Ferroptosis-mediated primary open-angle glaucoma: Insight from gene signature and identification of potential small-molecule drugs

Ferroptosis-mediated primary open-angle glaucoma: Insight from gene signature and identification of potential small-molecule drugs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ferroptosis-mediated primary open-angle glaucoma: Insight from gene signature and identification of potential small-molecule drugs Xiaoyu Zhou, Xinyue Zhang, Jiahao Xu, Ping Wu, Xuanchu Duan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5869894/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Recent studies have found that ferroptosis may be involved in the process of trabecular meshwork injury in glaucoma. This study aims to reveal ferroptosis-related gene signature in primary open-angle glaucoma (POAG) and identify small molecule drugs as new direction of therapy. Methods Ferroptosis-related indicators in POAG patients and chronic ocular hypertension (COH) rats were detected by ELISA kits. The dataset (GSE27276) from GEO database and ferroptosis-related genes from FerrDb were downloaded for analysis. Small molecule drugs targeting ferroptosis-related signature components were predicted via CMap database and CB-Dock2. H 2 O 2 -induced human trabecular meshwork cells (HTMCs) oxidative stress model was constructed to validate the expression of hub genes and efficacy of drugs. Digoxin was made into eye drops to verify its intraocular pressure (IOP) lowering effect in vivo. Results Ferroptosis levels were enhanced in POAG patients and COH eyes of rats. A total of 14 ferroptosis-related differentially expressed genes were identified. PPI analysis and in vitro experiments showed HBA1, SLC2A3 and SCD played an important role in ferroptosis-mediated POAG. CMap and molecular docking indicated that ATPase inhibitors digoxin might be considered as potential therapeutic drugs for POAG. Digoxin administration alleviated H 2 O 2 -induced HTMCs ferroptosis and lowered IOP of COH eyes. Conclusions This study clarified the ferroptosis-related gene signature in the pathogenesis of POAG, which provide a theoretical basis for the prevention and early diagnosis of POAG. Translation of specific small molecule drugs will propose new ideas for therapy of POAG. Primary open-angle glaucoma Ferroptosis Small-molecule drugs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Glaucoma has emerged as the foremost cause of irreversible blindness globally. Among its various forms, Primary Open Angle Glaucoma (POAG) stands out as a multifaceted disease marked by optic nerve atrophy and progressive visual field loss. Elevated intraocular pressure (IOP), stemming from augmented aqueous humor outflow resistance, has been pinpointed as the primary risk factor driving POAG onset and its progression towards blindness[ 1 ]. The underlying dysfunction of trabecular meshwork cells (TMCs) and the resultant imbalance between extracellular matrix (ECM) synthesis and degradation are pivotal in this heightened outflow resistance, albeit the precise pathophysiological mechanisms remain elusive[ 2 ]. Oxidative stress has been established as a critical player in trabecular meshwork dysfunction and damage to retinal ganglion cells (RGCs) and the optic nerve[ 3 ]. The resultant lack of antioxidant mechanisms within TMCs leads to the accumulation of reactive oxygen species (ROS), fostering chronic inflammatory infiltration, TMC apoptosis, rearrangement, and ultimately, elevated IOP[ 4 , 5 ]. When ROS encounters the membrane-rich polyunsaturated fatty acids (PUFAs), lipid peroxidation (LPO) occurs, primarily in plasma and organelle membranes[ 6 ]. This LPO acts as a cell death signal, capable of inducing regulated cell death pathways, such as ferroptosis. Notably, the primary, secondary, and final LPO products have been found to accumulate within trabecular meshwork tissue[ 7 ]. In glaucoma patients, increased expression levels of LPO markers, including 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), have been identified in both trabecular meshwork tissue and aqueous humor[ 8 ]. Furthermore, genes associated with LPO, such as cytochrome P450 family member CyP1B1 and thioredoxin reductase 2 (TXNRD2), are implicated in trabecular meshwork injury during glaucoma[ 9 , 10 ]. These findings collectively underscore the intimate relationship between oxidative stress, lipid metabolism disruptions, and glaucoma pathogenesis. Ferroptosis, a novel form of regulated cell death, arises from the accumulation of iron-dependent LPO. During ferroptosis, iron metabolism homeostasis is disrupted, leading to increased intracellular free iron levels and the production of excessive lipid ROS via iron-dependent oxidase. This excessive LPO accumulation ultimately results in plasma membrane destruction[ 11 ]. Various studies have developed ferroptosis index scoring systems utilizing ferroptosis-related gene expression signatures to predict diagnosis and prognosis across diverse diseases[ 12 – 14 ]. However, the specific ferroptosis-related gene expression patterns in POAG pathogenesis remain uncharted. Prior research has demonstrated that glutamate receptor-mediated ferroptosis of RGCs is a significant contributor to glaucoma-induced blindness, with iron chelating agents significantly mitigating neuronal injury[ 15 ]. Ajay Ashok et al. have shown that in TGF-β2-induced TMCs, hepcidin orchestrates a self-sustaining feedforward loop through iron-catalyzed ROS. Notably, hepcidin antagonists and antioxidants can partially inhibit this harmful TGFβ2-hepcidin loop[ 16 ]. A prior study also revealed that glaucoma patients exhibit significantly higher serum iron levels compared to the normal population[ 17 ]. Recently, miR-93 and miR-141 have been implicated in POAG pathogenesis by targeting Nrf2, a crucial ferroptosis regulator[ 18 ]. By modulating ferroptosis, Nrf2 may contribute to trabecular meshwork injury. Ferroptosis has also been implicated in ECM remodeling in pulmonary and liver fibrosis, where inhibiting iron accumulation and ferroptosis attenuates fibrosis models[ 19 , 20 ]. These studies hint at ferroptosis's potential role in trabecular meshwork injury in glaucoma, suggesting that targeting TMC ferroptosis could be an effective strategy to reduce IOP. In our current study, we delved into the expression characteristics of ferroptosis-related genes in TMCs during glaucoma pathogenesis. Furthermore, we screened specific small molecule inhibitors based on these ferroptosis-related genes. Our findings aim to pave new avenues for glaucoma prevention, early diagnosis, and therapy targeting trabecular meshwork injury. Methods 1. Ethical statement All experiments associated with human participants in this study were approved by the Ethics Committee of the Changsha Aier Eye Hospital (No. KYPJ012) and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All animal protocols are based on the experimental animal welfare ethics guide drawn up by the Chinese Association for Laboratory Animal Sciences. Animal care and experimental protocol were approved by the Animal Ethical and Welfare Committee of Beijing Gene Line Bioscience Co., Ltd (No. JL-ZXY-20230101). 