Scutellarin Protects Retinal Ganglion Cells in Glaucoma by Targeting the Hsp90aa1-MAPK Pathway to Inhibit Apoptosis

Abstract

Purpose

Glaucoma, a major cause of irreversible blindness, is characterized by progressive retinal ganglion cells (RGCs) degeneration. Existing treatments can merely delay disease progression, failing to prevent RGC loss or visual field defects. For the management of ophthalmic diseases, Scutellarin (Scu) has demonstrated neuroprotective potential, but its efficacy and mechanisms in glaucoma remain unexplored. This study aims to evaluate the neuroprotective role of Scu in glaucoma and to reveal its underlying mechanisms.

Methods

Cell viability was assessed using CCK-8 and Hoechst 33342/propidium iodide staining. RGC damage and visual function were evaluated via flash visual evoked potential, immunofluorescence and hematoxylin-eosin staining. The potential targets and pathways were identified by network pharmacology, whereas the Scu-target binding affinity was evaluated by molecular docking and cellular thermal shift assay. Expression of key genes was silenced using siRNA, and protein levels were analyzed by Western blotting.

Results

Scu significantly mitigated the damage in a glutamate-induced retinal excitotoxicity glaucoma model. Network pharmacology and molecular docking identified Hsp90AA1 as a key target. Kyoto Encyclopedia of Genes and Genomes analysis associated the protective effects of Scu with the MAPK pathway, which was further supported by GO analysis that highlights its role in apoptosis regulation. Overall, Scu was found to mitigate apoptosis by modulating the Hsp90aa1-MAPK pathway, evidenced by the fact that Hsp90aa1 knockdown abolished the protective effects of Scu.

Conclusions

Scu exerts neuroprotective effects in glaucoma by targeting the Hsp90aa1-MAPK pathway, thereby suppressing apoptosis. This offers a potential therapeutic strategy for glaucoma management.

Glaucoma, as the leading cause of irreversible blindness that affects millions of individuals worldwide,¹ is featured by the selective loss of retinal ganglion cells (RGCs) and damage to their axons, eventually resulting in visual field defects.² At present, reduction of IOP serves the only established therapy to delay or partially prevent the progression of this disease.³ However, this approach fails to completely prevent RGC death and visual impairment, making it crucial to explore effective strategies for protecting RGCs from degeneration in glaucoma.

Scutellarin (Scu) is a natural flavonoid derived from a variety of plants. Toxicological studies have demonstrated that Scu is either slightly toxic or non-toxic, establishing it as a promising candidate for therapeutic use. Owing to its low toxicity and widespread availability, Scu possesses multiple pharmacological activities, including the therapeutic effects for cardiovascular⁴ and cerebrovascular disease,^5,6 anticancer properties^7–9 and neuroprotective effects.¹⁰ More specifically, Scu exhibits significant anti-fibrotic, anti-apoptotic, and anti-inflammatory effects, among other biological activities. In ophthalmic research, oral Scu treatment has been shown to preserve retinal structure and visual function in mice induced with chronic IOP elevation.¹¹ In models of acute IOP elevation and retinal hypoxia, Scu was found to inhibit inflammatory responses by modulating the NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway both in vivo and in vitro.¹² However, the protective effects of Scu in glaucoma and its underlying mechanisms remain underexplored.

Network pharmacology provides a powerful tool to identify key therapeutic targets by analyzing the structural and functional similarities between drugs and their interactions with disease-related genes while considering the complex interactions among target molecules.¹³ Therefore this study aims to investigate how Scu prevents RGC death in glaucoma by using network pharmacology and to validate the predicted results in cell and animal models. The goal is to establish an experimental foundation for the clinical application of Scu in preventing RGC degeneration in glaucoma.

Methods

Cell Culture

For all in vitro experiments, the R28 cell line—generously provided by the Department of Anatomy, School of Basic Medicine, Central South University—was used exclusively.^14–16 The cells were kept in low-glucose DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Newzerum, Christchurch, New Zealand) at 37°C under a humidified atmosphere containing 5% CO₂.

Animals

In vivo experiments were conducted using male C57BL/6J mice (seven weeks old) housed in the SPF barrier facility of the Central South University Laboratory Animal Center. The animals were maintained in strictly controlled environment (22°C ± 2°C, normal light/dark cycle), allowing water and feed access. All procedures had been approved by the Institutional Animal Care and Use Committee of Central South University.

Drug Administration

In Vitro

The R28 cells were exposed to one of three conditions: Control (no treatment), Glutamate (Glu) (Abcam, Cambridge, MA, USA) treatment, or Glu + Scu (HY-N0751; MedChemExpress, Monmouth Junction, NJ, USA) treatment for different durations. After an additional incubation for 24 hours, the cells were harvested for downstream assays.

