, Q3] 14.75% [12.64%, 20.48%]) and RGC viability (mean ± SE 63.74 ± 5.13%),whereas (p < 0.05 and p < 0.05) at 0.5 mM ERG’s ratio (35.43% [24.35%, 43.08%]) andRGC’s density (74.34 ± 5.10%) blunted the ischemia-associated significant (p < 0.05 andp < 0.01) reduction in ERG b-wave amplitude (6.89% [4.24%, 10.40%]) and RGC cell viabil-ity (45.64 ± 3.02%). Catalpol 0.5 mM also significantly protected against retinal ischemiasupported by the increased amplitude ratio of ERG a-wave and oscillatory potential, alongInt. J. Mol. Sci. 2025, 26, 4019 https://doi.org/10.3390/ijms26094019Int. J. Mol. Sci. 2025, 26, 4019 2 of 26with recovering a delayed a-/b-wave response time ratio. When contrasted with DKK1or Lucentis, catalpol exhibited similar protective effects against retinal ischemia via sig-nificantly (p < 0.05) blunting the ischemia-induced overexpression of β-catenin, VEGF, orangiopoietin-2. Moreover, ischemia-associated significant increases in apoptotic cells inthe inner retina, inflammatory biomarker MCP-1, and ischemic indicator HIF-1α weresignificantly nullified by catalpol. Catalpol demonstrated antiapoptotic, anti-inflammatory,anti-ischemic (in vivo retinal ischemia or in vitro OGD), and antioxidative (in vitro) proper-ties, counteracting retinal ischemia via suppressing upstream Wnt/β-catenin and inhibitingdownstream HIF-1α, VEGF, and angiopoietin-2, together with its decreasing TUNEL apop-totic cell number and inflammatory MCP-1 concentration.Keywords: catalpol; H2O2; OGD; catalpol; retinal ischemia; neuroprotection; Chinesemedicine; β-catenin; VEGF; angiopoietin-2; HIF-1α; MCP-11. IntroductionRetinal ischemia, characterized by an insufficient blood supply to the retina and/orchoroid, is a seminal factor in the pathological processes behind vision-threatening retinaldisorders. As a highly metabolic tissue, the retina relies on a continuous blood supplyproviding nutrients and oxygen to sustain cellular function; during ischemic events, thissupply is disrupted, leading to cellular stress and tissue damage. Retinal ischemia is centralto conditions such as central retinal artery occlusion (CRAO), central retinal vein occlu-sion (CRVO), proliferative diabetic retinopathy, glaucoma, and hereditary retinal diseases(e.g., familial exudative vitreoretinopathy, Coats disease, Norrie disease) [1–5].Under the conditions of ischemia—oxygen and nutrient deprivation—retinal cellsenter a state of metabolic stress, activating adaptive mechanisms to conserve energy andmaintain essential cellular functions. As oxygen levels further decline, cells shift towardsanaerobic respiration to maintain ATP production. While this switch provides short-termmetabolic support, it rapidly becomes maladaptive, as prolonged ischemia initiates acascade of damaging processes [6]. Reduced ATP availability impairs energy-dependention pumps, disrupting ionic gradients that lead to cellular swelling and dysfunction. Thisdisruption increases intracellular calcium levels, initiating excitotoxicity—a harmful processof cellular overstimulation that undermines structural integrity, while anaerobic respirationleads to lactic acid buildup, intensifying cellular stress [7].As ischemia persists, mitochondrial function is compromised, further contributing tothe buildup of reactive oxygen species (ROS). With a hampered oxygen supply, the electrontransport chain (ETC) is disrupted, leading to electron leakage that generates ROS, such assuperoxide anions. Additionally, upon reperfusion, the sudden influx of oxygen creates anoxidative burst, intensifying ROS production [8]. Moreover, the ischemia-induced immuneresponse further contributes to increased ROS production. These cumulative responsesdamage lipids, proteins, and DNA, exacerbating retinal cell degeneration [9].To compensate for oxygen deficiency, the retina initiates an adaptive neovascular re-sponse. Under hypoxic conditions, the cellular response is mediated by several key players, in-cluding hypoxia-inducible factor-1α (HIF-1α), placental growth factor (PLGF), retinoblastoma-binding protein 2 (RBP2), and vascular endothelial growth factor (VEGF) [5]. HIF-1α actsas a transcriptional regulator, activating genes that facilitate angiogenesis through increasedVEGF expression [10]. While the angiogenic response aims to restore oxygen supply, it oftenbecomes maladaptive in the retinal context. The newly formed vessels are structurally fragileand prone to leakage and rupture, resulting in complications, such as macular edema andInt. J. Mol. Sci. 2025, 26, 4019 3 of 26hemorrhage [11]. These are hallmarks of diseases, such as retinal vein/artery occlusion,diabetic macular edema, and age-related macular degeneration.While these pathways form the classical framework of pathological response in retinalischemia, recent research highlights the Wnt/β-catenin signaling pathway as a pivotalplayer with both adaptive and maladaptive roles in ischemic retinal diseases [12]. TheWnt signaling pathway is essential for the normal development of retinal vessels; however,in ischemic conditions, it becomes dysregulated and accelerates disease progression. Inthe presence of hypoxia, the Wnt/β-catenin pathway is activated as a survival mecha-nism; β-catenin (unphosphorylated), in turn, becomes overexpressed and interacts withdownstream transcriptional factors, such as HIF-1α [13], initiating a shift toward inflam-matory and proangiogenic activity. This shift amplifies VEGF production, further drivingneovascularization (NV) [14].Building upon this pathway, angiopoietin-2 (Ang-2) expression has emerged as a crucialdownstream mediator and a novel biomarker in ischemic retinal disease [15]. Ang-2, knownfor its role in destabilizing blood vessels, becomes overexpressed in response to hypoxicstress. In a balanced vascular environment, angiopoietin-1 (Ang-1) binds to the Tie-2 receptorto promote vessel stability pericyte modulation. However, as Ang-2 levels rise in ischemicconditions, it competitively inhibits Ang-1’s stabilizing effect on the Tie2 receptor, increasingvascular permeability and enhancing vessel fragility [16]. The destabilizing effect of Ang-2 ex-acerbates the pathological angiogenic cycle, especially in conjunction with elevated VEGF [15].The co-expression of Ang-2 with VEGF has shown accelerated NV compared to VEGF alone,exacerbating the fragility and abnormality of the immature vessels [17]. Without intervention,this cascade results in severe complications, including ocular hemorrhage, subretinal andcystoid macular edema, and eventual retinal cell death with vision compromise.Despite notable advancements in therapeutic treatments, such as intravitreal mono-clonal antibody injections targeting vascular endothelial growth factor (anti-VEGF) and/orangiopoietin-2—including Ranibizumab (Lucentis) and Aflibercept (Eylea)—retinal is-chemia remains to be a challenge in the field of ophthalmology [18]. Whilst these treatmentsgenerally help stabilize vision and reduce NV, certain severe or chronic ischemic conditionsstill do not adequately respond [19]. This limits the effectiveness of existing monotherapiesand has driven the development of combined therapeutic approaches, such as Vabysmo,which targets both VEGF and Ang-2 to enhance treatment outcomes [20]. Human vitreousextraction studies have shown that patients with exudative age-related macular degen-eration (eAMD), proliferative diabetic retinopathy, and retinal artery or vein occlusionexhibit elevated levels of Ang-2—a multifunctional cytokine involved in both angiogenesisand the regulation of inflammation [21]. Inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6,IL-10, and MCP-1) might be related to retinal ischemia-related disorders, such as exudativeAMD [21,22]. New scientific research in this field, including genetic and inflammatorystudies in the treatment of wet macular degeneration, indicate that co-inhibiting Ang-2and VEGF pathways leads to a significant reduction in blood vessel leakage and tumor-associated angiogenesis, offering a synergistic benefit over single-agent therapy [23]. Forexample, Canonica et al. (2023) reported that the dual inhibition of Ang-2 and VEGF-Anotably suppressed retinal inflammation in JR5558 mice [24]. This was demonstrated by asubstantial reduction in Iba1-positive microglia and macrophages clustered around CNVlesions, an outcome not achieved to the same extent with the single inhibition of eitherAng-2 or VEGF-A [24]. Other genetic research on Ang-2−/− phenotype mice showed lessvascular leakage in response to VEGF, while wild-type mice experienced increased leakageof VEGF and no change when treated with NaCl [25].The recognition that retinal ischemia involves complex and multiple biological path-ways highlights the need for novel treatment strategies that target multiple mechanisms.Int. J. Mol. Sci. 2025, 26, 4019 4 of 26Such treatments could improve outcomes for patients who are resistant to standardmonotherapies. Additionally, the requirement for frequent injections—often adminis-tered monthly or bi-monthly—presents challenges related to patient discomfort, potentialside effects, and long-term adherence [26]. These limitations underscore the necessity ofpursuing innovative strategies that adopt multi-targeted approaches.Catalpol, chemically known as 1,6,7-trihydroxyxanthone glycoside, is a naturallyoccurring, water-soluble, iridoid glycoside sourced from Rehmannia glutinosa, a plant com-monly referred to as the Chinese foxglove or “Di Huang” in Traditional Chinese Medicine(TCM). Valued for its anti-inflammatory and antioxidative effects, catalpol has been shownto possess anti-ischemic potential, particularly shown through its neuroprotective effects incerebral ischemia and anti-inflammatory effects in myocardial ischemia [27,28]. In vitro,catalpol has demonstrated antioxidative effects against H2O2-induced oxidative stress inastrocytes [29] and anti-ischemic-like effects against astrocytes under OGD [30]. In vivo,catalpol has demonstrated protective effects against renal ischemia/reperfusion injurythrough the downregulation of inflammatory markers [31]. Catalpol has also demonstratedneuroprotective effects against strokes in rats. Moreover, the administration of catalpolmitigated the impairment of the neovascular unit post-ischemic stroke [32].While much of the research on catalpol focuses on cardiovascular and cerebrovasculardiseases, its potential applications for retinal protection are promising, given the retina’sneuronal similarities to the brain. Recent research underscores catalpol’s antioxidative andantiapoptotic properties, particularly in relevance to ischemic conditions [27–41]. Studieshave demonstrated that catalpol can enhance the survival of neurons through neurogen-esis activation and promote vascular integrity following ischemic events [30,33,35,39,41].Consistently, catalpol has been found to increase cerebral blood flow and stimulate stroke-induced STAT3 activation, subsequently restoring STAT3 activity by facilitating its bindingto VEGF [38]. Moreover, catalpol treatment was revealed to increase the expression ofGAP-43 and p-S6 [37], contributing to pro-axonal regeneration and its neurorestorativeeffects. Further demonstrating its efficacy, catalpol has been shown to promote vascular in-tegrity amidst neovascularization following corneal ischemia [36]. Despite these promisingfindings, research on catalpol’s specific effects in retinal ischemia remains limited, makingour present study a novel contribution that could extend the understanding of catalpol’spotential as a retinal therapeutic agent.As aforementioned, inflammatory markers play a critical role in ischemic-related oculardisorders, such as age-related macular degeneration (AMD), central retinal vein occlusion(CRVO), and normal-tension glaucoma. Key cytokines, including TNF-α, IL-6, and IL-1β [36],have been extensively studied in the context of ischemia reperfusion injury, with previousresearch demonstrating the anti-inflammatory effects of catalpol. Studies have shown thatcatalpol can suppress VEGF and TNF-α levels in animal models, thereby reducing pathologicalneovascularization and dampening inflammatory responses. While these cytokines havebeen the primary focus of prior research, monocyte chemoattractant protein-1 (MCP-1) isan equally critical inflammatory chemokine that warrants further investigation. Traditionalstudies on ischemia reperfusion injury have predominantly centered on TNF-α, IL-6, andIL-1β in the context of catalpol’s effects, as previously mentioned [36]. To build upon thisexisting knowledge, we have incorporated an innovative approach by exploring MCP-1’santi-inflammatory pathway. Despite its established role in retinal inflammation, MCP-1has received comparatively less attention in this context. It plays a pivotal role in immunecell recruitment, particularly macrophages, which drive oxidative stress, inflammation, andvascular dysfunction. Given its significance in retinal pathology, our study aimed to expandthe understanding of MCP-1’s role in ischemic retinal conditions, highlighting its importancealongside TNF-α, IL-6, and IL-1β in the inflammatory cascade.Int. J. Mol. Sci. 2025, 26, 4019 5 of 26The main aim of this study is to investigate the potential neuroprotective and anti-ischemic effects of catalpol in the context of retinal ischemia, based on preliminary datapreviously presented as a poster and oral presentation at the 26th European Associationfor Vision and Eye Research (EVER) Congress [42]. This study will employ a multifacetedapproach combining in vivo and in vitro studies to comprehensively assess the impactof catalpol in retinal ischemia. In vitro investigations included the use of a cell injurymodel, including H2O2-induced oxidative stress mode, OGD model, and MTT cell viabilityassay. In vivo studies included electroretinogram (ERG), the retrograde labeling of retinalganglion cells (RGCs), TUNEL assay, Western blot, and mRNA expression studies. Thepresent study measured the expression levels of critical retinal ischemic or related inflam-matory biomarkers, such as MCP-1, β-catenin, VEGF, and Ang-2, to gain insights into themechanisms of action of catalpol against retinal ischemia.2. Results2.1. Oxidative StressCellular viability was assessed by examining cell density under an optical mi-croscopy (Figure 1, n = 6). In the control group (Figure 1A; RGC-5 cells cultured inDMEM), a notable population of RGC-5 cells were observed. Conversely, exposure tooxidative stress induced by the incubation of RGC-5 cells with 1 mM H2O2 (Figure 1B)for 24 h resulted in a prominent reduction in RGC-5 cell count. Notably, 15 min pre-administration of catalpol did not exhibit a therapeutic effect at 0.25 mM against 1 mMH2O2 (Figure 1C), with a more pronounced protective effect against oxidative stress ob-served at 0.5 mM (Figure 1D). In Figure 1E, in comparison with the RGC-5 cells culturedin DMEM (control = 100%; n = 6), exposure to 1 mM of H2O2 for 24 h led to a signifi-cant (*** p < 0.001) reduction in cell viability, which was not affected by pretreatment withvehicle (vehicle + H2O2 1 mM = 55.14 ± 2.19%; n = 6). Furthermore, when compared toRGC-5 cells subjected to 24 h H2O2-induced oxidative stress, 15 min pre-administrationof 0.5 mM catalpol resulted in a statistically significant (†† p = 0.003) amelioration of theH2O2-induced decrease in cell viability (catalpol 0.5 mM + H2O2 1 mM = 64.15 ± 2.41%;n = 6). Notably, preincubation with 0.25 mM catalpol did not exhibit an improvement incell viability (catalpol 0.25 mM + H2O2 1 mM = 53.85 ± 1.28%; n = 6).