2. Data source and preparation We searched the Gene Expression Omnibus (GEO) database to obtain the sequencing data of trabecular meshwork (TM) tissues from POAG patients. We used the following key words “POAG” “trabecular meshwork” and “Home sapiens” (organism). Finally, we selected GSE27276 dataset for subsequent analysis, GSE27276 dataset compared genome-wide expression of trabecular meshwork in 13 controls and 15 POAG subjects based on the GPL2507 platform. A total of 19 control TM samples and 17 POAG TM samples were collected. Six controls and one POAG cases had the expression performed from both left and right eyes. One technical replicate was done between two cases. Control TM samples were obtained from donor eyes without a clinical history of glaucoma. POAG TM samples were obtained during trabeculectomy surgery. There were 5 African American and 10 Caucasian POAG patients, 3 African American and 10 Caucasian controls. Their ages ranged from 40 to 94 years[21]. The detailed information can be obtained from GEO database. GEO2R was used to analyze the differentially expressed genes (DEGs) between control and POAG TM samples. |logFC| >1 and adjusted P＜0.05 were used as threshold for identifying DEGs. And logFC >0 represents up-regulated genes; logFC <0 represents down-regulated genes. 567 ferroptosis-related genes (FRGs) including ferroptosis driver, marker, suppressor and unclassified regulator were downloaded from FerrDb (http://www.zhounan.org/ferrdb/current/). Venn diagram was used to analyze the overlap between FRGs and DEGs. Heat map showing expression patterns of differentially expressed FRGs between controls and patients with POAG were generated using “ggplot2” R package. “RCircos” package was used to display the location of FRGs on chromosomes. 3. Screening key genes Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online database (http://stringdb.org/) was utilized to construct the protein-protein interaction (PPI) network of differentially expressed FRGs. PPI network helps to construct the interactions among various proteins, which is beneficial for understanding the function and biochemical processes of proteins. It can also reveal disease and drug patterns. Correlation analysis of differentially expressed FRGs expression and visualization were conducted by “corrplot” R package and “ggplot2” R package, respectively. Nomograms of multivariable models related to differentially expressed FRGs were generated with “rms” and “rmda”R package. Importance score of differentially expressed FRGs was generated by “randomForest” R package. The operating characteristic curves (ROC) curves were generated to validate the potential predictive effectiveness of differentially expressed FRGs as biomarkers for POAG through “pROC” R package. 4. Functional enrichment analysis The “clusterProfiler” “org.Hs.eg.db” “enrichplot” “circlize” “RColorBrewer” “dplyr” “ggpubr” “ComplexHeatmap” and “ggplot2” R packages were used to perform Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analyses and visualization based on the differentially expressed FRGs. The analysis threshold was used: adjusted P <0.05. 5. Identification of small molecular compounds Ferroptosis-related DEGs were uploaded into the connectivity map (CMap) database (https://clue.io). TOP 10 candidate small molecular compounds and mechanisms of action were discovered by CMap mode-of-action (MoA) analysis and visualized by Hiplot (https://hiplot-academic.com/). 6. Molecular docking The 3D structure of digoxin was obtained from the PubChem database. The 3D structure of the receptor proteins were obtained from the Protein Data Bank (PDB) or UniProtKB. Then uploaded the structural files of protein receptors and ligand small molecules to the CB-Dock2 website (https://cadd.labshare.cn/cb-dock2). Structure-based blind docking method and Vina score were adopted to predict and evaluate the binding interactions between small molecules (ligands) and target proteins (receptors). 7. Cell culture and treatments Primary human trabecular meshwork cells (HTMCs) were isolated from corneal rims of volunteers without glaucoma who donated their cornea. After penetrating keratoplasty, TM tissue strips containing pigment were carefully dissected free. HTMCs from TM tissues were cultured in DMEM/F12 medium with 10% fetal bovine serum and 1% penicillin and streptomycin. And they were placed in a humidified incubator at 37℃, 5% CO 2 . 3% Hydrogen peroxide (H 2 O 2 ) was purchased from Sigma Aldrich. When the cell density reached approximately 70%, HTMCs were pre-treated with digoxin for 24h followed by 1.13mM H 2 O 2 for 1 h. Finally, all cells were collected and the relevant indicators were detected. 8. Quantitative Real-time PCR Analysis Total RNA was extracted and purified from HTMCs with Trizol reagent (Transgen #ER501), and RNA was reverse-transcribed to cDNA using reverse transcriptase kit (Transgen #AU341). qPCR was performed to realize gene amplification and quantification in the help of SYBR Green qPCR Super Mix (Transgen #AQ601). 