In Vivo

A mouse model of NMDA-induced excitotoxicity was established via intravitreal injection. The mice were randomly assigned to receive either saline, NMDA (HY-17551; MedChemExpress) alone (20 mm, 1 µL), or NMDA + Scu at the concentrations of 5, 10, 20, or 40 µM. After anesthetization with 1% pentobarbital sodium, and standard preoperative procedures—including tropicamide-induced mydriasis, povidone-iodine disinfection, and ocular surface lubrication—were performed on the animals. Then, under an operating microscope, a 33 G microneedle was inserted 1 mm posterior to the limbus to create a self-sealing tunnel, through which 1 µL of the assigned solution was slowly delivered over ≥30 seconds; the needle was left in place for one minute to prevent reflux. On post-injection Day 5, the mice were euthanized to collect ocular tissues were collected for histological and molecular analyses.

Cell Viability Assay

The R28 cells were seeded into 96-well plates. After replacing the medium with fresh complete medium at varying concentrations of Glu alone or Glu + Scu, the cells were subjected to an additional incubation for 24 hours. Then, 10 µL of CCK-8 reagent (NCM Biotech, Newport, RI, USA) was dropped to each well, and the cells were further incubated in the dark for one hour. Subsequently, a microplate reader was used to detect the absorbance at 450 nm, with blank wells used for background correction.

Hoechst 33342/Propidium Iodide (PI) Dual Staining Assay

The cells were treated with specified drugs for 24 h first, then washed with PBS. Thereafter, a staining solution containing Hoechst 33342 and PI was then added, followed by incubation at 4 °C in the dark for 30 minutes. Images were immediately captured using an inverted fluorescence microscope (Nikon Inc., Melville, NY, USA). The total (Hoechst-positive) and dead (PI-positive) cells were counted, and the percentage of cell death was calculated as (PI-positive cells / total cells) × 100%. At least six fields of view were selected per sample for analysis.

Hematoxylin and Eosin (H&E) Staining

After being euthanized, both eyes were enucleated and fixed in 4% paraformaldehyde for 24 hours. After serial dehydration, the eyeballs were paraffin-embedded and serially sectioned coronally at 4 µm thickness with the optic nerve used as a landmark. The sections were stained with HE, mounted with neutral balsam, and scanned with a light microscope (Leica, Wetzlar, Germany) for subsequent morphometric analysis.

Immunofluorescence Staining of Retinal Whole‐Mounts

The eyes were fixed in 4% paraformaldehyde for one hour. The retinas were then radially cut into four equal quadrants. After isolation, the retinal cups were rinsed in 1 × PBS. The samples were blocked in 5% BSA/0.5% Triton X-100 for one hour, then incubated at 4°C overnight with either anti-RBPMS (ab152101, 1:500; Abcam) or BRN3A (ab245230, 1:500; Abcam). Then washed with PBS, the samples were further incubated with the appropriate fluorescence-conjugated secondary antibodies (488-conjugated AffiniPure Goat Anti-Rabbit IgG [H+L], 1:2000; Jackson ImmunoResearch Labs, West Grove, PA, USA) for one hour, protected from light. After additional PBS washes, the retinas were flat-mounted on glass slides, covered with the antifade mounting medium, and sealed with coverslips. Whole-mount images were acquired using a fluorescence microscope (Leica) for subsequent quantitative analysis.

Flash Visual Evoked Potential Analysis

Five days after intravitreal injection, the mice were deeply anesthetized, and their pupils were fully dilated with 0.5% tropicamide. When the pupil diameter reached ≥2 mm, the animals were placed in a prone position on a 37°C heated platform. Three silver-chloride electrodes were inserted as follows: the reference electrode placed subcutaneously 2 mm anterior to the bregma; the active electrode placed at the occipital protuberance; and the ground electrode placed at the tail base. After covering the contralateral eye, recordings were initiated.

Western Blot (WB) Analysis

Proteins were extracted from the R28 cells and retinal tissues using RIPA buffer (Epizyme Inc., Boston, MA, USA) supplemented with a Protease Inhibitor Cocktail and a Phosphatase Inhibitor Cocktail (APExBIO Technology, Houston, TX, USA). The retinas were homogenized on ice for 15 minutes, followed by centrifugation (12,000g) at 4°C for 20 minutes. The protein concentration was detected using a bicinchoninic acid assay assay (Beyotime Institute of Biotechnology, Jiangsu, China). The proteins were then mixed with 5× loading buffer and heated at 95°C for 10 minutes and were further separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked with 5% skim milk for one hour, followed by overnight incubation at 4°C with the following primary antibodies: anti-BCL-2 (ET1702-53, 1:1000; Huaan Biotechnology Co., Ltd., Hangzhou, China), anti-BAX (R380709, 1:1000; ZenBio, Inc., Durham, NC, USA), anti-Phospho-ERK1/2 (4370, 1:2000; Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (1:2000; Proteintech, Rosemont, IL, USA), anti-HSP90AA1 (HY-P80714, 1:500, MedChemExpress, Monmouth Junction, NJ, USA), anti-P38 (ET1702-65, 1:1000; Huaan Biotechnology Co., Ltd.), anti-Phospho-P38 (HA722150, 1:1000; Huaan Biotechnology Co., Ltd.), anti-JNK (680353, 1:2000, ZenBio, Inc.), anti-Phospho-JNK (9255, 1:500; Cell Signaling Technology), anti-ALB (R30030, 1:500; ZenBio, Inc.), anti-cleaved CASP3 (66470-2-lg, 1:1000; Proteintech), anti-TNFα (WL01581, 1:1000; Wanleibio Co., Ltd., Shenyang, China) and anti-β-TUBULIN (66240-1-Ig, 1:20,000; Proteintech). Thereafter, the membranes were washed with PBST (10 minutes × 3 times) and then incubated with the peroxidase-conjugated secondary antibody (1:20,000; Jackson ImmunoResearch) for one hour. After additional washes, the proteins were visualized using enhanced chemiluminescence reagents (Advansta, San Jose, CA, USA) with a chemiluminescence imager (Bio-Rad Laboratories, Ann Arbor, MI, USA). The integrated optical density values of specific proteins was quantified in ImageJ software, with β-tubulin serving as the internal control.