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 5 of 28 pathology, our study aimed to expand the understanding of MCP-1’s role in ischemic ret-inal conditions, highlighting its import ce alongside TNF-α, IL-6, and IL-1β in the in-flammatory cascade. The main aim of this study is to investigate the potential neuroprotective and anti-ischemic effects of catalpol in the context of retinal ischemia, based on preliminary data previously presented as a poster and oral presentation at the 26th European Association for Vision and Eye Research (EVER) Congress [42]. This study will employ a multifaceted approach combining in vivo an in vitro studies to comprehensively assess the impact of catalpol i retinal ischemia. In vitro inve tigation included he use of a cell injury model, including H2O2-induced oxidative stress mode, OGD model, and MTT cell viability assay. In vivo studies included electroretinogram (ERG), the retrograde labeling of retinal gan-glion cells (RGCs), TUNEL assay, Western blot, and mRNA expression studies. The pre-sent study measured the expression levels of critical retinal ischemic or related inflamma-tory biomarkers, such as MCP-1, β-catenin, VEGF, and Ang-2, to gain insights into the mechanisms of action of catalpol against retinal ischemia. 2. Results 2.1. Oxidative Stress Cellular viability was assessed by examining cell density under an optical micros-copy (Figure 1, n = 6). In the control group (Figure 1A; RGC-5 cells cultured in DMEM), a notable population of RGC-5 cells were observed. Conversely, exposure to oxidative stress induced by the incubation of RGC-5 cells with 1 mM H2O2 (Figure 1B) for 24 h resulted in prominent reduction in RGC-5 cell count. Notably, 15 min re-administration of catalpol did not exhibit a therapeutic effect at 0.25 mM against 1 mM H2O2 (Figure 1C), with a more pronounced protective effect against oxidative stress observed at 0.5 mM (Figure 1D). In Figure 1E, in comparison with the RGC-5 cells cultured in DMEM (control = 100%; n = 6), exposure to 1 mM of H2O2 for 24 h led to a significant (*** p < 0.001) reduction in cell viability, which was not affected by pretreatment with vehicle (vehicle + H2O2 1 mM = 55.14 ± 2.19%; n = 6). Furthermore, when compared to RGC-5 cells subjected to 24 h H2O2-induced oxidative stress, 15 min pre-administration of 0.5 mM catalpol resulted in a st -tistically signific nt (†† p = 0.003) amelioration of the H2O2-induced decrease in cell viability (catalpol 0.5 mM + H2O2 1 mM = 64.15 ± 2.41%; n = 6). Notably, preincubation with 0.25 mM catalpol did not exhibit an improvement in cell viability (catalpol 0.25 mM + H2O2 1 mM = 53.85 ± 1.28%; n = 6). Figure 1. Oxidative stress model. Optical microscopy and cell viability. (A) Control (RGC-5 cells in DMEM) displayed numerous cells. (B) Incubation with 1 mM H2O2 for 24 h drastically reduced cell Figure 1. Oxidative stress model. Optical microscopy and cell viability. (A) Control (RGC-5 cellsin DMEM) displayed numerous cells. (B) Incubation with 1 mM H2O2 for 24 h drastically reducedcell count. (C,D) Fifteen min pre-incubation with catalpol did not exhibit a therapeutic effect against1 mM H2O2 ((C); at 0.25 mM) but resulted in a prominent protection of the cells against oxidativestress ((D); at 0.5 mM). (E) Compared to control, 24 h incubation of cells with 1 mM H2O2 significantly(*** p < 0.001) reduced cell viability. However, 15 min pre-incubation with 0.5 mM catalpol significantly(†† p < 0.01) ameliorated the H2O2-induced significant (*** p < 0.001) decrease in cell viabil-ity. Scale bar = 50 µm. Abbreviations: RGC—retinal ganglion cell; DMEM—Dulbecco’s modifiedEagle medium.Int. J. Mol. Sci. 2025, 26, 4019 6 of 262.2. Oxygen–Glucose Deprivation (OGD)The assessment of RGC-5 cell count was performed via light microscopy (Figure 2).The normal group consisted of cells cultured in DMEM (Figure 2A), while cells subjectedto OGD with pre-administration of DMSO were defined as vehicle + OGD (Figure 2B).A noticeable reduction in cell count compared to the normal group was observed in thevehicle + OGD group. Subsequently, the impact of 15 min pre-administration of 0.25 mM(Figure 2C) and 0.5 mM (Figure 2D) catalpol on cells subjected to OGD was examined.Specifically, the protective effect of the pre-OGD administration of catalpol on the OGDwas demonstrated by increased cell viabilities at 0.5 mM. The effect of 0.5 mM catalpol wasfound to be greater when administered 15 min before OGD (Figure 2D), with a lesser effectobserved at 0.25 mM catalpol (Figure 2C).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 6 of 28 count. (C,D) Fifteen min pre-incubation with catalpol did not exhibit a therapeutic effect against 1 mM H2O2 ((C); at 0.25 mM) but resulted in a prominent protection of the cells against oxidative stress ((D); at 0.5 mM). (E) Compared to control, 24 h incubation of cells with 1 mM H2O2 signifi-cantly (*** p < 0.001) reduced cell viability. However, 15 min pre-incubation with 0.5 mM catalpol significantly (†† p < 0.01) ameliorated the H2O2-induced significant (*** p < 0.001) decrease in cell viability. Scale bar = 50 µm. Abbreviations: RGC—retinal ganglion cell; DMEM—Dulbecco’s modi-fied Eagle medium. 2.2. Oxygen–Glucose Deprivation (OGD) The assessment of RGC-5 cell count was performed via light microscopy (Figure 2). The normal group consisted of cells cultured in DMEM (Figure 2A), while cells subjected to OGD with pre-administration of DMSO were defined as vehicle + OGD (Figure 2B). A noticeable reduction in cell count compared to the normal group was observed in the ve-hicle + OGD group. Subsequently, the impact of 15 min pre-administration of 0.25 mM (Figure 2C) and 0.5 mM (Figure 2D) catalpol on cells subjected to OGD was examined. Specifically, the protective effect of the pre-OGD administration of catalpol on the OGD was demonstrated by increased cell viabilities at 0.5 mM. The effect of 0.5 mM catalpol was found to be greater when administered 15 min before OGD (Figure 2D), with a lesser eff ct observed at 0.25 mM catalpol (Fig 2C). In Figure 2E, cell viability was significantly (*** p < 0.001) reduced in the vehicle + OGD group (60.84 ± 4.57%; n = 3) compared to the normal group (100%; n = 3). Moreover, compared to the vehicle + OGD group, the pre-OGD administration of 0.5 mM catalpol (catalpol 0.5 mM + OGD = 77.63 ± 8.59%; n = 3) exhibited a significant (* p = 0.012) prot c-tive effect against OGD. How ver, the 15 min pre-ad i istr ti 0.25 mM (ca-talpol 0.25 mM + OGD = 63.46 ± 3.30%; n = 3) did not have significant (p = 0.55) increase in cell viabilities gainst OGD. Figure 2. OGD model. Light microscopy was utilized to examine RGC-5 cell count. (A) Normal cells in DMEM. (B) Cells subjected to 8 h OGD and pre-administered with DMSO (vehicle + OGD) showed a considerable reduction in the cell number compared to normal. Pre-administration of ca-talpol demonstrated increased cell viability with greater effect at 0.5 mM (D) and lesser at 0.25 mM (C). As demonstrated in Figure (E), cell viability was significantly (*** p < 0.001; n = 3) reduced (60.84 ± 4.57%) following OGD. This reduction was significantly († p = 0.012) ameliorated following 15 min pre-administration of 0.5 mM catalpol (77.63 ± 8.59%). However, 15 min pre-administration of 0.25 mM catalpol (63.46 ± 3.30%) did not significantly protect cells against the OGD. The control was normalized as 100%. Scale bar = 50 µm. Abbreviations: OGD—oxygen–glucose deprivation; DMEM—Dulbecco’s modified Eagle medium. Figure 2. OGD model. Light microscopy was utilized to examine RGC-5 cell count. (A) Normalcells in DMEM. (B) Cells subjected t 8 h OGD and pre-administered with DMSO (vehicle + OGD)showed a considerable reduction in the cell number compared to normal. Pre-administrationof catalpol demonstrated increased cell viability with greater effect at 0.5 mM (D) and lesser at0.25 mM (C). As demonstrated in Figure (E), cell viability was significantly (*** p < 0.001; n = 3)reduced (60.84 ± 4.57%) following OGD. This reduction was significantly († p = 0.012) amelioratedfollowing 15 min pre-administration of 0.5 mM catalpol (77.63 ± 8.59%). However, 15 min pre-administration of 0.25 mM catalpol (63.46± 3.30%) did not significantly protect cells against the OGD.The control was normalized as 100%. Scale bar = 50 µm. Abbreviations: OGD—oxygen–glucosedeprivation; DMEM—Dulbecco’s modified Eagle medium.In Fig re 2E, cell viability was significantly (*** p < 0.001) reduced in the vehicle + OGDgroup (60.84 ± 4.57%; n = 3) compared to the normal group (100%; n = 3). Moreover,compared to the vehicle + OGD group, the pre-OGD administration of 0.5 mM catalpol(catalpol 0.5 mM + OGD = 77.63 ± 8.59%; n = 3) exhibited a significant (* p = 0.012)protective effect against OGD. However, the 15 min pre-administration of catalpol 0.25 mM(catalpol 0.25 mM + OGD = 63.46 ± 3.30%; n = 3) did not have significant (p = 0.55) increasein cell viabilities against OGD.2.3. Electroretinogram (ERG)The ERG wavefront averages were utilized to examine the retinal electrophysiologicalresponse (Figure 3). As depicted in Figure 3A, the ERG b-wave amplitude was found to be2.04 mV in the retinas of normal rat eyes. Following retinal ischemia, a drastic reductionin b-wave amplitude was observed, unaffected by pre-ischemia treatment with vehicle(vehicle + I/R: 0.06 mV). However, pre-ischemia treatment with 0.25 or 0.5 mM catalpol(Cata 0.25 mM + I/R or Cata 0.5 mM + I/R) mitigated the ischemia-induced reduction inb-wave amplitude, raising the amplitudes to 0.32 and 0.75 mV, respectively.Int. J. Mol. Sci. 2025, 26, 4019 7 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 7 of 28 2.3. Electroretinogram (ERG) The ERG wavefront averages were utilized to examine the retinal electrophysiologi-cal response (Figure 3). As depicted in Figure 3A, the ERG b-wave amplitude was found to be 2.04 mV in the retinas of normal rat eyes. Following retinal ischemia, a drastic re-duction in b-wave amplitude was observed, unaffected by pre-ischemia treatment with vehicle (vehicle + I/R: 0.06 mV). However, pre-ischemia treatment with 0.25 or 0.5 mM catalpol (Cata 0.25 mM + I/R or Cata 0.5 mM + I/R) mitigated the ischemia-induced reduc-tion in b-wave amplitude, raising the amplitudes to 0.32 and 0.75 mV, respectively. As shown in Figure 3B and Table 1 (n = 6), compared to the control group, the b-wave ratio exhibited a significant (* p < 0.05) decrease in the vehicle + I/R group. Notably, pre-ischemic catalpol significantly († p < 0.05; at Cata 0.5 mM + I/R group) reduced the ische-mia-induced b-wave ratio. Figure 3. Electroretinogram (ERG): the effect of pre-ischemic catalpol intravitreous injection (IVI) on retinal ischemia plus reperfusion (I/R). (A) HIOP induced ischemia, and subsequent reperfusion drastically reduced ERG b-wave amplitude (vehicle + IR) compared to normal. Counteraction of this reduction was observed with pretreatment of catalpol, especially at 0.5 mM catalpol. (B) The vehicle + I/R group showed a significant decrease (* p < 0.05) in the b-wave ratio compared to the normal group. Pretreatment with catalpol was significant at 0.5 mM († p < 0.05) mitigated the ischemia-induced decrease in b-wave ratio. (C,D) ERG a-wave/oscillatory potential. The vehicle + I/R group showed a significant decrease (* p < 0.05) in the a-wave/oscillatory potential amplitude ratios com-pared to those of the normal group. Pretreatment with catalpol significant († p < 0.05) at 0.5 mM mitigated the ischemia-induced decrease in a-wave/oscillatory potential amplitude ratio. (E,F) ERG a-/b-wave response time. ERG a-/b-wave response time ratio was significantly delayed in the vehicle + I/R group compared to the normal group (* p < 0.05), indicating delayed nerve impulse conduction after I/R injury. Treatment with catalpol 0.25 mM + I/R counteracted delayed response time but did not reach statistical significance compared to the vehicle + I/R group (* p > 0.05). However, catalpol 0.5 mM + I/R significantly resuscitated prolonged response time compared to the vehicle + I/R group († p < 0.05) and approached normal levels. Abbreviations: ischemia/reperfusion, I/R. Figure 3. Electroretinogram (ERG): the effect of pre-ischemic catalpol intravitreous injection (IVI)o retinal ischemia plus rep rfusion (I/R). (A) HIOP induced ischemia, and subseque t reperfusiondrastically reduced ERG b-wave amplitude (vehicle + IR) compared to normal. Counteraction ofthis reduction was observed with pretreatment of catalpol, especially at 0.5 mM catalpol. (B) Thevehicle + I/R group showed a significant decrease (* p < 0.05) in the b-wave ratio compared to thenormal grou . Pretreatme t with catalpol was significant at 0.5 mM († p < 0.05) mitigated t e ischemia-induced decrease in b-wave ratio. (C,D) ERG a-wave/oscillatory potential. The vehicle + I/R groupshowed a significant decrease (* p < 0.05) in the a-wave/oscillatory potential amplitude ratios comparedto those of the normal group. Pretreatment with catalpol significant († p < 0.05) at 0.5 mM mitigatedthe ischemia-induced decrease in a-wave/oscillatory potential amplitude ratio. (E,F) ERG a-/b-waverespons time. ERG a-/b-wave response time rat o was significa ly delayed in th veh cle + I/R groupcompared to the normal group (* p < 0.05), indicating delayed nerve impulse conduction after I/Rinjury. Treatment with catalpol 0.25 mM + I/R counteracted delayed response time but did not reachstatistical significance compared to the vehicle + I/R group (* p > 0.05). However, catalpol 0.5 mM + I/Rsignificantly resuscitated prolonged response time compared to the vehicle + I/R group († p < 0.05) andapproached normal levels. Abbreviations: ischemia/reperfusion, I/R.As shown in Figure 3B and Table 1 (n = 6), compared to the control group, the b-waveratio exhibited a significant (* p < 0.05) decrease in the vehicle + I/R group. Notably,pre-isch mic c talpol significantly († p < 0.05; at Cata 0.5 mM + I/R group) reduced theischemia-induced b-wave ratio.Table 1. (A) Electroretinogram b-wave. The effect of catalpol on retinal ischemia/reperfusion 1. (B) Elec-troretinogram a-wave ratio. The effect of catalpol on retinal ischemia/reperfusion 2. (C) Electroretinogramoscillatory potential ratio. The effect of catalpol on retinal ischemia/reperfusion 3. (D) Electroretinograma-wave response time ratio. The effect of catalpol on retinal ischemia/reperfusion 4. (E) Electroretinogramb-wave response time ratio. The effect of catalpol on retinal ischemia/reperfusion 5.