9. Cell viability and cytotoxicity HTMCs were stimulated with H 2 O 2 to induce cell injury. Lactate dehydrogenase (LDH) assay was released from injured cells. HTMCs were seeded in 96-well plates and stimulated by corresponding intervention. The cell supernatant was collected and measured by LDH cytotoxicity assay kit (Beyotime) at the absorbance of 490nm and 600nm according to the manufacturer’s instructions. The cells were incubated with the CCK-8 (APExBIO) solution for 2 h. Then the cell viability was detected by measuring the optical density at 450 nm. 10. Western-blot HTMCs proteins were extracted using 98% radio immunoprecipitation assay (RIPA) lysis buffer, 1% protease inhibitor and 1% phosphatase inhibitor. Lysates were centrifuged at 15,000 rpm for 15 min, and sediment was removed. Protein expression (50 µg/lane) was detected by SDS PAGE (12–20% gel). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and then incubated by blocking with PBST containing 5% skim milk blocking buffer at room temperature for 2 h. Subsequently, the immunoblots were washed and incubated with antibodies at 4°C for 24 h. These primary antibodies includes GAPDH (HUABIO, EM1901-57, 1:1000), Hemoglobin α (HBA1) (Zen-bioscience, R25675, 1:1000), Glut3 (SLC2A3) (Santa Cruz, sc-74399, 1:1000), SCD (Zen-bioscience, R25675, 1:1000), and α-SMA (Abcam, ab5694, 1:1000). Then, the membranes were washed and incubated with appropriate secondary antibody (HUABIO, HA1006 and HA1001) at room temperature for 2 h. Bands of the proteins were visualized with enhanced chemiluminescence luminescence (ECL) reagents and scanned with Bio-Rad Imager. 11. Animal experiments 6 healthy male Brown Norway rats aged 6 weeks were adapted to feeding schedules for 2 weeks. From 1 week before surgery to 4 weeks after the surgery, IOP of the rats was measured 6 times a day using Icare rebound tonometer. The average value was taken as the preoperative IOP. Intravenous injection of hypertonic saline on episcleral veins in right eyes were used as the chronic ocular hypertension (COH) group, while left eyes as control group. The specific protocol was described in previous studies[22]. After maintaining stable IOP for 4 weeks, rats were sacrificed. Aqueous humor and corneoscleral limbus were collected for ELISA and histological staining detection. 12. Digoxin administration These experiments were conducted in two consecutive steps. Firstly, right eyes of 10 healthy male Brown Norway rats were induced by intravenous injection of hypertonic saline to construct COH model as described above. Left eyes were used as control. The second step was performed 4 weeks after COH modeling. These successfully created COH rat models with stable elevated IOP were randomly assigned for two groups : digoxin administration group and vehicle group. Digoxin and vehicle were prescribed to drip rats’ eyes twice a day for 4 weeks. 13. Histological staining After sacrifice, the corneoscleral limbus tissues obtained from rats were fixed in 4% paraformaldehyde and then embedded in paraffin wax. The sections were stained with hematoxylin and eosin (H&E). For tissue immunofluorescence, the sections were incubated overnight with HBA1, SCD, SLC2A3, α-SMA and Collagen I (COL-1) (Affinity, AF7001) antibody at 4 ℃. The sections were subsequently incubated with goat anti rabbit IgG (H+L)-Cy3 secondary antibody for 1 h at room temperature followed by DAPI staining for 5min. For immunohistochemistry (IHC), after antigen-repairing, the sections were incubated overnight with 4-Hydroxynonenal (4-HNE) antibody (BIOSS, bs-6313R) at 4 ℃. The sections were subsequently incubated with polymer-HRP goat anti rabbit secondary antibody for 40 min at room temperature followed by DBA staining for 10min. Images were obtained with a scan platform. 14. Detection of MDA, GPX4, GSH and iron ion levels in aqueous humor MDA (Jianglai biology, Shanghai, JL-11466 for human, JL13297 for rat) and glutathione peroxidase 4 (GPX4) (Jianglai biology, Shanghai, JL46163 for human, JL48671 for rat) levels in aqueous humor were detected using corresponding species ELISA kit according to the instructions. Glutathione (GSH) (Jianglai biology, Shanghai, JL-T0906) and iron ion (Jianglai biology, Shanghai, JL-T1116) levels were detected using corresponding biochemical kits. GSH reacts with DTNB to form a complex with a characteristic absorption peak at 412nm. Its absorbance is directly proportional to the GSH content. Iron dissociates from the complex in an acidic medium, is then reduced to divalent iron by a reducing agent, and reacts with ferrous zine to form purplish red compound. This colored substance has a characteristic absorption peak at 562nm, which can be used to calculate the iron content The optical density was measured by microplate reader. 15. Statistical analysis All data were analyzed with stats [4.2.1], car [3.1-0] packages of R software (4.2.1) and GraphPad Prism software. All data were given as means ± SEM. MDA and GPX4 levels in aqueous humor of rats were analyzed using paired sample t test. Student’s t-test (satisfied normality test and homogeneity of variance), Welch t' test (satisfied normality test and not satisfied homogeneity of variance) or Wilcoxon rank sum test (not satisfied normality test, nonparametric test) were used to compare two groups. One-way ANOVA was used to compare four groups. P -values < 0.05 was considered statistically significant. Results 1. Enhanced ferroptosis levels in POAG patients and chronic ocular hypertension rat model To investigate the potential role of ferroptosis in POAG, we conducted an analysis of ferroptosis indicators in the aqueous humor of POAG patients and control individuals. Our findings revealed that, compared to the control group, patients with POAG exhibited elevated levels of iron ions and MDA in their aqueous humor, which are indicative of elevated lipid peroxidation (Figure 1A, B). Conversely, the expression levels of GPX4 and GSH, key antioxidants that counteract ferroptosis, were notably lower in the POAG group compared to the control group (Figure 1C, D). To further elucidate the role of ferroptosis in glaucoma, we developed a COH rat model by injecting hypertonic saline into the episcleral vein of the right eye, while using the left eye as a control. After one week of modeling, the IOP remained stable and elevated for more than four weeks (Figure 1E). Consistent with our findings in POAG patients, the MDA content in the aqueous humor of the COH eyes was increased, while the GPX4 content was decreased compared to the control eyes (Figure 1F, G). H&E staining displayed the morphological structure of the trabecular meshwork in both control and COH eyes (Figure 1H). Furthermore, IHC and immunofluorescence detection revealed that the lipid peroxidation marker 4-HNE was significantly overexpressed in the COH eyes. These results suggest that the imbalance in redox homeostasis, characterized by increased levels of iron ions and MDA, and decreased levels of GPX4 and GSH, indicates a potential mechanism through which ferroptosis may contribute to glaucoma pathogenesis. 