Quantitative RT‐PCR

The total RNA was isolated from retinal tissues was isolated using the TransZol Up Plus RNA Kit (TransGen Biotech Co., Beijing, China). After reverse transcription with HiScript III RT SuperMix for quantitative PCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China), real-time quantitative PCR was performed on a Prism 7500 system (Applied Biosystems, Foster City, CA, USA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.). All primers were designed and supplied by Sangon Biotech (Shanghai, China), including Alb (forward: 5′-TGCTTTTTCCAGGGGTGTGTT-3′, reverse: 5′-TTACTTCCTGCACTAATTTGGCA-3′), Tnf (forward: 5′-CCCTCACACTCAGATCATCTTCT-3′, reverse: 5′-GCTACGACGTGGGCTACAG-3′), Casp3 (forward: 5′-TACCTATGTCTTGCCCGTGG-3′, reverse: 5′-TAGCAGGTCGTCATCATCCC-3′), Hsp90aa1 (forward: 5′-TGTTGCGGTACTACACATCTGC-3′, reverse: 5′-GTCCTTGGTCTCACCTGTGATA-3′), and Gapdh (forward: 5′-ACTTTGGCATTGTGGAAGGG-3′, reverse: 5′-AGTGGATGCAGGGATGATGT-3′). The transcript levels were calculated relative to Gapdh by using the 2^−ΔΔCT approach. Each sample was tested in triplicate and the whole experiment was replicated three times.

Network Pharmacology

To identify the pharmacological targets of Scu, an integrated computational approach was used with three bioinformatics platforms: SEA (sea.bkslab.org), PharmMapper (lilab-ecust.cn), and SwissTargetPrediction (swisstargetprediction.ch). The predicted interactions were validated against the UniProt database to assess their relevance to human disease pathways, enabling systematic identification of the potential therapeutic mechanisms of Scu.

For glaucoma-associated genetic factors, data were extracted from GeneCards (genecards.org), DisGeNET (disgenet.org), and OMIM (omim.org) using "GLAUCOMA" as the search term. The retrieved data were integrated and subjected to quality control to eliminate duplicates, if any, and ensure reliability.

To identify novel therapeutic targets, intersectional analysis of Scu-related genetic markers and glaucoma-associated pathways was performed using the bioinformatics computational platform (bioinformatics.com.cn). Protein-protein interaction (PPI) networks were built using STRING (string-db.org) and analyzed in Cytoscape with the cytoHubba plugin to identify key interaction hubs based on topological parameters (degree centrality, betweenness centrality, and closeness centrality).

Functional annotation and pathway analysis were conducted using Metascape (metascape.org), including Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (Supplementary Material S2). The results were visualized using bubble plots generated on the bioinformatics computational platform.

For molecular docking, the three-dimensional structure of the primary therapeutic target was prepared using AutoDock Tools, and the ligand structures were optimized after retrieval from PubChem. Docking simulations were performed using AutoDock VINA (Supplementary Material S2), with analysis focused on identifying the ligand-receptor complex with the lowest binding free energy (indicating optimal interaction stability).

Cellular Thermal Shift Assay (CETSA)

The target cells were cultured to approximately 80 % confluency and treated with the compound of interest for 24 hours. After washes with PBS (twice), the cells were then detached with trypsin-EDTA, neutralized with complete medium containing 10% fetal bovine serum, and acquired by centrifugation (1000g) for five minutes. Furthermore, the cell pellets were resuspended in PBS supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail, and the cell suspensions were aliquoted into PCR tubes. The samples were precisely heated for three minutes across a temperature gradient from 37°C to 62°C and then immediately transferred to ice for five minutes to arrest thermal denaturation. Three freeze–thaw cycles in liquid nitrogen were performed to ensure complete cell lysis. The lysates were spun in a centrifuge (20,000g) at 4°C for 20 minutes, and the supernatants were mixed with 4 × SDS-PAGE loading buffer and denatured at 95°C for 10 minutes. Last, the thermal stability of the target proteins was subsequently analyzed by WB.