(A)Gr Normal Vehicle + I/R Cata 0.25 mM + I/R Cata 0.5 mM + I/RM 100% 6.89% * 14.75% 35.43% †Q1 100% 4.24% 12.64% 24.35%Q3 100% 10.40% 20.48% 43.08%Int. J. Mol. Sci. 2025, 26, 4019 8 of 26Table 1. Cont.(B)Gr Normal Vehicle + I/R Catalpol 0.25 mM + I/R Catalpol 0.5 mM + I/RM 100% 20.00% * 52.00% 92.00% †Q1 90% 10.00% 42.00% 82.00%Q3 110% 30.00% 62.00% 102.00%(C)Normal Vehicle + I/R Catalpol 0.25 mM + I/R Catalpol 0.5 mM + I/R100.00 ± 2.82% 24.59 ± 2.83% * 52.06 ± 3.37% † 77.68 ± 6.15% †(D)Normal Vehicle + I/R Catalpol 0.25 mM + I/R Catalpol 0.5 mM + I/R100.00 ± 14.95% 236.28 ± 26.46% * 211.46 ± 10.71% 135.76 ± 19.02% †(E)Normal Vehicle + I/R Catalpol 0.25 mM + I/R Catalpol 0.5 mM + I/R100.00 ± 6.81% 189.58 ± 9.24% * 175.00 ± 10.76% 110.42 ± 9.24% †(A) 1 In comparison with the control group, the b-wave ratio exhibited a significant (* p < 0.05) decrease in the vehi-cle + I/R group at I/R day 1. Of note, pre-ischemic catalpol mitigated the ischemia-induced b-wave ratio reduction,with a modest effect observed at 0.25 mM and a significant effect at 0.5 mM († p < 0.05; Cata 0.5 mM + I/R group).Abbreviations: M, median; Q1, first quartile; Q3, third quartile; I/R, ischemia/reperfusion. (B) 2 In comparisonwith the control group, the a-wave ratio exhibited a significant (* p < 0.05) decrease in the vehicle + I/R groupat I/R day 1. Of note, pre-ischemic catalpol alleviated the ischemia-induced a-wave ratio reduction, with alesser effect observed at 0.25 mM and a significant improvement at 0.5 mM († p < 0.05; catalpol 0.5 mM + I/Rgroup). Abbreviations: M, median; Q1, first quartile; Q3, third quartile; I/R, ischemia/reperfusion. (C) 3 Incomparison with the control group, the oscillatory potential ratio exhibited a significant (* p < 0.05) decrease in thevehicle + I/R group at I/R day 1. Of note, pre-ischemic catalpol alleviated the ischemia-induced reduction inoscillatory potential ratio, with a modest effect observed at 0.25 mM and a significant effect at 0.5 mM († p < 0.05;catalpol 0.5 mM + I/R group). Abbreviations: I/R, ischemia/reperfusion. The results are presented as mean± SE.(D) 4 In comparison with the control group, the a-wave response time ratio was significantly (* p < 0.05) prolongedin the vehicle + I/R group at I/R day 1. Of note, pre-ischemic catalpol attenuated the ischemia-induced elongationof the a-wave response time ratio, with a smaller effect at 0.25 mM and a significant effect at 0.5 mM († p < 0.05;catalpol 0.5 mM + I/R group). Abbreviations: I/R, ischemia/reperfusion. The results are presented as mean ± SE.(E) 5 In comparison with the control group, the b-wave response time ratio was significantly (* p < 0.05) delayedin the vehicle + I/R group at I/R day 1. Notably, pre-ischemic catalpol mitigated the ischemia-induced delayin b-wave response time ratio, with a less pronounced effect at 0.25 mM and a significant effect at 0.5 mM(† p < 0.05; catalpol 0.5 mM + I/R group). Abbreviations: I/R, ischemia/reperfusion. The results are presentedas mean ± SE.As demonstrated in Figure 3C, ERG a-wave amplitude ratio was significantly reduced inthe vehicle + I/R group (median [Q1,Q2]: 20.0% [10.00%, 30.00%]) compared to the normalgroup (median [Q1, Q2]: 100% [90%, 110%]; p < 0.05), indicating substantial retinal dysfunctionfollowing I/R injury. Treatment with catalpol 0.25 mM + I/R (median [Q1, Q2]: 52.00%[42.00%, 62.00%]) and catalpol 0.5 mM + I/R (median [Q1, Q2]: 92.000% [82.00, 102.00%]) ledto significant (p < 0.05; at 0.5 mM) recovery compared to the vehicle + I/R group.As revealed in Figure 3D, ERG oscillatory potential amplitude ratio was signifi-cantly reduced in the vehicle + I/R group (24.59 ± 2.83%) compared to the normal group(100.00 ± 2.82%; p < 0.05). Treatment with both catalpol 0.25 mM + I/R (52.06 ± 3.37%)and catalpol 0.5 mM + I/R (77.68 ± 6.15%) led to significant (p < 0.05; 0.5 mM) resuscitationcompared to the vehicle + I/R group.As reported in Figure 3E,F, the ERG a-/b-wave response time ratio was significantlyprolonged in the vehicle + I/R group (236.28± 26.46%/189.58± 9.24%) compared to the normalgroup (100.00± 14.95%/100.00± 6.81%; p < 0.05), indicating delayed nerve impulse conductionafter I/R injury. Treatment with catalpol 0.25 mM + I/R (211.46± 10.71%/175.00± 10.76%)and catalpol 0.5 mM + I/R (135.76± 19.02%/110.42± 9.24%) led to less effect at 0.25 mM andimportantly significant (p < 0.05; at 0.5 mM) recovery compared to the vehicle + I/R group.Int. J. Mol. Sci. 2025, 26, 4019 9 of 262.4. The Effect of Catalpol on Retrograde Fluorogold Immunolabeled Retinal GanglionCells DensityAssessing RGC density using fluorogold retrograde labeling (Figure 4; n = 6) revealedthe density of the control group (normal, Figure 4A,E) to be 425.57 ± 31.47 cells/field.Compared to the control group, there was a significant (*** p < 0.001) reduction inRGC density (194.54 ± 18.85 cells/field) in animals subjected to retinal ischemia withpre-administered vehicle (vehicle + I/R, Figure 4B,E). Furthermore, this decrease wassignificantly (at 0.5 mM; †† p < 0.01) mitigated when the animals underwent retinalischemia with the pre-administration of catalpol (Cata 0.50 mM + I/R, Figure 4D,E:308.50 ± 12.10 cells/field). However, a non-significant effect was observed with pretreat-ment with a lower concentration of 0.25 mM catalpol (Cata 0.25 mM + I/R, Figure 4C,E:269.82 ± 25.57 cells/field).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 9 of 28 As demonstrated in Figure 3C, ERG a-wave amplitude ratio was significantly re-duced in the vehicle + I/R group (median [Q1,Q2]: 20.0% [10.00%, 30.00%]) compared to the normal group (median [Q1, Q2]: 100% [90%, 110%]; p < 0.05), indicating substantial retinal dysfunction following I/R injury. Treatment with catalpol 0.25 mM + I/R (median [Q1, Q2]: 52.00% [42.00%, 62.00%]) and catalpol 0.5 mM + I/R (median [Q1, Q2]: 92.000% [82.00, 102.00%]) led to significant (p < 0.05; at 0.5 mM) recovery compared to the vehicle + I/R group. As revealed in Figure 3D, ERG oscillatory potential amplitude ratio was significantly reduced in the vehicle + I/R group (24.59 ± 2.83%) compared to the normal group (100.00 ± 2.82%; p < 0.05). Treatment with both catalpol 0.25 mM + I/R (52.06 ± 3.37%) and catalpol 0.5 mM + I/R (77.68 ± 6.15%) led to significant (p < 0.05; 0.5 mM) resuscitation compared to the vehicle + I/R group. As reported in Figure 3E,F, the ERG a-/b-wave response time ratio was significantly prolonged in the vehicle + I/R group (236.28 ± 26.46%/189.58 ± 9.24%) compared to the normal group (100.00 ± 14.95%/100.00 ± 6.81%; p < 0.05), indicating delayed nerve impulse conduction after I/R injury. Treatment with catalpol 0.25 mM + I/R (211.46 ± 10.71%/175.00 ± 10.76%) and catalpol 0.5 mM + I/R (135.76 ± 19.02%/110.42 ± 9.24%) led to less effect at 0.25 mM and importantly significant (p < 0.05; at 0.5 mM) recovery compared to the vehicle + I/R group. 2.4. The Effect of Catalpol on Retrograde Fluorogold Immunolabeled Retinal Ganglion Cells Density Assessing RGC density using fluorogold retrograde labeling (Figure 4; n = 6) revealed the density of the control group (normal, Figure 4A,E) to be 425.57 ± 31.47 cells/field. Com-pared to the control group, there was a significant (*** p < 0.001) reduction in RGC density (194.54 ± 18.85 cells/field) in animals subjected to retinal ischemia with pre-administered vehicle (vehicle + I/R, Figure 4B,E). Furthermore, this decrease was significantly (at 0.5 mM; †† p < 0.01) mitigate when the animals underwent retinal isc e ia w th th pre-administration of catalpol (Cata 0.50 mM + I/R, Figure 4D,E: 308.50 ± 12.10 cells/field). However, a non-significant effect was observed with pretreatment with a lower concen-tration of 0.25 mM catalpol (C ta 0.25 mM + I/R, Figure 4C,E: 269.82 ± 25.57 cells/field). Figure 4. Fluorogold-labeling. The microscopic images demonstrated the density of retinal ganglion cells (RGCs). (A) The normal retina (normal). (B) Ischemic retina with 15 min pretreatment with vehicle (vehicle + I/R). (C-D) Fifteen-minute pretreatment of the ischemic retina with 0.25 mM (Cata 0.25 mM + I/R, (C)) and 0.5 mM (Cata 0.5 mM + I/R, (D)) catalpol. (E) Compared to normal, the density of RGCs was significantly (*** p < 0.001) reduced in rats subjected to retinal ischemia (vehicle Figure 4. Fluorogold-labeling. The microscopic images demonstrated the density of retinal ganglionc ls (RGCs). (A) The n rmal retina (normal). (B) Isch mic r tina with 15 min pretreatment withvehicle (vehicle + I/R). (C,D) Fifteen-minute pretreatment of the ischemic retina with 0.25 mM(Cata 0.25 mM + I/R, (C)) and 0.5 mM (Cata 0.5 mM + I/R, (D)) catalpol. (E) Compared to normal,the density of RGCs was significantly (*** p < 0.001) reduced in rats subjected to retinal ischemia(vehicle + I/R). This reduction was significant (†† p < 0.01) at 0.5 mM pretreatment of catalpol. Singlefield area ≈ 0.17 mm2. Scale bar = 50 µm.2.5. Protein and mRNA Analysis2.5.1. Western BlotRegarding the ratio of β-catenin/β-actin protein, in Figure 5A and Table 2 (n = 10), thedifference between normal and vehicle + I/R was significant (* p < 0.05). Moreover, thedifference between vehicle + I/R and catalpol 0.5 mM + I/R and that between vehicle + I/Rand DKK + I/R were significant († p < 0.05). However, the difference between vehicle + I/Rand catalpol 0.25 mM + I/R, Lucentis + I/R, and Eylea + I/R, respectively, was not significant.About the VEGF/β-actin protein, in Figure 5B and Table 3 (n = 6), the differencebetween normal and vehicle + I/R was significant (* p < 0.05). Moreover, the differencebetween vehicle + I/R and catalpol 0.5 mM + I/R and that between vehicle + I/R andLucentis + I/R were significant († p < 0.05). However, the difference between vehicle + I/Rand catalpol 0.25 mM + I/R, I/R + catalpol 0.5 mM, Eylea + I/R, and DKK + I/R, respec-tively, was not significant.Int. J. Mol. Sci. 2025, 26, 4019 10 of 26Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 10 of 28 + I/R). This reduction was significant (†† p < 0.01) at 0.5 mM pretreatment of catalpol. Single field area ≈ 0.17 mm2. Scale bar = 50 µm. 2.5. Protein and mRNA Analysis 2.5.1. Western Blot Regarding the ratio of β-catenin/β-actin protein, in Figure 5A and Table 2 (n = 10), the difference between normal and vehicle + I/R was significant (* p < 0.05). Moreover, the difference between vehicle + I/R and catalpol 0.5 mM + I/R and that between vehicle + I/R and DKK + I/R were significant († p < 0.05). However, the difference between vehicle + I/R and catalpol 0.25 mM + I/R, Lucentis + I/R, and Eylea + I/R, respectively, was not signifi-cant. Figure 5. Western blot and protein expression analysis of β-catenin (A), VEGF (B), and angiopoietin-2 (C) relative to β-actin or α-tubulin. (A–C) Ischemia/reperfusion (I/R) insult significantly (* p < 0.05) increased protein expression of β-catenin, VEGF, and angiopoietin-2. This overexpression was dose-dependently mitigated with catalpol pretreatment, showing a significant reduction at 0.5 mM († p < 0.05 at 0.5 mM) and a lesser effect at 0.25 mM. Pre-administration with DKK1 (1 µg/4 µL, Wnt in-hibitor) significantly († p < 0.05) attenuated β-catenin overexpression, while Lucentis (40 µg/4 µL, VEGF antibody) significantly († p < 0.05) inhibited the upregulation of VEGF. β-actin (42 kDa) or α-tubulin (52 kDa) were used as housekeeping proteins. Protein molecular weights: β-catenin (92 kDa), VEGF (43.6 kDa), and angiopoietin (57 kDa). A thin black line in (C) indicates where the gel was spliced to exclude irrelevant information—the marker and other experimental groups. (D) MCP-1 protein measured by ELISA. As compared to retinas subjected to pressure-induced retinal ischemia pretreated with vehicle (vehicle + I/R) to the normal retinas (control; n = 4), vehicle + I/R (n = 4) resulted in a significant (* p < 0.05) increase in MCP-1 inflammatory biomarker concentrations. This increase was significantly († p < 0.05) nullified by 15 min of pre-administration with 0.25 mM (n = 4) and 0.5 mM catalpol (n = 4). (E) Real-time polymerase chain reaction analysis measuring the expression of HIF-1α and β-actin. Twenty-four hours after ischemia plus reperfusion (I/R), whole retinal extracts were isolated from the normal eyes (control) or the ischemic eyes (subjected to 60-Figure 5. Western blot and protein expression analysis of β-catenin (A), VEGF (B), and angiopoietin-2(C) relative to β-actin or α-tubulin. (A–C) Ischemia/reperfusion (I/R) insult significantly (* p < 0.05)increased protein expression of β-catenin, VEGF, and angiopoietin-2. This overexpression wasdose-dependently mitigated with catalpol pretreatment, showing a significant reduction at 0.5 mM(† p < 0.05 at 0.5 mM) and a lesser effect at 0.25 M. Pre-administration with DKK1 (1 µg/4 µL, Wntinhibitor) significantly († p < 0.05) attenu ted β-catenin overexpr ssion, while Lucentis (40 µg/4 µL,F antibody) significantly († p < 0.05) inhibit d the upregulation of VEGF. β-actin (42 kDa) orα-t bulin (52 kDa) w re used as housekeeping proteins. Protein molecular weights: β-catenin(92 kDa), VEGF (43.6 kDa), and angiopoietin (57 kDa). A thin black line in (C) indicates wherethe gel was spliced to exclude irrelevant infor ation—the marker and other experimental groups.