2. Ferroptosis-related genes patterns in POAG A total of 194 DEGs were identified in TM tissue between POAG and control samples, with 14 of these genes being associated with ferroptosis (Figure 2A). A heatmap was utilized to visualize the expression patterns of these 14 FRGs, which included 5 ferroptosis drivers, 5 ferroptosis suppressors, and 4 unclassified genes (Figure 2B). Notably, most of these genes were down-regulated, with only FADS2, SCD, and HBA1 showing up-regulation. The chromosomal locations of these FRGs were depicted in Figure 2C. GO and KEGG enrichment analysis revealed that these 14 DEGs were significantly enriched in processes related to oxygen levels, glucocorticoid response, one-carbon compound transport, gas transport, water transport, and iron ion binding (Figure 2D, E). To further investigate the interactions among these FRGs, a PPI network was constructed (Figure 3A). Within this network, SLC2A3 emerged as a hub, participating in the majority of connections. However, HBA1 did not display known PPIs with other genes in the network. Despite this, correlation analysis demonstrated that HBA1 exhibited a strong expression correlation with the other 13 genes (Figure 3B). Gene importance scoring was performed, and it was found that HBA1, SLC1A5, and FADS2 (with importance scores of ≥2) might play critical roles in POAG (Figure 3C). In a nomogram, HBA1 also demonstrated excellent predictive performance for POAG (Figure 3D). Additionally, Receiver ROC curves were established to assess the potential diagnostic efficiency of the 14 hub genes as biomarkers for POAG (Figure 3E). HBA1 showed exceptional ability to distinguish POAG patients from controls, with an Area Under the Curve (AUC) of 0.997. The other 13 hub genes also exhibited favorable accuracy as biomarkers for POAG. These findings collectively reveal a ferroptosis-related gene signature in TM tissues of POAG patients, highlighting the potential importance of these genes in the pathogenesis of the disease.. 3. Verification of hub genes expression levels We developed an oxidative stress model in HTMCs induced by H 2 O 2 , which is commonly employed as a cellular model for POAG. To stimulate the HTMCs, various concentrations of H 2 O 2 (ranging from 0 to 20mM, including 0.1, 0.5, 1, 2, 5, and 10mM) were applied for a duration of 1 hour. Cell viability was assessed using the CCK-8 kit, revealing that the median effective concentration (EC50) was 1.13mM, as depicted in Figure 4A. Consequently, a concentration of 1mM H 2 O 2 was selected for subsequent experimental procedures. Subsequently, the expression levels of 14 hub genes were quantified through qPCR. Compared to the control group, three hub genes (LCN2, HILPDA, and HBA1) exhibited increased expression, while five hub genes (SLC2A3, SCD, FADS2, DDIT4, and PROM2) showed decreased expression in H 2 O 2 -induced HTMCs (Figure 4B~O). Notably, the aberrant expressions of HBA1, DDIT4, PROM2, and SLC2A3 align with the sequencing results. However, no significant differences were observed in the expression levels of six hub genes (AQP3, AQP5, GDF15, SLC1A5, ZFP36, and DUSP1) between the control and H 2 O 2 -treated groups. These findings highlight the pivotal role of HBA1-mediated ferroptosis in the pathogenesis of POAG. 4. Potential small-molecule compounds screening To identify potential small-molecule compounds for the treatment of POAG, we uploaded ferroptosis-related DEGs to the Connectivity Map small-molecule drug database for annotation of chemical compounds and mechanisms of action. The top 10 potential small-molecule drugs identified were LDN-193189, BX-795, everolimus, ingenol, XMD-892, cycloheximide, digoxin, proscillaridin, calmidazolium, and digitoxin. Additionally, eight mechanisms of action (MOA) were identified: serine/threonine kinase inhibitor, IκB kinase (IKK) inhibitor, mammalian target of rapamycin (mTOR) inhibitor, protein kinase C (PKC) activator, mitogen-activated protein (MAP) kinase inhibitor, protein synthesis inhibitor, calcium channel blocker, and ATPase inhibitor, as shown in Figure 5A. These results suggest that digoxin and other ATPase inhibitors may exhibit therapeutic potential in POAG. Furthermore, we conducted molecular docking between digoxin and proteins encoded by eight differentially expressed hub genes in H 2 O 2 -induced HTMCs (Figure 5B). Based on structure-based blind docking, the Vina score results indicated that digoxin is more likely to regulate ferroptosis in HTMCs by targeting HBA1, SCD, and SLC2A3 (Vina score < -10), as detailed in Table 1. Table 1 CurPocket between digoxin and corresponding target in CB-dock2 Protein Ligand Vina score Cavitym volume(A³) Center (x, y, z) Docking size (x, y, z) DDIT4 Digoxin -9.2 485 -2, 9, -9 33, 33, 33 FADS2 -9.4 7782 -1, 7, -1 33, 33, 33 HBA1 -11.1 24166 67, 66, 61 33, 33, 33 HILPDA -6.9 6 0, -1, -3 33, 33, 33 SLC2A3 -10.5 1814 -22, -5, -11 33, 33, 33 PROM2 -9.4 468 -55, 2, 38 33, 33, 33 SCD -11.1 3063 24, 65, 45 33, 33, 33 LCN2 -9.5 493 41, 84, 45 33, 33, 33 5. Digoxin protect HTMCs from ferroptosis To validate the potential therapeutic benefits of digoxin in treating HTMCs ferroptosis, various concentrations of digoxin were utilized to treat HTMCs exposed to H 2 O 2 . Notably, the enhanced LDH release into the supernatant, a hallmark of cell death induced by H 2 O 2 , was inhibited by 0.1 nM and 1 nM digoxin (Figure 6A). Moreover, digoxin reversed the H 2 O 2 -induced increases in MDA and iron ions, as well as the decrease in GPX4 levels in the cell culture medium (Figure 6B, C, D). While statistical analysis did not reveal significant effects of digoxin on GSH content, the trends in GSH level changes were consistent with expectations (Figure 6E). Western blot analysis further demonstrated that digoxin reversed the H 2 O 2 -induced downregulation of SLC2A3 and SCD, as well as the upregulation of HBA1 (Figure 6F). Additionally, digoxin suppressed the H 2 O 2 -induced upregulation of α-SMA, which is indicative of extensive ECM remodeling. These findings provide evidence that digoxin may protect HTMCs from ferroptosis by targeting SLC2A3, SCD, and HBA1. 