Hsp90aa1 Knockdown in Mice

To investigate the role of Hsp90aa1 in retinal injury, we employed intravitreal siRNA injection was used to achieve transient knockdown in vivo. According to the manufacturer's instructions (RiboBio, China), 1nmol of siRNA was dissolved in 1 µL saline solution, and 1 µL of the mixed solution was then delivered into the vitreous cavity through a syringe. Three days after injection, the mice were euthanized, and the retinas were harvested for qRT-PCR and WB analyses to confirm Hsp90aa1 silencing. On verification of efficient knockdown, drug administration was performed on Day 3 after siRNA injection to ensure temporal alignment between gene silencing and experimental interventions.

Statistical Analysis

All animal experiments were randomized by a third party using a random-number table, and investigators remained blinded throughout the study. Intravitreal injections were performed by two independent operators: one prepared coded solution (NMDA, Scu, or vehicle) and the other administrated injections, both kept blinded of group identities. For histological evaluation, sectioning, staining, and imaging were carried out by different technicians. RGC counts and ganglion cell complex (GCC) thickness measurements were performed independently by two blinded investigators using ImageJ, and the averaged values were used for further analysis to minimize bias.

The data were expressed as mean ± SD from ≥ three independent runs. GraphPad Prism 9.0 was used for all tests: one-way ANOVA with Tukey's post-hoc for comparisons among multiple groups and unpaired two-tailed t-tests for pairwise comparisons, followed by Bonferroni correction; P < 0.05 was considered statistically significant.

Results

Scu Alleviates Retinal Damage and Enhances Visual Conduction Function in Mice With NMDA-Induced Injury

NMDA exerts its neurotoxic effects by binding to the NMDA-type glutamate receptors expressed on RGCs, leading to excitatory damage in retinal tissues.¹⁷ To examine this pathological mechanism, an experimental glaucoma model was developed in mice via intravitreal administration of NMDA solution.^18,19 The potential neuroprotective effects of Scu were evaluated by administering the compound at varying concentrations to NMDA-treated animals. Then the retinal whole-mount preparations were processed for immunohistochemical analysis using the anti-Brn3a or anti-RBPMS antibody, enabling specific identification and quantitative assessment of RGC populations.

Five days after intravitreal injection, compared with controls, the RGC counts were markedly reduced in the central, middle, and peripheral retina (Figs. 1a–d). Scu was found to provide region-dependent protection against NMDA-induced RGC loss: in the central and middle zones, Scu at concentrations of 5, 10, and 20 µM significantly enhanced RGC survival, whereas in the peripheral zone, only 10 µM Scu exerted an obvious neuroprotective effect. Notably, 40 µM Scu failed to rescue RGCs in any retinal region (Figs. 1a–d).

Figure 1.

Scutellarin alleviates retinal damage and enhances visual conduction function in vitro. (a–d) Immunofluorescence staining and quantitative analysis of the effects of Scu on NMDA-induced RGC injury in mice (n = 3). ****P < 0.0001: Control (CON) versus NMDA, #P < 0.05, ##P < 0.01, ####P < 0.0001: NMDA versus NMDA + Scu group. (e–g) H&E staining and quantitative analysis of total retinal thickness and GCC thickness in retina tissue harvested at post intravitreal injection of NMDA or NMDA + Scu (n = 3). The thickness was measured in the area 300 µm around the optic nerve. Scale bar: 10 µm. ****P < 0.0001: CON versus NMDA; #P < 0.05, ##P < 0.01, ####P < 0.0001: NMDA versus NMDA + Scu group. (h–j) Flash visual evoked potentials of mice five days after intravitreal injection of NMDA and Scu (n = 6). ****P < 0.0001: CON versus NMDA, ##P < 0.01, ###P < 0.001: NMDA versus NMDA + Scu group. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer. The data were expressed as mean ± SD.

Histological evaluation of the retinal architecture was conducted via HE staining. Quantitative analysis revealed a significant reduction in ganglion cell layer (GCL) nuclear density within a 300 µm retinal segment at five days post-injection (Fig. 1e, 1g). Administration of Scu exhibited dose-dependent neuroprotective effects, with 10 µM concentration showing the maximal preservation of GCL cellularity (Figs. 1e, 1g). Additionally, the thickness of the retinal GCC was measured at 300 µm from the center of the optic disc. It was found that the GCC was significantly thinner in the NMDA group than in controls (Figs. 1e, 1f), while Scu treatment prevented GCC thinning, with statistically significant improvements (Fig. 1e, 1f). Collectively, these data establish 10 µM Scu as the optimal concentration for mitigating NMDA-mediated excitotoxicity under the present experimental paradigm.