(D) MCP-1 protein measured by ELISA. As compared to retinas subjected to pressure-induced retinalischemia pretreated with vehicle (vehicle + I/R) to the normal retinas (control; n = 4), vehicle + I/R(n = 4) resulted in a significant (* p < 0.05) increase in MCP-1 inflammatory biomarker concentrations.This increase was significantly († p < 0.05) nullified by 15 min of pre-administration with 0.25 mM(n = 4) and 0.5 mM catalpol (n = 4). (E) Real-time polymerase chain reaction analysis measuring theexpression of HIF-1α and β-actin. Twenty-four hours after ischemia plus reperfusion (I/R), wholeretinal extracts were isolated from the normal eyes (control) or the ischemic eyes (subjected to 60-minhigh intraocular pressure) pre-administered before I/R with intravitreous vehicle (DMEM) or catalpol(0.25 or 0.5 mM). Ischemia reperfusion (I/R) injury resulted in a significant increase in HIF-1α proteinlevels (* p < 0.05). This elevation was significantly reduced († p < 0.05) after 15 min pretreatmentwith 0.25 and 0.5 mM catalpol. Data are presented as the mean ± SD (n = 3). Abbreviations: VEGF,vascular endothelial growth factor; HIF-1α, hypoxia-inducible factor-1α.Table 2. Western blotting. The ratios of the protein levels of β-catenin divided by that of β-actin §.Gr 1 2 3 4 5 6 7 8M 100% 200.3% * 41.3% † 156.9% 62.5% † 129.0% 173.4% 168.1%Q1 100% 159.9% 20.7% 40.4% 46.9% 101.0% 119.6% 100.2%Q3 100% 382.3% 97.1% 159.4% 88.8% 138.3% 265.6% 227.1%§ There were eight groups (n = 6 each): (1) normal, (2) vehicle + OGD, (3) catalpol 0.5 mM + OGD, (4) catalpol0.25 mM + OGD, (5) DKK + OGD, (6) OGD + catalpol 0.5 mM, (7) Eylea + OGD, and (8) Lucentis + OGD.The differences in median values between the normal and vehicle + OGD groups, as well as between thevehicle + OGD and catalpol 0.5 mM + OGD groups, were greater than would be expected by chance and werestatistically significant (* p < 0.05 and † p < 0.05, respectively). A significant difference († p < 0.05) was alsoobserved between the vehicle + OGD and DKK + OGD groups. Abbreviations: M, median; Q1, first quartile;Q3, third quartile; OGD, oxygen–glucose deprivation.Int. J. Mol. Sci. 2025, 26, 4019 11 of 26Table 3. Western blotting. The ratios of the protein levels of VEGF divided by that of β-actin.Gr 1 2 3 4 5 6 7 8M 100% 1866.3% * 87.6% † 139.2% 333.7% 251.9% 126.6% † 258.1%Q1 100% 674.1% 73.3% 92.4% 176.7% 23.2% 51.1% 225.6%Q3 100% 2499.3% 93.1% 359.1% 645.1% 612.7% 307.2% 316.6%There were 8 groups (n = 6), i.e., 1 (normal), 2 (vehicle + OGD), 3 (catalpol 0.5 mM + OGD), 4 (catalpol0.25 mM + OGD), 5 (OGD + catalpol 0.5 mM), 6 (Eylea + OGD), 7 (Lucentis + OGD), and 8 (DKK + OGD). Thedifferences in the median values between the normal and vehicle + OGD groups and between the vehicle + OGD andcatalpol 0.5 mM + OGD groups were greater than would be expected by chance; there was a statistically significantdifference (* p < 0.05 and † p < 0.05). A significant difference († p < 0.05) also existed between vehicle + OGD andLucentis + OGD. Abbreviations: M, median; Q1, first quartile; Q3, third quartile; OGD, oxygen–glucose deprivation.Regarding the angiopoietin-2/α-tubulin protein, in Figure 5C and Table 4 (n = 6),the difference between normal/vehicle + normal and vehicle + I/R or I/R was significant(* p < 0.05). Furthermore, pre-ischemic catalpol dose-dependently significantly († p < 0.05at 0.5 mM, Cata 0.5 mM + I/R) counteracted the I/R-upregulated ang-2 levels, which wasnot affected by vehicle (vehicle + I/R).Table 4. Western blotting. The ratios of the protein levels of angiopoietin-2 divided by thatof α-tubulin.Gr 1 2 3 4 5 6M 100% 127.5% 88.4% † 169.2% 609.8% 626.1% *Q1 100% 101.8% 42.5% 89.8% 561.9% 572.9%Q3 100% 140.0% 110.9% 627.6% 653.8% 703.6%There were 8 groups (n = 6), i.e., 1 (normal), 2 (vehicle + normal), 3 (catalpol 0.5 mM + OGD), 4 (catalpol0.25 mM + OGD), 5 (OGD), and 6 (vehicle + OGD). The differences in the median values between the normaland vehicle + OGD groups and between the vehicle + OGD and catalpol 0.5 mM + OGD groups were greaterthan would be expected by chance; there was a statistically significant difference (* p < 0.05 and † p < 0.05).Abbreviations: M, median; Q1, first quartile; Q3, third quartile; OGD, oxygen–glucose deprivation.The impact of 0.5 mM catalpol on the presence of apoptotic cells in the inner retinallayer was assessed using the TUNEL assay. As presented in Table 1 and Figure 6 (n = 5),no TUNEL-positive cells were observed in the control group (normal; 0 cells/field). How-ever, one day after pressure-induced retinal ischemia followed by vehicle treatment, therewas a significant increase in TUNEL-positive cells (vehicle + I/R; 1.80 ± 0.37 cells/field).This marked increase in apoptosis was significantly reduced upon treatment with 0.5 mMcatalpol (0.5 mM catalpol + I/R; 0.40 ± 0.25 cells/field).Int. J. Mol. Sci. 2025, 26, x FOR PEER REVIEW 12 of 28 Q1 100% 101.8% 42.5% 89.8% 561.9% 572.9% Q3 100% 140.0% 110.9% 627.6% 653.8% 703.6% There were 8 groups (n = 6), i.e., 1 (normal), 2 (vehicle + normal), 3 (catalpol 0.5 mM + OGD), 4 (catalpol 0.25 mM + OGD), 5 (OGD), and 6 (vehicle + OGD). The differences in the median values between the normal and vehicle + OGD groups and between the vehicle + OGD and catalpol 0.5 mM + OGD groups were greater than would be expected by chance; there was a statistically significant diff rence (* p < 0.05 and † p < 0.05). Abbre iations: M, median; Q1, first quartile; Q3, third quartile; OGD, oxygen–glucose deprivation. The impact of 0.5 mM catalpol on the presence of apoptotic cells in the inner retinal layer was assessed using the TUNEL assay. As presented in Table 1 and Figure 6 (n = 5), no TUNEL-positive cells were observed in the control group (normal; 0 cells/field). How-ever, one day after pressure-induced retinal ischemia followed by vehicle treatment, there was a significant increase in TUNEL-positive cells (vehicle + I/R; 1.80 ± 0.37 cells/field). This marked increase in apoptosis was significantly reduced upon treatment with 0.5 mM catalpol (0.5 mM catalpol + I/R; 0.40 ± 0.25 cells/fi ld). Figure 6. TUNEL assay. (A) Representative histopathological image of TUNEL-stained apoptotic cells (white arrow). (B) Quantification of apoptotic cells (TUNEL-positive cells) across different ex-perimental groups. A significant increase (* p < 0.05) in the number of apoptotic cells was observed in the ischemic group compared to the normal group. Pre-administration of 0.5 mM catalpol signif-icantly († p < 0.05) reduced the number of apoptotic cells. Scale bar 25 µm. Abbreviations: TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling. 2.5.2. Measurement of MCP-1 Protein Using ELISA In Figure 5D (n = 4) and Table 5, when comparing retinal cells subjected to retinal ischemia induced by HIOP with the normal retinas (287.77 ± 4.68 pg/mL; control), retinal ischemia (441.87 ± 19.58) led to significant (* p < 0.001) elevated MCP-1 inflammatory pro-tein levels. This elevation was significantly († p < 0.001) attenuated by 15 min of pretreat-ment with 0.25 mM (285.58 ± 2.92) and 0.5 mM catalpol (251.54 ± 11.15). Table 5. Analysis of the expression of MCP-1 protein levels (pg/mL) by ELISA. Group Normal Vehicle + I/R 0.25 mM Catalpol + I/R 0.5 mM Catalpol + I/R MCP-1 287.77 ± 4.68 441.87 ± 19.58 * 285.58 ± 2.92 † 251.54 ± 11.15 † Analysis of the expression of MCP-1 protein levels by ELISA. Cells were extracted and isolated from the normal retinas (control) or the ischemic retinas pre-administered with vehicle or 0.25/0.5 mM of catalpol. Retinal ischemia resulted in a significant (* p < 0.05) upregulation of MCP-1 protein levels. This significant elevation was significantly († p < 0.05) attenuated by 15 min of pretreatment with 0.25 or 0.50 mM catalpol. Results are displayed as the mean ± SE (n = 4). Figure 6. TUNEL assay. (A) Representative histopathological image of TUNEL-stained apoptoticcells (white arrow). (B) Quantification of apoptotic cells (TUNEL-positive cells) across different exper-imental groups. A significant increase (* p < 0.05) in the number of apoptotic cells was observed in theischemic group compared to the normal group. Pre-adm istration of 0.5 mM catalpol significantly(† p < 0.05) reduced the number of apoptotic cells. Scale bar 25 µm. Abbreviations: TUNEL—terminaldeoxynucleotidyl transferase dUTP nick end labeling.Int. J. Mol. Sci. 2025, 26, 4019 12 of 262.5.2. Measurement of MCP-1 Protein Using ELISAIn Figure 5D (n = 4) and Table 5, when comparing retinal cells subjected to retinalischemia induced by HIOP with the normal retinas (287.77 ± 4.68 pg/mL; control), retinalischemia (441.87 ± 19.58) led to significant (* p < 0.001) elevated MCP-1 inflammatoryprotein levels. This elevation was significantly († p < 0.001) attenuated by 15 min ofpretreatment with 0.25 mM (285.58 ± 2.92) and 0.5 mM catalpol (251.54 ± 11.15).Table 5. Analysis of the expression of MCP-1 protein levels (pg/mL) by ELISA.Group Normal Vehicle + I/R 0.25 mM Catalpol + I/R 0.5 mM Catalpol + I/RMCP-1 287.77 ± 4.68 441.87 ± 19.58 * 285.58 ± 2.92 † 251.54 ± 11.15 †Analysis of the expression of MCP-1 protein levels by ELISA. Cells were extracted and isolated from the normalretinas (control) or the ischemic retinas pre-administered with vehicle or 0.25/0.5 mM of catalpol. Retinalischemia resulted in a significant (* p < 0.05) upregulation of MCP-1 protein levels. This significant elevation wassignificantly († p < 0.05) attenuated by 15 min of pretreatment with 0.25 or 0.50 mM catalpol. Results are displayedas the mean ± SE (n = 4).2.5.3. The Effect of Catalpol on HIF-1α mRNA in the RetinaIn Figure 5E (n = 4) and Table 6, compared with the normal retina (HIF-1α = 0.76 ± 0.07)],the ratios for HIF-1α (1.62± 0.09) in the vehicle-pretreated ischemic retina was significantly up-regulated (p < 0.05) after I/R. When compared with the vehicle-pretreated ischemic retina, theischemic retina pretreated with 0.25 mM/0.5 mM catalpol (HIF-1α = 0.88 ± 0.02/0.79 ± 0.02)showed a significant (p < 0.05) counteraction of the I/R-induced HIF-1α upregulation.Table 6. The ratios of mRNA expression levels of HIF-1α to that of β-actin.Group Normal Vehicle + I/R 0.25 mMCatalpol + I/R0.5 mMCatalpol + I/RHIF-1α/β-actin 0.76 ± 0.07 1.62 ± 0.09 * 0.88 ± 0.02 † 0.79 ± 0.02 †Total mRNA was extracted and isolated from the normal retinas (control) or the ischemic retinas pre-administeredwith vehicle, or 0.25/0.5 mM of catalpol. Quantitative analysis is presented at 1 day after I/R. * and † denotesignificant differences between the normal and vehicle + I/R groups, as well as between the vehicle + I/R and0.25/0.5 mM catalpol + I/R groups, respectively. The findings are presented as the mean± SE (n = 4).2.6. TerminalDeoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL).The impact of 0.5 mM catalpol on the presence of apoptotic cells in the inner retinallayer was assessed using the TUNEL assay. As presented in Table 7 and Figure 6 (n = 5),no TUNEL-positive cells were observed in the control group (normal; 0 cells/field). How-ever, one day after pressure-induced retinal ischemia followed by vehicle treatment, therewas a significant increase in TUNEL-positive cells (vehicle + I/R; 9.40 ± 0.87 cells/field).This marked increase in apoptosis was significantly (p < 0.05) reduced upon treatment with0.5 mM catalpol (0.5 mM catalpol + I/R; 3.40 ± 0.51 cells/field).Table 7. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.Group Control Vehicle + I/R 0.5 mM Catalpol + I/RApoptotic cell No. (n = 5) 0 9.40 ± 0.87 * 3.40 ± 0.51 †No TUNEL-positive cells were observed in the normal (control) group. In contrast, the number of apoptotic cells wassignificantly increased at 1 day after ischemia/reperfusion (I/R) with vehicle pretreatment (vehicle + I/R) comparedto the control group. Pretreatment with 0.5 mM catalpol (0.5 mM catalpol + I/R) significantly reduced the number ofapoptotic cells in the inner retinal layer compared to the vehicle + I/R group. Quantitative analysis is presented at1 day after I/R. * and † denote significant differences between the normal and vehicle + I/R groups, as well as betweenthe vehicle + I/R and 0.5 mM catalpol + I/R groups, respectively. Abbreviation: TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.3. DiscussionThe prevailing notion within the scientific community suggests the possibility of ashared mechanism of action across different retinal ischemic diseases. While anti-VEGFInt. J. Mol. Sci. 2025, 26, 4019 13 of 26antibodies have exhibited significant efficacy in addressing ocular hemorrhage and macularedema over the past two decades, it is noteworthy that suboptimal visual outcomes persistin a subset of patients despite these treatments. Hence, the emergence of alternative drugstargeting different pathways becomes imperative.Recent advancements in the treatment of ischemic eye diseases have led to the develop-ment of drugs that simultaneously target multiple pathways. As a result, the managementof retinal ischemia increasingly involves a combination of compounds, highlighting theimportance of novel therapeutic agents that extend beyond VEGF inhibition. As afore-mentioned, catalpol has garnered attention recently due to its antioxidative, antiapoptotic,and anti-ischemic properties. Catalpol has shown its anti-ischemic effects in protectingastrocytes, cardiac endothelium, cerebral vasculature, and corneal vasculature from is-chemic damage. However, given that catalpol’s effects on retinal ischemia remain largelyunexplored, our study aimed to investigate its potential impact on retinal ischemia.3.1. Catalpol’s Protective Effect Against I/R, OGD, and Oxidative StressRetinal ischemia is fundamentally driven by oxygen and glucose deprivation, whichleads to energy failure and subsequent cell death/apoptosis [43]. This is consistent withthe present study’s results. The OGD model employed revealed a significant reduction inRGC-5, retinal progenitor, cellular viability, underscoring the critical impact of ischemiaon retinal cells, such as RGCs. Notably, pretreatment with catalpol ameliorated thesereductions, with the 0.5 mM concentrations yielding a statistically significant protectiveeffect. Catalpol’s stabilization of cell viability under metabolically compromised conditionshighlights its neuroprotective potential in ischemic/hypoxic environments.The ischemic environment along with ischemic reperfusion drives a pro-inflammatorycascade that induces oxidative stress [44], further compounding cellular damage andaccelerating retinal cell injury. Ischemia in retinal cells causes a shift to anaerobic respiration,leading to ATP depletion, disrupted ion gradients, and excitotoxicity. This results in cellularswelling and