6. IOP lowering effect of digoxin for POAG To assess the effect of digoxin on IOP reduction in POAG in vivo, COH rat model was established as previously described, with IOP stabilized for a month. Subsequently, COH rats received twice-daily topical administrations of either 1 mM digoxin or vehicle for an additional month (Figure 7A). As anticipated, digoxin significantly decreased IOP in COH eyes compared to the vehicle-treated group (Figure 7B). Furthermore, digoxin mitigated the elevation of MDA levels in the aqueous humor of COH eyes (Figure 7C) and enhanced the reduced GPX4 levels in the same (Figure 7D). Histological staining of the eyeballs revealed that digoxin inhibited the COH-induced upregulation of 4-HNE in the TM tissue (Figure 8). Additionally, digoxin reversed the COH-induced downregulation of SLC2A3 and SCD, as well as the upregulation of HBA1, while also inhibiting COH-induced ECM remodeling, as indicated by α-SMA and COL-1 expression. These findings suggest that digoxin reduces IOP by inhibiting ferroptosis in the trabecular meshwork. Discussion In the current study, we conducted an exhaustive analysis of the clinical manifestations of ferroptosis patterns in Primary Open-Angle Glaucoma (POAG). Despite accumulating evidence pointing to ferroptosis as a pivotal factor in glaucoma pathogenesis, the precise mechanism underlying ferroptosis-induced trabecular meshwork injury remains elusive. Our GO and KEGG analysis revealed that ferroptosis is triggered and executed through hypoxia, iron ion binding, and carbon metabolism pathways, aligning with prior research findings. Notably, we observed significant enrichment in the responses to glucocorticoid and water transport, which are congruent with the reported pathogenic mechanisms of POAG. Our study unveiled several genes that mediate ferroptosis in POAG, including SLC2A3, HBA1, and SCD. Notably, SLC2A3 has been recognized as a crucial biomarker of ferroptosis in various diseases [23, 24]. It primarily functions in glucose transmembrane transport and may contribute to ferroptosis by modulating glycolysis and immune infiltration[25]. Abhishek's research highlighted the 12p13.3 CNV locus overlapping the SLC2A3 gene as a vital determinant for IOP control[26]. Furthermore, Xuan's study demonstrated that inhibiting FADS2 disrupts lipid peroxidation, leads to mitochondrial dysfunction, and triggers ferroptosis by directly downregulating GPX4[27]. Additionally, HBA1 regulates ferroptosis through GSH-related pathways and influences the transport of p-pyruvate and p-serine[28]. Intriguingly, HbA1c levels exhibit a positive correlation with IOP in patients with non-proliferative diabetic retinopathy[29]. These findings provide valuable insights into the complex interplay between ferroptosis and POAG, suggesting potential therapeutic targets for future interventions. HBA1 stands out as a distinctive gene in our study, displaying a notable lack of direct interactions while exhibiting a robust correlation in expression with other key genes. This gene demonstrated the highest importance score and significant diagnostic potential for POAG. Remarkably, HBA1 is the sole gene that consistently appeared in sequencing results from TM tissues, PCR results from HTMCs, and histological sections of a COH rat model. Despite these promising findings, the link between these hub genes, ferroptosis, and the underlying pathophysiological mechanisms of POAG remains unclear due to a lack of sufficient evidence. Our data strongly indicate that these hub genes, including HBA1, hold great promise for diagnosing POAG. However, further clinical and basic research is imperative to validate their pivotal role in the disease process. By delving deeper into the mechanisms by which these genes influence ferroptosis and POAG pathogenesis, we may uncover novel therapeutic targets and improve our understanding of this blinding condition. Furthermore, potential small-molecule compounds for the treatment of POAG have been identified, offering a theoretical foundation for the development of therapeutic strategies targeting TM injury. Currently, the effective treatment of glaucoma faces numerous challenges, with most anti-glaucoma medications focusing on lowering IOP by either increasing aqueous humor outflow or reducing its production through the uveoscleral outflow pathway. Traditional anti-glaucoma medications suffer from issues such as limited potency, poor adherence to long-term use, ocular surface damage, surgical failure, and other adverse reactions[30, 31]. Prostaglandin analogs targeting extracellular matrix remodeling to increase aqueous humor outflow through uveoscleral pathway are currently the most widely used IOP lowering drugs[32]. Another relatively new type of anti-glaucoma medication, Rho kinase inhibitor, reduces IOP by directly targeting traditional outflow pathway and simultaneously relaxing trabecular meshwork cells[33]. Since the drainage of aqueous humor through the TM channel accounts for 50% to 75% of the total outflow, developing medications that target TM injury to promote increased aqueous humor outflow is currently the most promising approach for glaucoma treatment. In this study, we screened eight pharmacological and genetic inhibitors of ferroptosis as potential anti-glaucoma medications. Notably, three of these inhibitors are Na+,K+-ATPase inhibitors: digoxin, proscillaridin, and digitoxin. Previous studies have shown that iron overload increases Na,K-ATPase activity in erythrocyte plasma membranes[34]. Na,K-ATPase can interact with Parkin which prevents Parkin translocating to the mitochondria and inactivates mitophagy, leading to ferroptosis[35]. On the other hand, Na,K-ATPase also interacts with SLC7A11 and inhibits ferroptosis in Parkinson's disease. So the relationships between Na,K-ATPase and ferroptosis are complicated and varies from different cell types and disease. Our results suggested a therapeutic role of digoxin in POAG. Consistent with our study, Adriana found that