To evaluate the visual conduction function, flash visual evoked potential analysis, a clinical test that measures nerve conduction and the integrity of the axonal myelin sheath,²⁰ was performed. Five days after NMDA injection, the latency of the P1 wave appeared to be prolonged and its amplitude was reduced compared with the control group, implying retinal dysfunction. Overall, Scu treatment partially restored the P1 wave, reflecting an improvement in visual conduction (Figs. 1h–j).

Scu Mitigates Glu-Induced Cytotoxicity in R28 Cells

In vivo results demonstrated that Scu strongly attenuated the NMDA-induced RGC loss and visual dysfunction (Fig. 1). To dissect the underlying cellular mechanisms and verify whether these protective effects are recapitulated in a controlled in vitro setting, the R28 retinal precursor cell line was exposed to Glu-induced excitotoxicity.

The R28 cellular model has been commonly employed as an in vitro tool for exploring the RGC neuroprotective mechanisms and disease pathways,^16,18,20 which was used to investigate the potential protective effects of Scu against Glu-mediated neurotoxicity in this study. The cellular viability after Glu exposure was quantified through CCK-8 assay after 24-hour treatment with varying concentrations, enabling assessment of the cytotoxic effects of Glu on R28 cells. The results demonstrated that Glu inhibited the R28 cell survival in a dose-dependent manner. Notably, intervention with 10 mm Glu significantly reduced cell viability compared with controls (Fig. 2a). Based on these findings, 10 mm Glu was selected for subsequent in vitro experiments. Furthermore, a series of Scu concentrations were tested to assess the effects of Scu on Glu-intervened R28 cells. It was found that treatment with 1 µM Scu markedly restored the cell viability compared with the Glu group (Fig. 2b); thus 1 µM Scu was selected for further studies. Moreover, Hoechst 33342/PI double staining was performed to assess cell mortality in R28 cells under varing treatment conditions. The results revealed that 24-hour Glu intervention significantly increased the cell death rate, whereas Scu effectively attenuated the Glu-induced elevation in cell death (Figs. 2c, 2d).

Figure 2.

Scutellarin mitigates Glu-induced cytotoxicity in vivo. (a) CCK-8 assay of R28 cells treated with different concentrations of Glu for 24 hours. **P < 0.001, ****P < 0.0001: Control (CON) versus Glu. (b) CCK-8 assay of R28 cells treated with 10 mm Glu and different concentrations of Scu for 24 hours. ***P < 0.001, ****P < 0.0001: Glu versus Glu + Scu. (c, d) Hoechst–PI dual staining assay and statistical analysis of PI-positive cells after 24 hours treatment with 10 mm Glu and 1 µM Scu. ****P < 0.0001: CON versus Glu, ####P < 0.0001: Glu versus Glu + Scu group. Scale bar: 100 µm. The results were recorded as mean ± SD from at least three independent experiments.

Network Pharmacology-Based Analysis of the Effects of Scu on Glaucoma

Because both animal and cellular models confirmed the Scu-mediated neuroprotection against excitotoxic injury, network pharmacology and molecular docking were then used to identify the key molecular targets and pathways that may consistently explain these observations across different experimental systems. Comprehensive analysis of disease-specific databases identified 2,030 potential molecular targets associated with glaucoma pathogenesis. Meanwhile, pharmacological target screening retrieved 298 protein targets that are potentially modulated by the bioactive components of Scu. Eventually, through integrative bioinformatics analysis, 91 common molecular targets were established as potential mediators for the therapeutic effects of Scu in glaucoma management (Fig. 3a).

Figure 3.

Network pharmacology-based analysis of the effects of Scutellarin on glaucoma. (a) The Venn map related to overlapping genes in Scu and glaucoma-related genes. (b) Scu-Glaucoma - target -pathway network diagram. (c) PPI network of overlapping genes. (d, e) The cluster analysis of the central 16 targets. The central four targets were identified as core targets based on degree values. (f–i) The key targets verification results of network pharmacology (n ≥ 6). The mRNA expression of (f) Alb, (g) Casp3, (h) Tnf, (i) Hsp90aa1. ***P < 0.001, ****P < 0.0001: Control (CON) versus NMDA; ####P < 0.0001: NMDA versus NMDA + Scu group. The data were expressed as mean ± SD.

Based on these 91 overlapping targets, a PPI network (90 nodes and 891 edges) was constructed (Figs. 3b, 3c). Specifically, the core genes were operationally defined as those with topological values simultaneously satisfying three criteria: degree centrality ≥ 19.8, betweenness centrality ≥ 86.13, and closeness centrality ≥ 0.0059. This stringent filtering approach yielded 16 hub proteins, among which CASP3, TNF, ALB and HSP90AA1 ranked the highest (Figs. 3d, 3e), underscoring their pivotal roles as putative mediators for the therapeutic effects of Scu in glaucoma.