increased stress from lactic acid buildup. Furthermore, during ischemicreperfusion, ROS production is exacerbated by the sudden reintroduction of oxygen, whichinteracts with the damaged mitochondria and ETC to produce an oxidative burst. Thissurge of ROS overwhelms the cell’s natural antioxidant defenses, leading to extensive lipidperoxidation, protein oxidation, and DNA damage, further compromising the retinal cellintegrity and function. This effect was reproduced in the H2O2-induced oxidative stressmodel, where RGC-5 cells demonstrated a marked decrease in viability. Consistent withthe effects in the OGD, catalpol 0.5 mM significantly improved cell survival, demonstratingits antioxidative properties against oxidative stress.The detrimental effects of ischemic damage are also prominent in vivo. Follow-ing I/R injury, the significant impairment of Müller/Bipolar cell’s electrophysiolog-ical function was observed, as evidenced by a significant decline or delay in ERGa-wave/b-wave/oscillatory potential amplitude (indexing photoreceptors/bipolars orMüllers/amacrines) or a-wave/b-wave response time (reflecting nerve impulse conduc-tion velocity), alongside a significant reduction in retrograde-labeled RGC viability andincreased apoptotic cells in the inner retinal layer. However, catalpol 0.5 mM was able tosignificantly preserve RGC density, exert antiapoptotic effects on inner retinal cells, andmaintain retinal electrophysiological function. These results underscore catalpol’s ability tosupport the survival of various cells, while maintaining the functional integrity of retinalcells affected by ischemic injury.From both the in vivo and in vitro results, it is evident that catalpol possesses prop-erties that defend against I/R, OGD and oxidative stress. This is possibly explained byits antioxidative capacity, supported by You et al. (2011), who demonstrated that culti-Int. J. Mol. Sci. 2025, 26, 4019 14 of 26vars of Rehmannia glutinosa (which contain catalpol) exhibit potent antioxidative activities,with an IC_50 of 205.8 mg/g for DDPH radical scavenging activity and 38.8 mg/g forhydroxyl radical scavenging activity [45]. These findings suggest that, during IR whereROS form, catalpol could dampen the further damage that ensued by ROS during ischemicinjury, through chemically reducing these ROS. This is further supported by our in vivoresults where we can see a demonstrated reduced RGC cell death and decreased ERGa-wave/b-wave/oscillatory potential amplitude reduction.The protective properties of catalpol are further supported by a comprehensive reviewby Zhang and colleagues (2023), which elucidated catalpol’s antioxidative and antiapop-totic properties against cardio-cerebrovascular diseases [34]. Additionally, a meta-analysisby Zheng et al. (2017) further demonstrated catalpol’s neuroprotective, antioxidative, anti-inflammatory, and antiapoptotic effects in models of cerebral ischemic stroke [35]. Moreover,Lin et al. (2024) demonstrated that catalpol was able to alleviate oxidative stress throughinhibiting neuronal apoptosis following oxidative stress [33]. In addition, a large-scale analysisby Bhattamisra et al. (2019) revealed that catalpol exhibits cardioprotective, neuroprotective,anti-inflammatory, and antioxidative effects through multiple molecular mechanisms [46]. Fur-thermore, catalpol has been shown to provide a neuroprotective effect against brain ischemiain rats by antioxidation, antiapoptosis, and modulation of angiogenesis and neurogenesis.Altogether, these findings suggest that catalpol may be able to counteract critical mechanismsin retinal ischemic injury—energy failure and oxidative stress—by enhancing cell viabilityand limiting oxidative damage in a dose-dependent manner.3.2. Catalpol’s Mechanism of Action: HIF-1α, β-Catenin, VEGF, Angiopoietin-2, the WntSignaling Pathway, and the Inflammatory Factor MCP-1 in the Ischemic RetinaA growing body of research suggests that the Wnt-signaling pathway plays a crucialrole not only in the early normal development of retinal vessels but also in the progression ofcertain developmental retinal vascular diseases [1,2,5,15,47,48]. Furthermore, this pathwayis closely associated with both upstream β-catenin and downstream angiopoietin-2/VEGFsignaling. Ang-2, recognized as a pivotal proangiogenic factor [14], plays a significant rolein this pathway.Under normoxic environments, β-catenin interacts with T-cell factor-4 (TCF-4) topromote cell proliferation and maintain tissue integrity [12]. However, under the is-chemic/hypoxic milieu, β-catenin undergoes a functional shift that contributes to bothadaptive and pathological outcomes in retinal cells [49]. Specifically, it activates down-stream hypoxia-inducible factor 1α (HIF-1α), where the overexpressed HIF-1α protein com-petes with TCF-4 for binding with cellular β-catenin. Upon binding to HIF-1α, β-cateninpromptly shifts its role from co-activating TCF-4 to triggering HIF-1α-associated tran-scriptional processes [50]. This upregulation of HIF-1α-mediated transcription underischemic situations leads to subsequent elevation in ang-2/VEGF levels. VEGF, a potentpro-angiogenic factor, plays a critical role in the formation of new vessels (neovasculariza-tion); however, under ischemic conditions, it often leads to pathological neovascularization.The newly formed vessels typically lack structural integrity, making them prone to leak-age and rupture. This compromised vasculature leads to complications, such as macularedema and hemorrhage, which are hallmarks of ischemic retinal diseases, such as diabeticretinopathy and retinal vein occlusion [5,51].Alongside VEGF, angiopoietin-2 plays a complementary but distinct role in regulat-ing vascular homeostasis. Ang-2 functions as a context-dependent antagonist of Ang-1,which stabilizes blood vessels through its interaction with the Tie-2 receptor on peri-cytes [52]. Under physiological conditions, Ang-1 activates the Tie-2 receptor to maintainvascular maturation, quiescence, and barrier integrity. However, under ischemia, Ang-2expression is significantly upregulated, disrupting the balance between Ang-1 and Ang-2Int. J. Mol. Sci. 2025, 26, 4019 15 of 26and inhibiting the stabilizing effects of Ang-1 [16]. This results in vessel destabilization,increased endothelial permeability, and enhanced sensitivity of the vasculature to VEGFsignaling [53]. Consequently, the combined action of VEGF and Ang-2 further exacerbatesthe fragility of immature pathological vessels.In our present study, catalpol demonstrated the ability to downregulate β-catenin, VEGF,and Ang-2 levels in retinal tissue. Specifically, catalpol at 0.5 mM significantly counteracted theupregulation of these pro-angiogenic factors. In our analysis, we compared β-catenin levels inthe presence of DKK (an established inhibitor of the Wnt/β-catenin pathway), as well as VEGFlevels in the presence of Lucentis (ranibizumab), a well-known anti-VEGF agent. Interestingly,catalpol exhibited a similar inhibitory effect on β-catenin levels as DKK, suggesting thatcatalpol may act as an effective modulator of the Wnt/β-catenin pathway. Furthermore,catalpol demonstrated comparable anti-VEGF activity to Lucentis, indicating its potential tomodulate VEGF activity, in addition to its ability to inhibit Ang-2 expression. In line withthese findings, Han and colleagues (2018) reported similar results in their investigation ofrat corneal neovascularization, where catalpol was shown to inhibit levels of VEGF andTNF-α, reducing neovascularization and dampening inflammation. In a previous publication,Zhu et al. (2015) indicated that the suppression of TNF-α, IL-1β, IL-6, and IL-10 activitieswas involved in the protective effect of catalpol on I/R injury [31]. Inflammation indeedplays a vital role in ischemia-related disorders. In the present evaluation of the specificproinflammatory cytokine, monocyte chemoattractant protein-1, MCP-1, the novel resultsproved the consequent elevation of MCP-1 after retinal I/R, where catalpol was demonstratedto downregulate the levels of MCP-1, alleviating inflammation and protecting against retinalischemia-related disorders, namely exudative AMD.Moreover, catalpol demonstrated the ability to downregulate angiopoietin-2 levelsin the retinal tissue. The modulation of Ang-2 by catalpol underscores its role in restor-ing vascular homeostasis under ischemic conditions. By attenuating Ang-2 expression,catalpol may help rebalance the Ang-1/Ang-2 axis, thereby mitigating the destabilizingeffects typically observed in pathological neovascularization. This reduction in Ang-2enhances pericyte support for the existing blood vessels and contributes to the improvedstructural integrity of newly formed vessels, ultimately reducing the risk of leakage (edema)and bleeding (hemorrhage). The combined mechanism of action of inhibiting upstreamβ-catenin, suppressing VEGF levels, and reducing Ang-2 protein levels likely contribute toits ability to protect retinal cells (i.e., retinal progenitors: RGC-5, retinal ganglion cells) andretinal electrophysiology under ischemic conditions and oxidative stress.The present protein analyses and results strongly support the hypothesis that catalpol exertsits anti-inflammatory, antioxidative, and anti-ischemic/hypoxic properties via mechanisms ofdownregulating the ischemia-associated overexpression of MCP-1, β-catenin, HIF-1α, VEGF,and angiopoietin-2. These observations are corroborated by recent studies [15,31,40,41,54] andunderscore the inhibition of MCP-1, anti-Wnt/β-catenin, anti-HIF-1α, anti-VEGF, and anti-angiopoietin-2 properties of catalpol.3.3. In Vitro Antioxidative/Anti-Ischemic-Like and In Vivo Anti-Ischemic/Antiapoptotic Effectsof Catalpol: Comparison with Other Compounds and Its Relation to MCP-1, HIF1α, β-Catenin,VEGF, and Angiopoietin-2Given the increasing interest in TCM compounds for ocular health, it is importantto understand how catalpol compares to other TCM compounds known for their neuro-protective and anti-inflammatory properties. While our research does not aim to identifya single compound capable of fully addressing retinal ischemia, it seeks to contributeto the broader understanding of how TCM compounds like catalpol can be integratedinto combination therapies for retinal ischemic disorders. Combination therapies are be-coming increasingly essential for managing complex and treatment-resistant conditions,Int. J. Mol. Sci. 2025, 26, 4019 16 of 26such as normal tension glaucoma, exudative age-related macular degeneration (AMD),or proliferative diabetic retinopathy unresponsive to conventional therapy. For instance,in normal tension glaucoma, neuroprotective agents, like β-blockers (e.g., betaxolol) and α2agonists (e.g., brimonidine), and carbonic anhydrase inhibitors (e.g., acetazolamide) maybe used to mitigate optic nerve damage. Meanwhile, in exudative AMD, combining anti-VEGF therapies like ranibizumab or aflibercept with angiopoietin-2 inhibitors (Vabysmo)offers a comprehensive approach to managing vascular leakage and neovascularization.Catalpol is distinguished by its ability to target the angiopoietin-2, Wnt/β-catenin, andVEGF pathways alongside modulating the inflammatory biomarker MCP-1. Its mechanismof action alleviates ischemic injury through downregulating β-catenin and consequentlydownregulating HIF-1α and VEGF; moreover, it further prevents vascular leakage andretinal cell damage through downregulating angiopoietin-2 and MCP-1.In contrast, moscatilin, a bibenzyl component of Dendrobium nobile Lindley, has beenshown to protect RGC-5/RGCs against oxidative stress in vitro/retina ischemia in vivo byupregulating Norrin/downregulating PKM2, RBP2, HIF-1α, VEGF, and PLGF [1]. What ismore, baicalein/mannitol “cytoprotects” hRPE (ARPE-19) against chronic/acute oxidativestress in vitro by downregulating VEGF/upregulating catalase [55]. Finally, S-allyl L-cysteineprovided a protective effect on RGC-5 cells against oxidative stress in vitro by downregulatingMCP-1, PKM2, and VEGF [56,57] and prevents RGCs from kainate-induced excitotoxicityin vivo through its antiapoptotic effect [4]. Presently, catalpol also protects the neurons in theinner retinas (e.g., RGCs) from retinal ischemia in vivo through its antiapoptotic effect [4].Considering its clinical relevance, catalpol presents a potential therapeutic avenue fora spectrum of retinal ischemic disorders, including retinal vascular occlusion, proliferativediabetic retinopathy, neovascular AMD, and possibly, retinal developmental anomalies,like Coats’ disease. These conditions are characterized by pathological processes, suchas angiogenesis, ocular hemorrhage, and macular edema, all associated with elevatedβ-catenin, HIF-1α, VEGF, angiopoietin-2, and MCP-1 levels. Catalpol’s unique action onmultiple angiogenic pathways, including β-catenin, HIF-1α, VEGF, and angiopoietin-2,and on inflammatory biomarkers (i.e., MCP-1) suggests that these mechanisms may playkey roles in treating retinal ischemia. By targeting distinct but complementary pathways in-volved in vascular stability and neuroprotection, catalpol could provide a novel therapeuticapproach. Furthermore, combining catalpol with current treatments, such as anti-VEGF,anti-PLGF/VEGF traps, and anti-angiopoietin-2 agents, may enhance their efficacy intreating complex retinal conditions.3.4. Side Effects, Limitations, and Future DirectionsThe safety assessment of any therapeutic compound is crucial, and extensive researchhas been conducted to evaluate the toxicity profile of catalpol [58–60]. Studies involv-ing acute toxicity in mouse models have provided insights into its safety parameters.Dong et al. (2009) demonstrated that the short-term oral administration of catalpol didnot result in any noticeable toxic symptoms [59]. Mice continued their normal feeding habitsand physical activities, showing no signs of distress or physiological abnormalities [59].For long-term evaluation, Jiang et al. (2008) investigated the effects of intravenous catalpolover a 90-day period, administering doses of 10, 20, and 40 mg/kg/day [60]. Findingsrevealed no significant changes in the biochemical markers or structural integrity of majororgans, indicating that extended exposure to catalpol does not present substantial toxicityconcerns [60]. Beyond systemic toxicity, potential effects on visual function were also ex-plored. Electroretinography (ERG) b-wave assessments, combined with Sigma plot analysis,were used to examine any impact on retinal health. Results