various digoxin derivatives exhibited great IOP lowering effects in animal ocular hypertension model[36]. It is supposed that the mechanism may be associated with the ability of digoxin to inhibit the production of aqueous humor in the ciliary body. In this study, we discovered the role of digoxin in TM protection. However, narrow therapeutic window and corneal endothelial toxicity limits digoxin application in ocular disease[37]. IKK inhibitor BX-795 has the potential to protect RGCs in glaucoma, while no research has shown its correlation with IOP[38]. Protein synthesis inhibitor cycloheximide was reported to reverse glucocorticoid-induced responsiveness in HTMCs, such as up-regulated MYOC[39, 40]. Several calcium channel blockers, such as verapamil and diltiazem, can significantly reduce IOP and improve ophthalmic blood flow[41, 42]. These novel small-molecule compounds are expected to be anticipated options for novel therapeutic strategies of glaucoma. While it still requires more basic experiments and clinical cohorts to further validate the therapeutic effect of these medications. This study provides clarity on the pivotal role of ferroptosis-related gene signatures in the underlying mechanisms of POAG. It presents a novel viewpoint regarding the clinical manifestations and ramifications of ferroptosis in glaucoma, thereby establishing a robust theoretical framework for the prevention and early identification of this condition. Additionally, the potential for developing tailored small molecule drugs targeting ferroptosis-related pathways holds promise for groundbreaking therapeutic approaches to glaucoma. Declarations Declaration of interests There was no conflict of interest. Funding This work was supported by National Natural Science Foundation of China [grant numbers 82401262 to Xiaoyu Zhou, 82471079 to Xuanchu Duan]; Hunan Engineering Research Center for Glaucoma with Artificial Intelligence in Diagnosis and Application of New Materials [grant numbers 2023TP2225 to Xuanchu Duan]; the Natural Science Foundation of Hunan Province, China [grant numbers 2023JJ40004 to Xiaoyu Zhou, 2023JJ70014 to Xuanchu Duan]; the Natural Science Foundation of Changsha [grant numbers kq2403205 to Jiahao Xu]; and the Science and Technology Foundation of Aier Eye Hospital Group, China [grant numbers AMF2406D02 to JiahaoXu]. Acknowledgements Not applicable. 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Invest Ophthalmol Vis Sci 54(5):3607–3612 Xuan Y, Wang H, Yung MM, Chen F, Chan WS, Chan YS, Tsui SK, Ngan HY, Chan KK, Chan DW (2022) SCD1/FADS2 fatty acid desaturases equipoise lipid metabolic activity and redox-driven ferroptosis in ascites-derived ovarian cancer cells. Theranostics 12(7):3534–3552 Zhang X, Du L, Qiao Y, Zhang X, Zheng W, Wu Q, Chen Y, Zhu G, Liu Y, Bian Z et al (2019) Ferroptosis is governed by differential regulation of transcription in liver cancer. Redox Biol 24:101211 Hymowitz MB, Chang D, Feinberg EB, Roy S (2016) Increased Intraocular Pressure and Hyperglycemic Level in Diabetic Patients. PLoS ONE 11(3):e0151833 Wang T, Cao L, Jiang Q, Zhang T (2021) Topical Medication Therapy for Glaucoma and Ocular Hypertension. Front Pharmacol 12:749858 Zhou X, Zhang X, Zhou D, Zhao Y, Duan X (2022) A Narrative Review of Ocular Surface Disease Related to Anti-Glaucomatous Medications. Ophthalmol Ther 11(5):1681–1704 Cai Z, Cao M, Liu K, Duan X (2021) Analysis of the Responsiveness of Latanoprost, Travoprost, Bimatoprost, and Tafluprost in the Treatment of OAG/OHT Patients. J Ophthalmol 2021:5586719 Clement Freiberg J, von Spreckelsen A, Kolko M, Azuara-Blanco A, Virgili G (2022) Rho kinase inhibitor for primary open-angle glaucoma and ocular hypertension. Cochrane Database Syst Rev 6(6):CD013817 Sousa L, Garcia IJ, Costa TG, Silva LN, Reno CO, Oliveira ES, Tilelli CQ, Santos LL, Cortes VF, Santos HL et al (2015) Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane. PLoS ONE 10(7):e0132852 Zhang X, Li G, Chen H, Nie XW, Bian JS (2024) Targeting NKAalpha1 to treat Parkinson's disease through inhibition of mitophagy-dependent ferroptosis. Free Radic Biol Med 218:190–204 Katz A, Tal DM, Heller D, Habeck M, Ben Zeev E, Rabah B, Bar Kana Y, Marcovich AL, Karlish SJ (2015) Digoxin derivatives with selectivity for the alpha2beta3 isoform of Na,K-ATPase potently reduce intraocular pressure. Proc Natl Acad Sci U S A 112(44):13723–13728 Mendoza-Moreira AL, Rodrigo-Rey S, Figuerola-Garcia MB, Gonzalez-Alonso A, Marcos-Parra MT, Perez-Santonja JJ (2022) Corneal Endothelial Dysfunction as a Manifestation of Digoxin Toxicity. Cornea 41(9):1174–1176 Minegishi Y, Nakayama M, Iejima D, Kawase K, Iwata T (2016) Significance of optineurin mutations in glaucoma and other diseases. Prog Retin Eye Res 55:149–181 Nguyen TD, Chen P, Huang WD, Chen H, Johnson D, Polansky JR (1998) Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem 273(11):6341–6350 Shepard AR, Jacobson N, Fingert JH, Stone EM, Sheffield VC, Clark AF (2001) Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 42(13):3173–3181 Ganekal S, Dorairaj S, Jhanji V, Kudlu K (2014) Effect of Topical Calcium Channel Blockers on Intraocular Pressure in Steroid-induced Glaucoma. J Curr Glaucoma Pract 8(1):15–19 Mayama C (2014) Calcium channels and their blockers in intraocular pressure and glaucoma. Eur J Pharmacol 739:96–105 Additional Declarations No competing interests reported. Supplementary Files graphicalabstract.