To verify the involvement of these key targets in Scu-mediated neuroprotection effects on glaucoma, their expression levels were analyzed through qRT-PCR. As illustrated in Figures 3f, 3g, and 3i, the expression of Alb, Casp3, and Tnf was significantly elevated in the NMDA group and was markedly reversed after Scu treatment. Conversely, the expression of Hsp90aa1 was significantly suppressed in the NMDA group and showed a notable increase after Scu treatment (Fig. 3h). Consistently, WB analysis, as illustrated in the Supplementary Figures S1g–l, confirmed these gene-expression changes at the protein level, further supporting the centrality of these targets in Scu-mediated neuroprotection. Overall, above findings indicate that Scu exerts its therapeutic effects by modulating the expression of the core genes associated with glaucoma.

Molecular Docking Analysis Identified Hsp90aa1 as a Potential Target for Scu in Mitigating Damage

In silico molecular docking simulations were performed to examine the interactions of Scu with four potential molecular targets (Fig. 4a). The ligand structure was acquired from the PDB, whereas the configurations of target protein (ALB, TNF, CASP3, and HSP90AA1) were downloaded from PubChem. After structural optimization, docking calculations were executed using AutoDock Vina. Computational analysis revealed that HSP90AA1 exhibited the most favorable binding affinity with Scu, as evidenced by the lowest calculated binding energy, establishing it as the principal molecular target of Scu. CETSA further corroborated this molecular-docking prediction: Scu significantly increased the heat stability of Hsp90aa1 at 47°C (Fig. 4b). These data indicate a high-affinity, specific interaction between Scu and Hsp90aa1. Immunoblot experiments demonstrated significant downregulation of Hsp90aa1 protein levels following NMDA/Glu exposure, which was effectively reversed by Scu treatment (Figs. 4c–f), providing additional evidence for the involvementof Hsp90aa1 in Scu-mediated neuroprotection against excitotoxic damage.

Figure 4.

Molecular docking analysis identified Hsp90aa1 as a potential target. (a) Binding energy heat map of Scu to core targets. (b) CETSA was used to determine the thermal instability of the interaction of Hsp90aa1 with Scu at a series of temperatures ranging from 37°C to 62°C under short drug exposure. (c, d) The expression of HSP90AA1 in vitro (n = 3). ***P < 0.001: Control (CON) versus NMDA; #P < 0.05: NMDA versus NMDA + Scu group. (e, f) The expression of HSP90AA1 in vivo (n = 3). ***P < 0.001: CON versus Glu ; ####P < 0.0001: Glu versus Glu + Scu group. The data were expressed as mean ± SD.

The MAPK Pathway Plays a Crucial Role in Mediating the Protective Effects of Scu

GO enrichment analysis, supported by clustering network results, indicated that the potential anti-glaucoma mechanisms of Scu may involve suppressing apoptotic pathway, modulating inflammatory responses, and regulating of reactive oxygen species metabolism (Supplementary Figure S1m). Based on the top 20 KEGG pathways (excluding cancer-related, lipid metabolism, and atherosclerosis pathways) identified by pathway enrichment analysis, Scu might participate in multiple signaling cascades, such as the MAPK signaling, relaxin signaling, longevity regulation, and p53-mediated pathways (Fig. 5a).

Figure 5.

The MAPK pathway plays a pivotal role in mediating the protective effects of Scutellarin. (a) KEGG pathway enrichment analysis of Scu in the treatment of glaucoma. Top 20 bars are shown. (b–g) The expression of apoptosis-related proteins and MAPK pathway components in vitro. **P < 0.001, ***P < 0.001, ****P < 0.0001: Control (CON) versus NMDA; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001: NMDA versus NMDA + Scu group (n = 3). (h–m) The expression of apoptosis-related proteins and MAPK pathway components in vivo. **P < 0.001, ***P < 0.001, ****P < 0.0001: CON versus Glu ; #P < 0.05, ##P < 0.01, ###P < 0.001: Glu versus Glu + Scu group (n = 3). The data were expressed as mean ± SD.

To investigate whether Scu alleviates Glu excitotoxicity-induced cell apoptosis via the MAPK signaling pathway, WB analysis was performed on apoptosis-related proteins and MAPK pathway components. The results showed that in both in vitro and in vivo models, the ratios of phosphorylated Erk (p-Erk)/Erk, phosphorylated p38 (p-p38)/p38 and phosphorylated JNK (p-JNK)/JNK, as well as the expression of Bax, an apoptosis maker, were significantly upregulated in the model group compared with controls (Figs. 5b–m). In contrast, Scu treatment significantly inhibited the activation of p-Erk, p-p38 and p-JNK, while markedly promoting the expression of the anti-apoptotic protein Bcl-2 (Figs. 5b–m). These findings suggest that Scu may mitigate Glu excitotoxicity-induced apoptosis by modulating the MAPK signaling pathway.