showed no significant differ-ences in ERG b-wave amplitudes between catalpol-treated subjects and controls. TheseInt. J. Mol. Sci. 2025, 26, 4019 17 of 26findings suggest that catalpol does not induce retinal toxicity and is unlikely to compromisevisual function under the tested conditions. These findings suggest that catalpol is a safecompound with limited toxicity, offering additional benefits due to its multimodal mecha-nisms, antioxidative, and anti-inflammatory properties, making it a promising candidate fortreating ischemia-related ocular disorders, such as age-related macular degeneration (AMD).The RGC-5 cell line has been widely used to study retinal ganglion cells (RGCs),but its characterization remains controversial. Originally identified as rat-derived, it waslater confirmed to originate from mice [61,62]. Additionally, inconsistencies in Thy-1expression raise concerns about its reliability as a true RGC model [61,63]. To address theselimitations, primary RGCs extracted from Wistar rat retinas were used instead, ensuringa biologically relevant model for studying key markers, such as VEGF, β-catenin, HIF-α,and MCP-1. RGC-5 cells were only used to assess viability following H2O2 exposureand oxygen–glucose deprivation (OGD), with results validated by the in vivo fluorogoldstaining of RGCs. Immunostaining confirmed Thy-1 localization in retinal ganglion cells,with strong reactivity in the inner plexiform layer (IPL) and ganglion cell layer (GCL) [57,64].RT-PCR further validated Thy-1 expression in extracted retinal cells from normal controls,demonstrating that the isolated RGCs retained key molecular markers characteristic ofauthentic RGCs in our in vivo study.The high intraocular pressure (HIOP) model was employed in the in vivo studies toinvestigate ischemia/reperfusion (I/R) injury, providing a controlled approach to acute reti-nal ischemia. However, it does not fully replicate chronic ischemic diseases [65,66]. Whileeffective, findings must be cautiously interpreted regarding long-term disease progression.To ensure experimental reliability, key surgical controls were maintained, including cornealhydration, anesthesia regulation, and body temperature stabilization [66]. Compared tovascular ligation, the pressure-induced ischemia model minimized unintended damage toadjacent ocular structures, making it a more precise method for studying retinal ischemicinjury. Despite its limitations, the HIOP model remains a valuable tool for investigatingthe complex mechanisms of retinal ischemia, offering critical insights into both cellularresponses and potential therapeutic interventions [66].In this study, catalpol was administered before I/R, representing a preventive ap-proach. Future investigations could explore the potential of catalpol as a post-I/R treatment,expanding its application in contexts where early intervention may not be feasible. Fur-thermore, optimizing dosing regimens, delivery methods (e.g., oral), combination therapy(catalpol plus anti-VEGF and verteporfin photodynamic therapy), and assessing long-termoutcomes could enhance our understanding of catalpol’s therapeutic potential and informits future clinical applications.4. Materials and Methods4.1. In Vitro Studies4.1.1. Cell Injury ModelSix-well plates were utilized to grow RGC-5 cells, with each well including 3× 105/well [55].The RGC-5 cell line was obtained from the American Type Culture Collection (ATCC, No.CRL-2302). The RGC-5 cell line represents a lineage of neuronal precursor cells [61]. RGC-5cells were cultured in Dulbecco’s modified Eagle medium (DMEM) solution (1.5 mL/well)at 37 ◦C for 1 day to achieve the required cell number (3 × 105/well) [67].4.1.2. Oxidative StressTo simulate the oxidative stress cells undergoing retinal ischemia, an H2O2-induced ox-idative stress model was employed [68–70]. The cells were then subjected to H2O2 (1 mM) for24 h [67], either with or without a 15 min pretreatment of catalpol. The groups were categorizedInt. J. Mol. Sci. 2025, 26, 4019 18 of 26into three, the normal control group (DMEM only incubation), the experimental control group(pre-incubation of vehicle with 1 mM H2O2 to simulate oxidative stress), and the treatmentgroups (pre-administration: catalpol 0.25 mM + H2O2 1 mM; catalpol 0.5 mM + H2O2 1 mM).Cell viability was assessed by counting the cells using a hemocytometer. To evaluate the im-pact of catalpol on oxidative stress, the cell viability was compared among the various groupsmentioned. Each experiment was carried out in triplicate.4.1.3. Oxygen–Glucose Deprivation (OGD)To simulate retinal ischemia, an OGD model was employed to induce oxygen andglucose deprivation stress on retinal cells ischemia [71–74]. RGC-5 cells were subjected toOGD for a duration of 8 h within a controlled simulating environment. This methodologywas adapted from several studies [5,75,76]. This involved culturing the cells in DMEMwithout glucose maintained at 37 ◦C and under 1% O2, 94% N2, and 5% CO2 (monitoredvia a Penguin incubator; Astec Company, Fukuoka, Japan).The study comprised of several experimental groups, including cells treated as follows:(i) DMEM with vehicle (normal control, n = 3), (ii) 15 min of pre-OGD application withvehicle (vehicle + OGD, n = 3), (iii) 15 min of pre-OGD application with 0.25 mM catalpol(Cata 0.25 mM + OGD, n = 3), and (iv) 15 min of pre-OGD application with 0.5 mM catalpol(Cata 0.5 mM + OGD, n = 3). Following 8 h of OGD, the cultured cells were transferred to anew DMEM for another day. Cell viability assessment was conducted utilizing MTT assays,and the cell number was counted by a hemocytometer or cellular morphology examinedunder optical microscopy.4.1.4. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) AssayFollowing incubation with either vehicle (control) or catalpol, the treated cells werewashed. MTT (0.5 mg/mL; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;Sigma-Aldrich, St. Louis, MA, USA) was then introduced to the 24-well plates containingthe original 400 µL cells (4 × 104/well) and left to incubate for 3 h at 37 ◦C, as instructedby Mosmann (1983) [77]. The reduced MTT (blue formazan) was then dissolved in 200 µLdimethyl sulfoxide. After agitation, the optical density (OD) of the solubilized formazanwas measured using an ELISA reader (Synergy H1 Multi-Mode Reader BioTek Instruments,Santa Clara, CA, USA) at 562 nm. Cell viability was determined by comparing the ODvalues to those of the control (100%; cells cultured in DMEM).4.2. In Vivo Studies4.2.1. AnimalsThe Institutional Animal Care and Use Committee at Cheng Hsin General Hospital(CHGH; Taipei, Taiwan; Approval No: CHIACUC 106-04) consented to all the animalexperiments that followed the Association for Research in Vision and OphthalmologyStatement for the Use of Animals in Ophthalmology and Vision Research. Wistar rats(200–250 g; equally sex distributed; National Laboratory Animal Center, Taipei, Taiwan)were housed with 40–60% humidity and at 19–23 ◦C. These rats were subjected to a 12 hlight/dark cycle with 12–15 air exchanges per hour. They had access to food and waterad libitum [1,2,55,56]. Animals were excluded from the study if they exhibited significantanatomical abnormalities, were pregnant, displayed abnormal physiology, or did notsurvive the procedure. The researchers were blinded to group allocation throughout theexperiment and data analysis to minimize bias.4.2.2. Drug AdministrationDrug or vehicle administration was conducted via intravitreal injection across vari-ous groups [2]. Animals were randomly assigned to each group. These groups includedInt. J. Mol. Sci. 2025, 26, 4019 19 of 26pre-ischemic administration with different concentrations of catalpol (Merck, Darmstadt,Germany; 50839; 96% purity HPLC), namely, 0.25 mM catalpol (Cata 0.25 mM + I/R) and0.5 mM catalpol (Cata 0.5 mM + I/R). Rats in the vehicle group, subjected to ischemia,received an identical volume of vehicle (vehicle + I/R) as the treatment groups. Addition-ally, certain groups of ischemic retinas received Lucentis (40 µg/4 µL; anti-VEGF antibody;ranibizumab; Novartis, Taipei, Taiwan), Eylea (160 µg/4 µL; anti-VEGF/PLGF antibody;aflibercept; Bayer, Taipei, Taiwan), and Wnt3a inhibitor Dkk1 (1 µg/4 µL; Abcam Inc.,Cambridge, UK, ab281791; 95% purity HPLC) 15 min prior to retinal ischemia [5]. Therats were then sacrificed with an overdose (at least 140 mg/kg) intraperitoneal injection ofpentobarbitone sodium; this was also carried out for the normal retina.4.2.3. Anesthesia and EuthanasiaAnimal anesthesia was induced through intraperitoneal injection of 100 mg/kg ofketamine (Pfizer, New York, NY, USA) with 5 mg/kg xylazine (Sigma-Aldrich, Saint Louis,MO, USA) to minimize animal suffering during retinal ischemia induction, ERG record-ings, and retrograde labeling of RGCs. The animals were sacrificed one day after retinalischemia/reperfusion [1]. Euthanasia was thoughtfully (Scientific Procedures Acts 1986)performed by the intraperitoneal injection of at least 140 mg/kg pentobarbitone sodium(SCI Pharmtech, Taoyuan, Taiwan).4.2.4. Ischemia InductionFollowing the administration of anesthesia, the Wistar rats were positioned within astereotaxic frame. The anterior chamber of the eye under study was cannulated with a30-gauge needle (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) connected to anelevated balanced salt solution reservoir (BSS® PLUS; Alcon, ZG, Zug, Switzerland). The saltreservoir was elevated at 163 cm to induce an intraocular pressure of 120 mmHg [66,78–80].The presence of a pale fundus confirmed the induction of an ischemic insult [1–5]. Theanimals were placed on 37 ◦C heating pads during the ischemia induction. To preventmicroleakage and ensure the lubrication of the eyes, hypromellose was applied to both eyes,with reapplication as required approximately every 30 min. No procedures were performedon the control eyes. IOP measurements were performed at baseline and 1 day post-I/Rusing a tonopen (Oculab, Inc., Glendale, CA, USA). Animals exhibiting leaks or significantdrops in IOP were excluded from this study; however, no such cases were encounteredin this investigation.4.2.5. ERG RecordingERG recordings were performed to evaluate the retinal electrophysiological response,specifically the function of the inner retinal layers, including Müller and bipolar cells. Priorto the ERG recordings [1,2,4,5], the Wistar rats were subjected to >8 h of dark adaptation.Pupil dilation was achieved with topical 1% tropicamide and 2.5% phenylephrine (Alcon,ZG, Switzerland), and topical 0.5% proparacaine (Alcon, ZG, Switzerland) was applied tonumb the ocular surfaces. Immediately before the ERG, anesthesia was administered, asmentioned in Section 4.2.3. of in vivo studies. ERG data were collected on all animals beforeand after ischemic insult (with or without intravitreal injections of vehicle or catalpol). Theanimal was then placed in a stereotaxic frame, and the body temperature was maintainedat 37 ◦C with a heated water jacket. A platinum wire loop was placed on the cornealsurface to act as the recording electrode. A ground electrode was connected to the scruffof the back, and a reference electrode to the tongue of the animal. A strobe positioned2 cm distal to the rat’s eye provided a stimulus at a frequency of 0.5 Hz and a brightnessof 2.1 candelas/m2. Fifteen consecutive responses were collected at 2 s intervals and at10 kHz; the responses were amplified using a P511/regulated power supply 107/stimulatorInt. J. Mol. Sci. 2025, 26, 4019 20 of 26PS22 (Grass-Telefactor; AstroNova, Brossard, QC, Canada). To facilitate the comparison,the oscillatory potential amplitude ratio together with a-wave and b-wave amplituderatio/response time ratio of the treated ischemic eye was compared and standardized tothat of the untreated contralateral normal eye.4.2.6. Retrograde Labeling of RGCsTo dye the RGC cells, fluorogold retrograde labeling was employed. The rats weresecured onto a stereotactic frame after anesthesia, as described in Section 4.2.3 of in vivostudies, then two small holes were made into the skull 1.5 mm lateral to the midlineand 6 mm posterior to the bregma. This was facilitated by a prior 2 cm incision to thescalp. Fluorogold (5%; 2 µL; Sigma-Aldrich) was injected through the burr holes via amicropipette at depths of 3.8, 4.0, and 4.2 mm below the skull [81].The micropipette was kept in each position for 3 min to prevent dye reflux. Af-ter injection, antibiotic ointment was applied to the wound, and the skin was sutured.Retinal ischemia induction was carried out on the studied eye 72 h following retrogradelabeling. The animals were sacrificed, as illustrated in Section 4.2.3, one day after retinalischemia/reperfusion.The eyes were subsequently enucleated utilizing a stitch at the 12 o’clock position toensure proper anatomical orientation. The eyes were then fixed in 4% paraformaldehydefor 60 min. Following the dissection of the anterior segment and the removal of the vitreousbody, the whole retina was meticulously isolated and washed with 0.1 M PBS. Followingfixation, the whole retina was mounted on a slide, separated evenly into four quadrants, andflat-mounted onto slides to prepare the retinal whole mounts. The slides were allowed toair-dry before being treated with DPx agent (Fluka Chemical, Ronkonkoma, NY, USA). Eachquadrant of the retina was subdivided into three regions, central, middle, and peripheral,located at distances of 1, 2, and 3 mm from the optic disc, respectively. Within each region,six microscopic areas of 0.430 × 0.285 mm2 each along the central line were analyzed. Intotal, 72 areas spanning the entire retina were analyzed. The average RGC density wascalculated and defined as the ratio of the total number of RGCs to the entire retinal surfacearea. Each microscopic field was saved to the computer, and cell numbers were manuallycounted on the screen by experienced technicians using a double-blind method and ahemocytometer [82]. The manual counts were also cross-referenced with automated cellcounting performed using ImageJ software (version 1.54b) [83].4.2.7. Protein and MRNA AnalysisWestern Blotting AnalysisThe retinas were harvested immediately after sacrifice. The retinal tissues were thenisolated and emulsified in the ultrasound in a lysis buffer containing a mammalian pro-tein extraction reagent (HyCell, Taipei, Taiwan). Identical amounts of denatured protein(40 µg/30 µL/well) were separated using sodium dodecyl sulphate polyacramide gelelectrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA) on a 10% separating gel anda 5% stacking gel containing 0.1% sodium dodecyl sulfate [55]. The separated proteinswere transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA,USA). The membranes underwent a blocking process for 1 h using 5% fat-free skimmedmilk (Fonterra, Taoyuan, Taiwan) in PBS. Following this, the membranes underwentovernight soaking at 4 ◦C with primary antibodies: rabbit anti-β-catenin monoclonal an-tibody (ab32572; 1:5000; Abcam Inc., Cambridge, UK), mouse anti-β-actin monoclonalantibody (MW: 42 kDa; ab6276; 1:10,000; Abcam Inc., Cambridge, UK), mouse mono-clonal α-tubulin (MW: 52 kDa; ab7291; 1:10,000; Abcam Inc., Cambridge, UK), rabbitanti-angiopoietin 2 polyclonal antibody (MW: 57 kDa; PA5-27297; 1:500; Thermo FisherInt. J. Mol. Sci. 2025, 26, 4019 21 of 26Scientific, Waltham, MA, USA), and mouse anti-VEGF monoclonal antibody (MW: 43.6 kDa;NB100-648; 1:1000; Novus Biologicals, Centennial, CO, USA). Subsequently, the blots weresoaked for 60 min at 37 ◦C with secondary antibodies: horseradish peroxidase-conjugatedgoat anti-rabbit, or anti-mouse IgG (1:2000; Santa Cruz Biotech, Santa Cruz, CA, USA) at37 ◦C for 60 min. A total of 4% fat-free skimmed milk was used to dilute the antibodies.UVP ChemStudio PLUS (Analytik Jena GmbH, Jena, Germany) was utilized to acquire andevaluate the immunoblot images. ImageJ version 1.53 t was utilized to conduct densitomet-ric analysis [2]. The levels of β-catenin, VEGF, and Ang-2 in each sample were measuredrelative to that of the control group (normalized to 100%).ELISA for MCP-1MCP-1 levels in retinal cell supernatants were quantified using enzyme-linked im-munosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN, USA; catalog no.DCP00). Following the manufacturer’s protocol, all assays were performed in triplicateto ensure accuracy. The average optical density of each sample was measured using aSynergy H1 Multi-Mode reader from BioTek Instruments, and MCP-1 concentrations wereexpressed in picograms per milliliter (pg/mL) based on the calculated standard curve.HIF-1α mRNA Levels via RT-PCRThe mRNA concentrations of retinal HIF-1α were measured by rtPCR [84]. One dayafter a retinal ischemic insult and pre-ischemia administration of the defined chemicals orthe normal (control) group, the animals were killed, and the retinal tissues were retrieved.This was followed by sonication in the presence of Tri Reagent (Sigma, St. Louis, MO, USA).Total retinal RNA was isolated, and first-strand complementary DNA (cDNA) synthesiswas performed on 2 µg deoxyribonuclease (DNase; 0.05 U/µL; Promega))-treated RNA us-ing High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Waltham, MA, USA).The first strand cDNA then underwent real-time PCR using Fast SYBRR Green MasterMix (Bio-protech, Gangwon-do, Republic of Korea). The PCR was initiated by incuba-tion at 95 ◦C for 20 s; then, 40 cycles of 95 ◦C for 3 s and 60 ◦C for 30 s were performed.Cycling was carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems,Waltham, MA, USA). Relative quantification (a comparative method) was performed us-ing the housekeeping gene β-actin as the internal standard. This procedure allowed forthe measurement of the normalized level of the target mRNA and considers the differ-ences in the quantity of total mRNA applied to each reaction (∆Ct = Ct target − Ct β-actin;cycle threshold, Ct). The relative HIF-1α expression changes caused by ischemia or the nor-mal (control) group were calculated as fold changes relative to the contralateral untreatedcontrol normal retina using the calibration equation (Ct = ∆Ct induced − ∆Ct normal).The relative quantification of gene expression was calculated according to the method2−∆∆Ct, as described in the manufacturer’s instructions, and was carried out by the ac-companying software (RQ, ver. 1.3). The PCR reagents, software, and machine werepurchased from AB Applied Biosystems. The data obtained for each treatment werepooled, and a total percentage change relative to the control was calculated. The PCRoligonucleotide primers were obtained from MISSION BIOTECH, Cambridge, MA, USA,as follows: β-actin: forward, 5′-GAACCGCTCATTGCCGATAGTG-3′; reverse, 5′-TTGTCCCTGTATGCCTCTGGTCG-3′; HIF-1α: forward, 5′-ACAGCTCCCCAGCAT TTCAC-3′;reverse, 5′-GGACAAACTCCCTCACCAAAAA-3′.4.2.8. TUNEL [Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick EndLabeling] AssayOne day after retinal ischemia, the eyes were enucleated for TUNEL staining (In situCell Death Detection Kit, Fluorescein; Roche; Mannheim, Germany) to detect cell apopto-Int. J. Mol. Sci. 2025, 26, 4019 22 of 26sis [81]. The tissue was then fixed with 10% formaldehyde for 24 h. The retinal sectionswere treated with proteinase K (25 g/mL) and incubated in H2O2/methanol for 5 min atroom temperature to inactivate endogenous peroxidases. Corresponding negative (withoutdUTP) and positive control (DNase-I-treated) sections were also measured. After rinsingwith Tris-buffered saline, the samples were incubated in a TdT enzyme/labeling reactionmix at 37 ◦C for 90 min. This reaction was based on the binding of digoxigenin–dUTP tothe 30’-OH end of DNA by TdT, followed by incubation with an anti-digoxigenin antibodyconjugated with peroxidase. Following the termination of the labeling reaction in stopbuffer, the sections were processed in a standard streptavidin–horseradish peroxidase(HRP) reaction with 3,3′ diaminobenzidine as the chromogenic peroxidase substrate andalso counterstained with methyl green. Assessment of the sections was undertaken at amagnification of 40 (Zeiss, Oberkochen, Germany). Six microscopic fields from each eyemade up of three adjacent areas on both sides of the optic nerve head from each eye (ONH;1 mm away from ONH) were used to count the TUNEL-positive cells in the inner retinallayer. The average number of TUNEL positive cells per field was used for the analysis [81].4.2.9. Statistical AnalysisThe graphs in Figures 1–6 were plotted using Sigma Plot 12.5 (Jandel Scientific,Corte Madera, CA, USA), and the program SPSS (Version 20.00) was utilized for the presentstatistical analysis. Analysis of variance (ANOVA) was performed to compare three or moreindependent groups. If the normality test indicated a normal distribution of the data, one-wayANOVA was used. The outcomes were presented as mean ± standard error. If not, non-parametric ANOVA was applied, in which the results were expressed as median and quartiles.Statistical significance was established at a probability level below 0.05 (p < 0.05).5. ConclusionsIn conclusion, our study highlights the emerging therapeutic potential of catalpol, par-ticularly its role in targeting the Wnt/β-catenin pathway to address retinal ischemia. Ourfindings indicate that ischemic insults significantly impact retinal apoptosis, viability, andfunction; however, the pre-administration of catalpol effectively attenuates these detrimentaleffects. Specifically, catalpol demonstrates antioxidative, anti-inflammatory, antiapoptotic,and anti-ischemic properties, preserving cell viability and maintaining retinal function inboth in vitro and in vivo models. Furthermore, our results suggest that catalpol exerts itsprotective effects, at least in part, via downregulating the upstream Wnt/β-catenin pathwayand subsequently reducing downstream HIF-1α/angiopoietin-2/VEGF/MCP-1 expressions.This multifaceted approach highlights the potential of targeting defined ischemic cascadealongside complementary therapies, such as catalpol, in managing retinal ischemic disorders.Author Contributions: Conceptualization, H.-M.C.; methodology, H.-M.C.; software, H.-M.C.;validation, H.-M.C., H.W.-H.C. and W.W.-J.C.; formal analysis, H.W.-H.C. and W.W.-J.C.; investigation,H.W.-H.C. and W.W.-J.C.; resources, H.-M.C.; data curation, H.W.-H.C. and W.W.-J.C.; writing—originaldraft preparation, H.W.-H.C. and W.W.-J.C.; writing—review and editing, H.-M.C., H.W.-H.C. andW.W.-J.C.; visualization, H.-M.C.; supervision, H.-M.C.; project administration, H.-M.C. All authors haveread and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: The study was conducted in accordance with the Declarationof Helsinki. The Institutional Animal Care and Use Committee at Cheng Hsin General Hospital(CHGH; Taipei, Taiwan; approval no: CHIACUC 106-04, date of approval: 2017/1/6) consented toall the animal experiments that followed the Association for Research in Vision and OphthalmologyStatement for the Use of Animals in Ophthalmology and Vision.Int. J. Mol. Sci. 2025, 26, 4019 23 of 26Informed Consent Statement: Not applicable.Data Availability Statement: The data that support the findings of this study are available from thecorresponding author upon reasonable request.Conflicts of Interest: The authors declare no conflicts of interest.References1. Chao, W.H.; Lai, M.Y.; Pan, H.T.; Shiu, H.W.; Chen, M.M.; Chao, H.M. Dendrobium nobile Lindley and its bibenzyl componentmoscatilin are able to protect retinal cells from ischemia/hypoxia by dowregulating placental growth factor and upregulatingNorrie disease protein. BMC Complement. Altern. Med. 2018, 18, 193. [CrossRef] [PubMed]2. Chao, H.W.; Chen, Y.K.; Liu, J.H.; Pan, H.T.; Lin, H.M.; Chao, H.M. Emodin protected against retinal ischemia insulted neuronsthrough the downregulation of protein overexpression of β-catenin and vascular endothelium factor. BMC Complement. Med.Ther. 2020, 20, 338. [CrossRef]3. Wu, J.H.; Liu, J.H.; Ko, Y.C.; Wang, C.T.; Chung, Y.C.; Chu, K.C.; Liu, T.T.; Chao, H.M.; Jiang, Y.J.; Chen, S.J.; et al. Haploinsuffi-ciency of RCBTB1 is associated with Coats disease and familial exudative vitreoretinopathy. Hum. Mol. Genet. 2016, 25, 1637–1647.[CrossRef] [PubMed]4. Chao, H.M.; Chen, I.L.; Liu, J.H. S-allyl L-cysteine protects the retina against kainate excitotoxicity in the rat. Am. J. Chin. Med.2014, 42, 693–708. [CrossRef]5. Tan, S.Q.; Geng, X.; Liu, J.H.; Pan, W.H.; Wang, L.X.; Liu, H.K.; Hu, L.; Chao, H.M. Xue-fu-Zhu-Yu decoction protects rats againstretinal ischemia by downregulation of HIF-1α and VEGF via inhibition of RBP2 and PKM2. BMC Complement. Altern. Med.2017, 17, 365. [CrossRef] [PubMed]6. Fortmann, S.D.; Grant, M.B. Molecular mechanisms of retinal ischemia. Curr. Opin. Physiol. 2019, 7, 41–48. [CrossRef]7. Rodríguez Villanueva, J.; Martín Esteban, J.; Rodríguez Villanueva, L.J. Retinal Cell Protection in Ocular Excitotoxicity Diseases.Possible Alternatives Offered by Microparticulate Drug Delivery Systems and Future Prospects. Pharmaceutics 2020, 12, 94.[CrossRef]8. Bonne, C.; Muller, A.; Villain, M. Free Radicals in Retinal Ischemia. Gen. Pharmacol. Vasc. Syst. 1998, 30, 275–280. [CrossRef]9. Su, L.J.; Zhang, J.H.; Gomez, H.; Murugan, R.; Hong, X.; Xu, D.; Jiang, F.; Peng, Z.Y. Reactive Oxygen Species-Induced LipidPeroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxidative Med. Cell. Longev. 2019, 2019, 5080843. [CrossRef]10. Ozaki, H.; Yu, A.Y.; Della, N.; Ozaki, K.; Luna, J.D.; Yamada, H.; Hackett, S.F.; Okamoto, N.; Zack, D.J.; Semenza, G.L.; et al.Hypoxia inducible factor-1alpha is increased in ischemic retina: Temporal and spatial correlation with VEGF expression. Investig.Ophthalmol. Vis. Sci. 1999, 40, 182–189.11. Penn, J.S.; Madan, A.; Caldwell, R.B.; Bartoli, M.; Caldwell, R.W.; Hartnett, M.E. Vascular endothelial growth factor in eye disease.Prog. Retin. Eye Res. 2008, 27, 331–371. [CrossRef] [PubMed]12. Kassumeh, S.; Weber, G.R.; Nobl, M.; Priglinger, S.G.; Ohlmann, A. The neuroprotective role of Wnt signaling in the retina. NeuralRegen. Res. 2021, 16, 1524–1528. [CrossRef]13. Chen, J.; Stahl, A.; Krah, N.M.; Seaward, M.R.; Dennison, R.J.; Sapieha, P.; Hua, J.; Hatton, C.J.; Juan, A.M.; Aderman, C.M.; et al.Wnt signaling mediates pathological vascular growth in proliferative retinopathy. Circulation 2011, 124, 1871–1881. [CrossRef][PubMed]14. Zhou, K.K.; Benyajati, S.; Le, Y.; Cheng, R.; Zhang, W.; Ma, J.X. Interruption of Wnt signaling in Müller cells amelioratesischemia-induced retinal neovascularization. PLoS ONE 2014, 9, e108454. [CrossRef] [PubMed]15. Scholz, A.; Plate, K.H.; Reiss, Y. Angiopoietin-2: A multifaceted cytokine that functions in both angiogenesis and inflammation.Ann. N. Y. Acad. Sci. 2015, 1347, 45–51. [CrossRef]16. Joussen, A.M.; Ricci, F.; Paris, L.P.; Korn, C.; Quezada-Ruiz, C.; Zarbin, M. Angiopoietin/Tie2 signalling and its role in retinal andchoroidal vascular diseases: A review of preclinical data. Eye 2021, 35, 1305–1316. [CrossRef]17. Oshima, Y.; Oshima, S.; Nambu, H.; Kachi, S.; Takahashi, K.; Umeda, N.; Shen, J.; Dong, A.; Apte, R.S.; Duh, E.; et al. Differenteffects of angiopoietin-2 in different vascular beds in the eye: New vessels are most sensitive. FASEB J. 2005, 19, 963–965.[CrossRef]18. Mohite, A.A.; Perais, J.A.; McCullough, P.; Lois, N. Retinal Ischaemia in Diabetic Retinopathy: Understanding and Overcoming aTherapeutic Challenge. J. Clin. Med. 2023, 12, 2406. [CrossRef]19. Cheng, J.-M.; Liu, X.-Q.; Liu, J.-H.; Pan, W.H.-T.; Zhang, X.-M.; Hu, L.; Chao, H.-M. Chi-Ju-Di-Huang-Wan protects rats againstretinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase. Chin. Med.2016, 11, 39. [CrossRef]20. Downey, L.; Sivaprasad, S.; Chhabra, R.; Bailey, C.; Chakrabarti, S.; Elsherbiny, S.; Patel, J.; Silvestri, G.; Watson, S.L.; Williams, G.; et al. Dualpathway inhibition with faricimab for previously treated neovascular age-related macular degeneration and diabetic macularoedema: Guidance from a UK panel of retina specialists. Eye 2024, 38, 3077–3086. [CrossRef]Int. J. Mol. Sci. 2025, 26, 4019 24 of 2621. Heier, J.S.; Singh, R.P.; Wykoff, C.C.; Csaky, K.G.; Lai, T.Y.Y.; Loewenstein, A.; Schlottmann, P.G.; Paris, L.P.; Westenskow, P.D.;Quezada-Ruiz, C. The Angiopoietin/Tie Pathway in Retinal Vascular Diseases: A Review. Retina 2021, 41, 1–19. [CrossRef]22. Fiedler, U.; Reiss, Y.; Scharpfenecker, M.; Grunow, V.; Koidl, S.; Thurston, G.; Gale, N.W.; Witzenrath, M.; Rosseau, S.; Suttorp, N.; et al.Angiopoietin-2 sensitizes endothelial cells to TNF-α and has a crucial role in the induction of inflammation. Nat. Med.2006, 12, 235–239. [CrossRef]23. Koh, Y.J.; Kim, H.Z.; Hwang, S.I.; Lee, J.E.; Oh, N.; Jung, K.; Kim, M.; Kim, K.E.; Kim, H.; Lim, N.K.; et al. Double antiangiogenicprotein, DAAP, targeting VEGF-A and angiopoietins in tumor angiogenesis, metastasis, and vascular leakage. Cancer Cell2010, 18, 171–184. [CrossRef]24. Canonica, J.; Foxton, R.; Garrido, M.G.; Lin, C.M.; Uhles, S.; Shanmugam, S.; Antonetti, D.A.; Abcouwer, S.F.; Westenskow, P.D.Delineating effects of angiopoietin-2 inhibition on vascular permeability and inflammation in models of retinal neovascularizationand ischemia/reperfusion. Front. Cell Neurosci. 