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5869894","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":405043800,"identity":"ef62f6ba-ca14-4d16-87b8-407887fca317","order_by":0,"name":"Xiaoyu Zhou","email":"","orcid":"","institution":"Aier Glaucoma Institute, Changsha Aier Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Zhou","suffix":""},{"id":405043803,"identity":"b139d296-6e4a-419e-83c8-fa9a406373be","order_by":1,"name":"Xinyue Zhang","email":"","orcid":"","institution":"Aier Glaucoma Institute, Changsha Aier Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Zhang","suffix":""},{"id":405043804,"identity":"42e03306-ae98-4267-919d-fc2229f27a8a","order_by":2,"name":"Jiahao Xu","email":"","orcid":"","institution":"Aier Glaucoma Institute, Changsha Aier Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiahao","middleName":"","lastName":"Xu","suffix":""},{"id":405043805,"identity":"b64831e0-3794-4b4f-bbd7-65020116a4dd","order_by":3,"name":"Ping Wu","email":"","orcid":"","institution":"Aier Glaucoma Institute, Changsha Aier Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Wu","suffix":""},{"id":405043811,"identity":"23ed7688-30f1-4242-a9c2-9dc168de1abc","order_by":4,"name":"Xuanchu Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDCCA1CSH8JlJkGLZAPJWgwOEKuF70bys8c8NXcSNx8//kyCocI6sYH97AG8WiRvpJkbzjj2LHHbmRwzCYYz6YkNPHkJeLUY3Egwk/jYcDhx2w0eNgnGtsOJDRI8BgS0pH+TSARq2TyD/ZkE4z+itORAbNkgwWAmwdhAhBbJM2/KJGccO2w840yOsUXCsXTjNp4c/Fr4jqdvk+apOSzb33784Y0PNday/exn8GtBBQlAzEaC+lEwCkbBKBgFOAAAgwRJwxhkGY8AAAAASUVORK5CYII=","orcid":"","institution":"Aier Glaucoma Institute, Changsha Aier Eye Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xuanchu","middleName":"","lastName":"Duan","suffix":""}],"badges":[],"createdAt":"2025-01-21 03:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5869894/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5869894/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74428871,"identity":"2e5ceaf0-67eb-4776-92da-fd616ad0abeb","added_by":"auto","created_at":"2025-01-22 08:24:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1953435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced ferroptosis in POAG patients and COH rat model. (A-D)\u003c/strong\u003e The levels of ferroptosis-related markers (MDA, GPX4, iron ion and GSH) in human aqueous humor, N=10. \u003cstrong\u003e(E) \u003c/strong\u003eElevated IOP in COH rats was induced significantly at 4 weeks, N=6. \u003cstrong\u003e(F, G)\u003c/strong\u003e The levels of MDA and GPX4 in aqueous humor of COH rats, N=6. \u003cstrong\u003e(H)\u003c/strong\u003e HE staining, 4-HNE IHC staining and immunofluorescence staining of COH models. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, scale bar=100μm (20×), scale bar=50μm (40×). The arrow refers to TM; TM: trabecular meshwork, SC: Schlemm’s canal, OS: left eyes, OD: right eyes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/53f5888addc3138768b89e87.png"},{"id":74428777,"identity":"be5aa7ee-ebcc-4849-9209-c6568804d119","added_by":"auto","created_at":"2025-01-22 08:24:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":529127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the candidate ferroptosis-related genes in TM of POAG.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eVenn diagram showed the intersection of FRGs and DEGs between POAG and control. \u003cstrong\u003e(B)\u003c/strong\u003e Heat map showed expression levels of hub genes; Red region represents up-regulation; Blue region represents down-regulation. \u003cstrong\u003e(C)\u003c/strong\u003e The location of these FRGs on chromosomes. \u003cstrong\u003e(D, E)\u003c/strong\u003e GO annotation and KEGG pathway functional enrichment analyses of FRGs in POAG.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/1cabfa0b8c9000ae11f9761a.png"},{"id":74428854,"identity":"96b9f5a3-9069-40f0-b918-a00935f239c8","added_by":"auto","created_at":"2025-01-22 08:24:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":663728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFerroptosis-related gene signature in POAG. (A) \u003c/strong\u003ePPI network of hub genes.\u003cstrong\u003e (B)\u003c/strong\u003eHeat map showed correlation of hub gene expression; Red region represents positive correlation; Blue region represents negative correlation, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003e(C)\u003c/strong\u003e Gene importance score of hub genes for POAG.\u003cstrong\u003e(D)\u003c/strong\u003e Nomogram of hub genes for POAG. \u003cstrong\u003e(E)\u003c/strong\u003e ROC curves predict the effects of hub genes in diagnosis of POAG.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/82844be1a6034c6f9bd39680.png"},{"id":74428875,"identity":"2d6b2b35-3374-436e-9618-4b88e38fbdd4","added_by":"auto","created_at":"2025-01-22 08:24:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of hub genes expression levels. (A) \u003c/strong\u003eEC50 of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on HTMCs was detected by CCK8 kit, N=5.\u003cstrong\u003e (M-O) \u003c/strong\u003eValidation of hub genes expression levels displayed by expression levels in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs. N=4, *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/cd16a02a39c1ea70c8938f42.png"},{"id":74428780,"identity":"34225c71-994d-49a9-afbd-d4637889483b","added_by":"auto","created_at":"2025-01-22 08:24:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1722961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of potential small-molecule compounds. (A)\u003c/strong\u003e Potential small molecule drugs targeting ferroptosis-related signature components by connectivity map (CMap) database. \u003cstrong\u003e(B)\u003c/strong\u003e The molecular docking between digoxin and corresponding target in CB-dock2.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/683b1afe894e7962724eb77b.png"},{"id":74428779,"identity":"5c29bb00-b4e8-4cbf-8d5e-35d95aba4355","added_by":"auto","created_at":"2025-01-22 08:24:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":593252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDigoxin inhibited oxidative stress-induced HTMCs ferroptosis. (A)\u003c/strong\u003e The effects of digoxin on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs cytotoxicity were detected by LDH kit, N=5. \u003cstrong\u003e(B-E) \u003c/strong\u003eThe content changes of ferroptosis-related markers (MDA, GPX4, iron ion and GSH) in cell culture medium under digoxin treatment, N=3. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001. \u003cstrong\u003e(F) \u003c/strong\u003eWestern-blot showed protein expression levels of HBA1, SLC2A3, SCD and α-SMA in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs after digoxin treatment.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/462e7ae23b08566d5b25ae0a.png"},{"id":74428870,"identity":"4de585a7-3cb3-4fc5-90cb-b268524c8cc9","added_by":"auto","created_at":"2025-01-22 08:24:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":784478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDigoxin lowered IOP in COH rat models. (A) \u003c/strong\u003eAnimal experiment flowchart.