Scu Suppresses RGC Apoptosis in NMDA-Treated Mice Through the Hsp90aa1-MAPK Pathway

To elucidate the mechanism of action of Scu, siRNA-mediated Hsp90aa1 gene silencing was performed on mouse models (Figs. 6⁷a–c). Post-knockdown analysis revealed that Scu administration did not significantly increase the RGC density compared with NMDA-treated controls (Figs. 6d–g), which was further supported by histological examination via HE staining, demonstrating that Scu failed to restore the GCL nuclear density or GCC thickness within 300 µm of the optic nerve head (Figs. 6h–j). Collectively, these findings establish Hsp90aa1 as a crucial mediator for the neuroprotective properties of Scu.

Figure 6.

Scutellarin inhibits apoptosis in NMDA-treated mice through the Hsp90aa1-MAPK signaling pathway. (a–c) WB and real-time PCR were used to assess changes in HSP90AA1 expression in mice following Hsp90aa1 knockdown (n = 8). ***P < 0.001: Control (CON) versus siRNA. (d–g) Immunofluorescence staining of retinal whole-mounts with RBPMS and quantification of RBPMS-positive RGCs at post intravitreal injection of NMDA or si-Hsp90aa1 + NMDA + Scu (n = 3). ***P < 0.001, ****P < 0.0001: CON versus NMDA, ###P < 0.001, ####P < 0.0001: CON versus si-Hsp90aa1 + NMDA + Scu group. Scale bar: 10 µm. (h–j) HE staining and quantitative analysis of total retinal thickness and GCC thickness in retina after intravitreal injection of NMDA or si-Hsp90aa1 + NMDA + Scu (n = 3). ****P < 0.0001: CON versus NMDA. ###P < 0.001, ####P < 0.0001: CON versus si-Hsp90aa1 + NMDA + Scu group. (k–p) Protein expression levels of MAPK pathway, BAX and BCL-2 were determined by WB, **P < 0.001, ***P < 0.001, ****P < 0.0001: CON versus NMDA group. #P < 0.05, ###P < 0.001, ####P < 0.0001: CON versus si-Hsp90aa1 + NMDA + Scu group (n = 3). INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer. The data were expressed as mean ± SD.

Figure 7.

Scutellarin provides substantial protection against Glu-induced excitotoxicity in a glaucoma model by mitigating RGC apoptosis. This effect is achieved through the upregulation of Hsp90aa1, resulting in the inhibition of MAPK signaling.

Under Hsp90aa1-deficient conditions, Scu treatment appeared to be ineffective at attenuating the NMDA-induced phosphorylation of Erk, p38 and JNK (Figs. 6k, 6n–p). Furthermore, the expression of the apoptotic marker Bax was elevated, accompanied by a reduced level of the anti-apoptotic factor Bcl-2, resulting in intensified cellular apoptosis (Figs. 6k–m). These experimental findings strongly suggest that the Hsp90aa1-MAPK signaling cascade plays a pivotal role in mediating the neuroprotective effects of Scu.

Discussion

This study demonstrates that Scu can effectively mitigate the Glu-induced RGC injury through anti-apoptotic mechanisms in both cellular and animal models. This neuroprotective activity is mediated through modulation of the Hsp90aa1-MAPK signaling cascade, which attenuates excitotoxicity-induced programmed cell death. These results establish a mechanistic foundation for the development of therapeutic strategies aimed at preventing RGC degeneration in glaucoma patients.

As a natural flavonoid derived from plants, Scu has long been used in China for managing chronic deseases.²¹ In recent years, its therapeutic potential in neurological disorders, including neurodegenerative diseases, has gained increasing attention.^22–24 Within the field of ophthalmology, studies have showed that Scu provides significant protection against diabetic retinopathy by inhibiting retinal ganglion cell pyroptosis; this effect is achieved through a multi-target, multi-pathway mechanism involving key molecules such as caspase-1, IL-1β, IL-18, GSDMD, and NLRP3.²⁵ Additionally, Scu has been found to suppress angiogenesis in diabetic retinopathy by modulating the VEGF/ERK/FAK/Src signaling pathways.²⁶ Our investigation indicates that Scu plays a protective role against Glu-induced excitotoxicity; however, its effect is lost at 40 µM (Fig. 2b). Importantly, intravitreal administration of Scu at concentrations up to 40 µM did not induce overt retinal toxicity in mice (Supplementary Figure S1a). Therefore we speculate that the lack of protection at 40 µM may result from a pro-oxidant shift in redox profile of Scu or a concomitant reduction in ocular bioavailability, rather than from intrinsic cytotoxicity.

In this study, a Glu excitotoxicity paradigm was used to demonstrate the neuroprotective efficacy of Scu in glaucoma. Nevertheless, clinical glaucoma is predominantly characterized by chronically elevated IOP, a feature absent in the NMDA model, which represents an inherent limitation of our approach. Recent investigations have shown that Scu can effectively improve the retinal thickness, visual function, and RGC survival in chronic ocular hypertension models.^11,27 Consistently, as shown in Supplementary Figure S1, Scu is also found to play an RGC protection role in an acute ocular hypertension paradigm, indicating that its neuroprotective effects extend beyond excitotoxic injury to encompass pressure-induced glaucomatous damage.