2023, 17, 1192464. [CrossRef]25. Benest, A.V.; Kruse, K.; Savant, S.; Thomas, M.; Laib, A.M.; Loos, E.K.; Fiedler, U.; Augustin, H.G. Angiopoietin-2 is critical forcytokine-induced vascular leakage. PLoS ONE 2013, 8, e70459. [CrossRef]26. Schargus, M.; Frings, A. Issues with Intravitreal Administration of Anti-VEGF Drugs. Clin. Ophthalmol. 2020, 14, 897–904.[CrossRef]27. Ge, H.; Lin, W.; Lou, Z.; Chen, R.; Shi, H.; Zhao, Q.; Lin, Z. Catalpol alleviates myocardial ischemia reperfusion injury byactivating the Nrf2/HO-1 signaling pathway. Microvasc. Res. 2022, 140, 104302. [CrossRef]28. El-Hanboshy, S.M.; Helmy, M.W.; Abd-Alhaseeb, M.M. Catalpol synergistically potentiates the anti-tumour effects of regorafenibagainst hepatocellular carcinoma via dual inhibition of PI3K/Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways. Mol.Biol. Rep. 2021, 48, 7233–7242. [CrossRef]29. Bi, J.; Jiang, B.; Liu, J.H.; Lei, C.; Zhang, X.L.; An, L.J. Protective effects of catalpol against H2O2-induced oxidative stress inastrocytes primary cultures. Neurosci. Lett. 2008, 442, 224–227. [CrossRef]30. Li, Y.; Bao, Y.; Jiang, B.; Wang, Z.; Liu, Y.; Zhang, C.; An, L. Catalpol protects primary cultured astrocytes from in vitroischemia-induced damage. Int. J. Dev. Neurosci. 2008, 26, 309–317. [CrossRef]31. Zhu, J.; Chen, X.; Wang, H.; Yan, Q. Catalpol protects mice against renal ischemia/reperfusion injury via suppressing PI3K/Akt-eNOS signaling and inflammation. Int. J. Clin. Exp. Med. 2015, 8, 2038–2044.32. Wang, J.; Zhang, Y.; Zhang, M.; Sun, S.; Zhong, Y.; Han, L.; Xu, Y.; Wan, D.; Zhang, J.; Zhu, H. Feasibility of Catalpol IntranasalAdministration and Its Protective Effect on Acute Cerebral Ischemia in Rats via Anti-Oxidative and Anti-Apoptotic Mechanisms.Drug Des. Devel Ther. 2022, 16, 279–296. [CrossRef]33. Lin, J.; Deng, L.; Qi, A.; Jiang, H.; Xu, D.; Zheng, Y.; Zhang, Z.; Guo, X.; Hu, B.; Li, P. Catalpol alleviates hypoxia ischemia-inducedbrain damage by inhibiting ferroptosis through the PI3K/NRF2/system Xc-/GPX4 axis in neonatal rats. Eur. J. Pharmacol.2024, 968, 176406. [CrossRef]34. Zhang, Z.; Dai, Y.; Xiao, Y.; Liu, Q. Protective effects of catalpol on cardio-cerebrovascular diseases: A comprehensive review.J. Pharm. Anal. 2023, 13, 1089–1101. [CrossRef]35. Zheng, X.-w.; Yang, W.-t.; Chen, S.; Xu, Q.-q.; Shan, C.-s.; Zheng, G.-q.; Ruan, J.-c. Neuroprotection of Catalpol for Experi-mental Acute Focal Ischemic Stroke: Preclinical Evidence and Possible Mechanisms of Antioxidation, Anti-Inflammation, andAntiapoptosis. Oxidative Med. Cell. Longev. 2017, 2017, 5058609. [CrossRef]36. Han, Y.; Shen, M.; Tang, L.Y.; Tan, G.; Yang, Q.C.; Ye, L.; Ye, L.H.; Jiang, N.; Gao, G.P.; Shao, Y. Antiangiogenic effects of catalpolon rat corneal neovascularization. Mol. Med. Rep. 2018, 17, 2187–2194. [CrossRef]37. Wang, J.; Wan, D.; Wan, G.; Wang, J.; Zhang, J.; Zhu, H. Catalpol induces cell activity to promote axonal regeneration via thePI3K/AKT/mTOR pathway in vivo and in vitro stroke model. Ann. Transl. Med. 2019, 7, 756. [CrossRef]38. Dong, W.; Xian, Y.; Yuan, W.; Huifeng, Z.; Tao, W.; Zhiqiang, L.; Shan, F.; Ya, F.; Hongli, W.; Jinghuan, W.; et al. Catalpol stimulatesVEGF production via the JAK2/STAT3 pathway to improve angiogenesis in rats’ stroke model. J. Ethnopharmacol. 2016, 191,169–179. [CrossRef]39. Zhu, H.F.; Shao, Y.; Qin, L.; Wang, J.H.; Feng, S.; Jiang, Y.B.; Wan, D. Catalpol Enhances Neurogenesis And Inhibits Apoptosis OfNew Neurons Via BDNF, But Not The BDNF/Trkb Pathway. Drug Des. Devel Ther. 2019, 13, 4145–4157. [CrossRef]40. Liu, Y.; Xue, Q.; Li, X.; Zhang, J.; Fu, Z.; Feng, B.; Chen, Y.; Xu, X. Amelioration of stroke-induced neurological deficiency bylyophilized powder of catapol and puerarin. Int. J. Biol. Sci. 2014, 10, 448–456. [CrossRef]41. Cai, Q.; Yao, Z.; Li, H. Catalpol promotes oligodendrocyte survival and oligodendrocyte progenitor differentiation via the Aktsignaling pathway in rats with chronic cerebral hypoperfusion. Brain Res. 2014, 1560, 27–35. [CrossRef]42. Chao, H.W.-H.; Chao, H.-M. Catapol might protect against retinal ischaemia by anti-oxidation, anti-ischaemia, Wnt pathwayinhibition and VEGF downregulation. In Proceedings of the Abstracts from the 2023 European Association for Vision and EyeResearch Festival, Valencia, Spain, 26–28 October 2023; Volume 102. [CrossRef]43. D’Onofrio, P.M.; Koeberle, P.D. What can we learn about stroke from retinal ischemia models? Acta Pharmacol. Sin. 2013, 34,91–103. [CrossRef]Int. J. Mol. Sci. 2025, 26, 4019 25 of 2644. Nebbioso, M.; Franzone, F.; Lambiase, A.; Bonfiglio, V.; Limoli, P.G.; Artico, M.; Taurone, S.; Vingolo, E.M.; Greco, A.; Polimeni, A.Oxidative Stress Implication in Retinal Diseases-A Review. Antioxidants 2022, 11, 1790. [CrossRef]45. You, B.-R.; Kim, H.-R.; Kim, H.-J.; Lee, J.-Y.; Lee, S.-Y.; Song, M.-R.; Park, J.-Y.; Kim, M.-R. Catalpol content and antioxidantactivities in various cultivars of Rehmannia glutinosa. J. Korean Soc. Food Sci. Nutr. 2011, 40, 481–485. [CrossRef]46. Bhattamisra, S.K.; Yap, K.H.; Rao, V.; Choudhury, H. Multiple Biological Effects of an Iridoid Glucoside, Catalpol and ItsUnderlying Molecular Mechanisms. Biomolecules 2019, 10, 32. [CrossRef]47. Kaidi, A.; Williams, A.C.; Paraskeva, C. Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. Nat.Cell Biol. 2007, 9, 210–217. [CrossRef]48. Braunger, B.M.; Ohlmann, A.; Koch, M.; Tanimoto, N.; Volz, C.; Yang, Y.; Bösl, M.R.; Cvekl, A.; Jägle, H.; Seeliger, M.W.; et al.Constitutive overexpression of Norrin activates Wnt/β-catenin and endothelin-2 signaling to protect photoreceptors from lightdamage. Neurobiol. Dis. 2013, 50, 1–12. [CrossRef]49. Chen, Q.; Ma, J.-x. Canonical Wnt signaling in diabetic retinopathy. Vision Res. 2017, 139, 47–58. [CrossRef]50. Bok, S.; Kim, Y.E.; Woo, Y.; Kim, S.; Kang, S.J.; Lee, Y.; Park, S.K.; Weissman, I.L.; Ahn, G.O. Hypoxia-inducible factor-1α regulatesmicroglial functions affecting neuronal survival in the acute phase of ischemic stroke in mice. Oncotarget 2017, 8, 111508–111521.[CrossRef]51. Yamada, K.H. A novel approach in preventing vascular leakage and angiogenesis in wet age-related macular degeneration.Neural Regen. Res. 2022, 17, 1751–1752. [CrossRef]52. Akwii, R.G.; Mikelis, C.M. Targeting the Angiopoietin/Tie Pathway: Prospects for Treatment of Retinal and Respiratory Disorders.Drugs 2021, 81, 1731–1749. [CrossRef]53. Larsen, H.O.; Grauslund, J.; Vergmann, A.S. Efficacy, Durability and Safety of Faricimab in Neovascular Age-Related MacularDegeneration and Diabetic Macular Oedema: Lessons Learned from Registration Trials. Ophthalmol. Ther. 2023, 12, 2253–2264.[CrossRef]54. Wu, C.; Chen, J.; Chen, C.; Wang, W.; Wen, L.; Gao, K.; Chen, X.; Xiong, S.; Zhao, H.; Li, S. Wnt/β-catenin coupled withHIF-1α/VEGF signaling pathways involved in galangin neurovascular unit protection from focal cerebral ischemia. Sci. Rep.2015, 5, 16151. [CrossRef]55. Liu, J.H.; Wann, H.; Chen, M.M.; Pan, W.H.; Chen, Y.C.; Liu, C.M.; Yeh, M.Y.; Tsai, S.K.; Young, M.S.; Chuang, H.Y.; et al. Baicaleinsignificantly protects human retinal pigment epithelium cells against H2O2-induced oxidative stress by scavenging reactiveoxygen species and downregulating the expression of matrix metalloproteinase-9 and vascular endothelial growth factor. J. Ocul.Pharmacol. Ther. 2010, 26, 421–429. [CrossRef]56. Chao, W.W.; Chao, H.W.; Lee, H.F.; Chao, H.M. The Effect of S-Allyl L-Cysteine on Retinal Ischemia: The Contributions of MCP-1and PKM2 in the Underlying Medicinal Properties. Int. J. Mol. Sci. 2024, 25, 1349. [CrossRef]57. Chen, Y.Q.; Pan, W.H.; Liu, J.H.; Chen, M.M.; Liu, C.M.; Yeh, M.Y.; Tsai, S.K.; Young, M.S.; Zhang, X.M.; Chao, H.M. The effectsand underlying mechanisms of S-allyl l-cysteine treatment of the retina after ischemia/reperfusion. J. Ocul. Pharmacol. Ther.2012, 28, 110–117. [CrossRef]58. Bai, Y.; Zhu, R.; Tian, Y.; Li, R.; Chen, B.; Zhang, H.; Xia, B.; Zhao, D.; Mo, F.; Zhang, D.; et al. Catalpol in Diabetes and itsComplications: A Review of Pharmacology, Pharmacokinetics, and Safety. Molecules 2019, 24, 3302. [CrossRef]59. Dong, W.; Jing, X.; Chen, L. Separation and identification of catalpol from Rehmannia and its acute toxicity test. J. Fudan Univ.2009, 48, 409–412.60. Tao, J.; Zhang, A.H.; Zhao, Z.G.; Jiang, B. Protective effect of catalpol in mice injuries induced by rotenone and evaluation of thesafety of catalpol. Prog. Mod. Biomed. 2008. [CrossRef]61. Van Bergen, N.J.; Wood, J.P.M.; Chidlow, G.; Trounce, I.A.; Casson, R.J.; Ju, W.-K.; Weinreb, R.N.; Crowston, J.G. Recharacterizationof the RGC-5 Retinal Ganglion Cell Line. Investig. Ophthalmol. Vis. Sci. 2009, 50, 4267–4272. [CrossRef]62. Sippl, C.; Tamm, E.R. What is the nature of the RGC-5 cell line? Adv. Exp. Med. Biol. 2014, 801, 145–154. [CrossRef]63. Wood, J.P.M.; Chidlow, G.; Tran, T.; Crowston, J.G.; Casson, R.J. A Comparison of Differentiation Protocols for RGC-5 Cells.Investig. Ophthalmol. Vis. Sci. 2010, 51, 3774–3783. [CrossRef]64. Chao, H.M.; Lin, D.E.; Chang, Y.; Hsu, W.M.; Lee, S.M.; Lee, F.L.; Chi, C.W.; Pan, W.H.; Liu, T.Y.; Lui, W.Y.; et al. Ferulic acid, butnot tetramethylpyrazine, significantly attenuates retinal ischemia/reperfusion-induced alterations by acting as a hydroxyl radicalscavenger. J. Ocul. Pharmacol. Ther. 2008, 24, 461–472. [CrossRef]65. Rosenbaum, D.M.; Rosenbaum, P.S.; Singh, M.; Gupta, G.; Gupta, H.; Li, B.; Roth, S. Functional and morphologic comparison oftwo methods to produce transient retinal ischemia in the rat. J. Neuroophthalmol. 2001, 21, 62–68. [CrossRef]66. Hartsock, M.J.; Cho, H.; Wu, L.; Chen, W.J.; Gong, J.; Duh, E.J. A Mouse Model of Retinal Ischemia-Reperfusion Injury ThroughElevation of Intraocular Pressure. J. Vis. Exp. 2016, 113, 54065. [CrossRef]67. Liu, J.-H.; Chen, M.-M.; Huang, J.-W.; Wann, H.; Ho, L.-K.; Pan, W.H.T.; Chen, Y.-C.; Liu, C.-M.; Yeh, M.-Y.; Tsai, S.-K.; et al.Therapeutic Effects and Mechanisms of Action of Mannitol During H2O2-Induced Oxidative Stress in Human Retinal PigmentEpithelium Cells. J. Ocul. Pharmacol. Ther. 2010, 26, 249–257. [CrossRef]Int. J. Mol. Sci. 2025, 26, 4019 26 of 2668. Coyle, C.H.; Martinez, L.J.; Coleman, M.C.; Spitz, D.R.; Weintraub, N.L.; Kader, K.N. Mechanisms of H2O2-induced oxidativestress in endothelial cells. Free Radic. Biol. Med. 2006, 40, 2206–2213. [CrossRef]69. Du, L.; Chen, J.; Xing, Y.Q. Eupatilin prevents H(2)O(2)-induced oxidative stress and apoptosis in human retinal pigmentepithelial cells. Biomed. Pharmacother. 2017, 85, 136–140. [CrossRef]70. Cia, D.; Vergnaud-Gauduchon, J.; Jacquemot, N.; Doly, M. Epigallocatechin Gallate (EGCG) Prevents H2O2-Induced OxidativeStress in Primary Rat Retinal Pigment Epithelial Cells. Curr. Eye Res. 2014, 39, 944–952. [CrossRef]71. Tasca, C.I.; Dal-Cim, T.; Cimarosti, H. In vitro oxygen-glucose deprivation to study ischemic cell death. Methods Mol. Biol.2015, 1254, 197–210. [CrossRef]72. Ding, W.; Shang, L.; Huang, J.-F.; Li, N.; Chen, D.; Xue, L.-X.; Xiong, K. Receptor interacting protein 3-induced RGC-5 cellnecroptosis following oxygen glucose deprivation. BMC Neurosci. 2015, 16, 49. [CrossRef]73. Dvoriantchikova, G.; Degterev, A.; Ivanov, D. Retinal ganglion cell (RGC) programmed necrosis contributes to ischemia-reperfusion-induced retinal damage. Exp. Eye Res. 2014, 123, 1–7. [CrossRef]74. Cervellati, F.; Cervellati, C.; Romani, A.; Cremonini, E.; Sticozzi, C.; Belmonte, G.; Pessina, F.; Valacchi, G. Hypoxia induces celldamage via oxidative stress in retinal epithelial cells. Free Radic. Res. 2014, 48, 303–312. [CrossRef]75. Park, H.-H.; Han, M.-H.; Choi, H.; Lee, Y.J.; Kim, J.M.; Cheong, J.H.; Ryu, J.I.; Lee, K.-Y.; Koh, S.-H. Mitochondria damaged byOxygen Glucose Deprivation can be Restored through Activation of the PI3K/Akt Pathway and Inhibition of Calcium Influx byAmlodipine Camsylate. Sci. Rep. 2019, 9, 15717. [CrossRef]76. Xu, S.; Li, Y.; Chen, J.-p.; Li, D.-Z.; Jiang, Q.; Wu, T.; Zhou, X.-z. Oxygen glucose deprivation/re-oxygenation-induced neuronalcell death is associated with Lnc-D63785 m6A methylation and miR-422a accumulation. Cell Death Dis. 2020, 11, 816. [CrossRef]77. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.Immunol. Methods 1983, 65, 55–63. [CrossRef]78. Büchi, E.R.; Suivaizdis, I.; Fu, J. Pressure-induced retinal ischemia in rats: An experimental model for quantitative study.Ophthalmologica 1991, 203, 138–147. [CrossRef]79. Kim, B.J.; Braun, T.A.; Wordinger, R.J.; Clark, A.F. Progressive morphological changes and impaired retinal function associatedwith temporal regulation of gene expression after retinal ischemia/reperfusion injury in mice. Mol. Neurodegener. 2013, 8, 21.[CrossRef]80. Wang, H.; Peng, Z.; Li, Y.; Sahn, J.J.; Hodges, T.R.; Chou, T.-H.; Liu, Q.; Zhou, X.; Jiao, S.; Porciatti, V.; et al. σ2R/TMEM97 inretinal ganglion cell degeneration. Sci. Rep. 2022, 12, 20753. [CrossRef]81. Peng, P.H.; Chao, H.M.; Juan, S.H.; Chen, C.F.; Liu, J.H.; Ko, M.L. Pharmacological preconditioning by low dose cobaltprotoporphyrin induces heme oxygenase-1 overexpression and alleviates retinal ischemia-reperfusion injury in rats. Curr. EyeRes. 2011, 36, 238–246. [CrossRef]82. Ko, M.L.; Hu, D.N.; Ritch, R.; Sharma, S.C. The Combined Effect of Brain-Derived Neurotrophic Factor and a Free RadicalScavenger in Experimental Glaucoma. Investig. Ophthalmol. Vis. Sci. 2000, 41, 2967–2971.83. Grishagin, I.V. Automatic cell counting with ImageJ. Anal. Biochem. 2015, 473, 63–65. [CrossRef] [PubMed]84. Chao, H.-M.; Chuang, M.-J.; Liu, J.-H.; Liu, X.-Q.; Ho, L.-K.; Pan, W.H.T.; Zhang, X.-M.; Liu, C.-M.; Tsai, S.-K.; Kong, C.-W.; et al.Baicalein Protects Against Retinal Ischemia by Antioxidation, Antiapoptosis, Downregulation of HIF-1α, VEGF, and MMP-9 andUpregulation of HO-1. J. Ocul. Pharmacol. Ther. 2013, 29, 539–549. [CrossRef] [PubMed]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
Article
eng
Reviewed
Vancouver : University of British Columbia Library
Multidisciplinary Digital Publishing Institute
CC BY 4.0
Faculty
Researcher
catalpol
H2O2
OGD
retinal ischemia
neuroprotection
Chinese medicine
β-catenin
VEGF
angiopoietin-2
HIF-1α
MCP-1
Catalpol Protects Against Retinal Ischemia Through Antioxidation, Anti-Ischemia, Downregulation of β-Catenin, VEGF, and Angiopoietin-2 : In Vitro and In Vivo Studies
Text