Although the R28 cell line is widely used as an in vitro model, as in our study, to mimic RGC injury,^16,18,19,28 it is not a bona fide RGC population and lacks key physiological attributes such as axonal projections and intact synaptic connectivity. Therefore it may not be capable of fully recapitulating the in vivo characteristics of RGCs. To address this issue, we complemented our in vitro findings with an in vivo NMDA-induced retinal excitotoxicity model to strengthen translational relevance (Fig. 1). Future studies will incorporate primary RGCs or iPSC-derived RGCs to more accurately assess the neuroprotective effects of Scu.

Moreover, network pharmacology revealed that Scu protects against glaucoma by suppressing MAPK pathway activation to mitigate RGC apoptosis. The MAPK signaling cascade is believed to associate with the regulation of numerous cellular functions, including differentiation, proliferation, apoptosis, inflammation, stress responses, and immune regulation.²⁹ Growing evidence suggests that MAPK pathways are involved in the initiation and progression of glaucoma.³⁰ In an NMDA-induced excitotoxic injury model of RGCs, JNK and p38 were recognized as key pro-apoptotic mediators in the retinal GCL, with c-Jun synthesis and phosphorylation implicated in NMDA-induced neuronal cell death.³¹ Notably, topical ocular administration p38 inhibitors, such as BIRB 796 and SB202190, has demonstrated neuroprotective effects in experimental glaucoma models.^32,33 These findings highlight the critical role of the MAPK pathway in Glu-induced retinal damage. Consistently, our network pharmacology analysis identified MAPK signaling as a core mechanism underlying the neuroprotective effects of Scu. Subsequent in vitro and in vivo investigations further confirmed that Scu exerts its protective effects through suppression of MAPK pathway activation, thereby reducing apoptosis in retinal cells.

Hsp90 is an abundant molecular chaperone that regulates the stability of a subset of proteins essential for various cellular processes.^34–37 Cytosolic Hsp90 exists in two closely related paralogs: Hsp90α, the stress-inducible form, and Hsp90β, the constitutively active form. Earlier research has shown that Hsp90α, encoded by the Hsp90AA1 gene, is abundantly expressed in the mouse retina,³⁸ whereas Hsp90β is present at lower levels. These findings suggest that Hsp90α is the predominant isoform of Hsp90 in the retina.³⁹ Hsp90 plays an essential role in retinal homeostasis, and prolonged inhibition of Hsp90 has been shown to induce photoreceptor cell death.⁴⁰ In Hsp90α knockout mice, retinal nuclear layer thinning, progressive photoreceptor cell deformation, and a marked decline in retinal electrophysiological responses were reported.³⁹ Despite the critical role of Hsp90 in cellular function, its potential involvement in ocular diseases remains incompletely explored. In this study, network pharmacology identified the MAPK signaling pathway as key to the neuroprotective effects of Scu in glaucoma, whereas molecular docking and CETSA confirmed a high-affinity interaction between Scu and Hsp90aa1. Based on these findings, we speculate that Scu exerts its neuroprotective effects by modulating the Hsp90aa1-MAPK signaling pathway. To validate this hypothesis, we conducted both in vitro and in vivo experiments, and the results indicated that Scu upregulated the Hsp90aa1 expression while alleviating Glu-induced neuronal damage. By inhibiting the MAPK pathway, Scu effectively mitigates apoptosis to provide neuroprotection. Furthermore, silencing Hsp90aa1 markedly diminished the protective effects of Scu, reinforcing the crucial role of Hsp90aa1 in mediating the therapeutic action of Scu. It is noteworthy that these findings agree with a 2025 study on diabetic retinopathy, where Hsp90aa1 was significantly downregulated and its pharmacological inhibition exacerbated retinal damage by potentiating the p-p38/p38 MAPK pathway,⁴¹ further confirming the cytoprotective role of Hsp90aa1.

The following limitations of this study should be pointed out. First, the neuroprotective effects of Scu are not mediated by a single pathway; network pharmacology suggests that it may act through a multi-target, multi-pathway mechanism. Because of the experimental timeline constraints, these targets and signaling axes have not been individually validated. Future work will focus on systematically analyzing all the potential candidate pathways. Second, although we observed the protective effects of Scu in models of chronic high IOP and Glu excitotoxicity models, its clinical translational value lacks evidence-based support. Conducting prospective clinical trials to assess the efficacy and safety of Scu in glaucoma patients will be a core focus of our next phase of research.

In summary, this study reveals that Scu alleviates apoptosis in a Glu-induced retinal excitotoxicity model of glaucoma by modulating the Hsp90aa1-MAPK signaling pathway. Our results emphasize the potential of Scu as a promising and innovative therapeutic approach for glaucoma treatment.