\u003cstrong\u003e (B) \u003c/strong\u003eDigoxin effectively reversed the IOP in COH rat models. \u003cstrong\u003e(C, D) \u003c/strong\u003eDigoxin reversed aberrant expressed MDA and GPX4 in aqueous humor of COH rat models. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/2b63f0f6541c2a5e3d0464f2.png"},{"id":74428857,"identity":"7f6a0990-2aa2-4910-8bdb-5dee19f7cd9f","added_by":"auto","created_at":"2025-01-22 08:24:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3178737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of Digoxin on ferroptosis in vivo. \u003c/strong\u003eH\u0026amp;E, 4-HNE, SLC2A3, HBA1, SCD, α-SMA and COL-1 staining in the representative COH models. The arrow refers to corresponding indicators; TM: trabecular meshwork, SC: Schlemm’s canal.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/0c2294d392eaa6e26716541d.png"},{"id":74646675,"identity":"fd1bfcea-f8a7-4719-bdad-8b4b425b81b0","added_by":"auto","created_at":"2025-01-24 10:02:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10635900,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/ac16b1aa-e06d-4c2a-b685-c21d5cc2b075.pdf"},{"id":74428855,"identity":"bd6be21a-712d-49d4-a0c0-06b9bfaf90fe","added_by":"auto","created_at":"2025-01-22 08:24:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":307080,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5869894/v1/0e82f2d7c43c219567342569.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ferroptosis-mediated primary open-angle glaucoma: Insight from gene signature and identification of potential small-molecule drugs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlaucoma has emerged as the foremost cause of irreversible blindness globally. Among its various forms, Primary Open Angle Glaucoma (POAG) stands out as a multifaceted disease marked by optic nerve atrophy and progressive visual field loss. Elevated intraocular pressure (IOP), stemming from augmented aqueous humor outflow resistance, has been pinpointed as the primary risk factor driving POAG onset and its progression towards blindness[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The underlying dysfunction of trabecular meshwork cells (TMCs) and the resultant imbalance between extracellular matrix (ECM) synthesis and degradation are pivotal in this heightened outflow resistance, albeit the precise pathophysiological mechanisms remain elusive[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress has been established as a critical player in trabecular meshwork dysfunction and damage to retinal ganglion cells (RGCs) and the optic nerve[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The resultant lack of antioxidant mechanisms within TMCs leads to the accumulation of reactive oxygen species (ROS), fostering chronic inflammatory infiltration, TMC apoptosis, rearrangement, and ultimately, elevated IOP[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. When ROS encounters the membrane-rich polyunsaturated fatty acids (PUFAs), lipid peroxidation (LPO) occurs, primarily in plasma and organelle membranes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This LPO acts as a cell death signal, capable of inducing regulated cell death pathways, such as ferroptosis. Notably, the primary, secondary, and final LPO products have been found to accumulate within trabecular meshwork tissue[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In glaucoma patients, increased expression levels of LPO markers, including 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), have been identified in both trabecular meshwork tissue and aqueous humor[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, genes associated with LPO, such as cytochrome P450 family member CyP1B1 and thioredoxin reductase 2 (TXNRD2), are implicated in trabecular meshwork injury during glaucoma[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These findings collectively underscore the intimate relationship between oxidative stress, lipid metabolism disruptions, and glaucoma pathogenesis.\u003c/p\u003e \u003cp\u003eFerroptosis, a novel form of regulated cell death, arises from the accumulation of iron-dependent LPO. During ferroptosis, iron metabolism homeostasis is disrupted, leading to increased intracellular free iron levels and the production of excessive lipid ROS via iron-dependent oxidase. This excessive LPO accumulation ultimately results in plasma membrane destruction[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Various studies have developed ferroptosis index scoring systems utilizing ferroptosis-related gene expression signatures to predict diagnosis and prognosis across diverse diseases[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the specific ferroptosis-related gene expression patterns in POAG pathogenesis remain uncharted. Prior research has demonstrated that glutamate receptor-mediated ferroptosis of RGCs is a significant contributor to glaucoma-induced blindness, with iron chelating agents significantly mitigating neuronal injury[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Ajay Ashok et al. have shown that in TGF-β2-induced TMCs, hepcidin orchestrates a self-sustaining feedforward loop through iron-catalyzed ROS. Notably, hepcidin antagonists and antioxidants can partially inhibit this harmful TGFβ2-hepcidin loop[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A prior study also revealed that glaucoma patients exhibit significantly higher serum iron levels compared to the normal population[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recently, miR-93 and miR-141 have been implicated in POAG pathogenesis by targeting Nrf2, a crucial ferroptosis regulator[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. By modulating ferroptosis, Nrf2 may contribute to trabecular meshwork injury. Ferroptosis has also been implicated in ECM remodeling in pulmonary and liver fibrosis, where inhibiting iron accumulation and ferroptosis attenuates fibrosis models[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These studies hint at ferroptosis's potential role in trabecular meshwork injury in glaucoma, suggesting that targeting TMC ferroptosis could be an effective strategy to reduce IOP.\u003c/p\u003e \u003cp\u003eIn our current study, we delved into the expression characteristics of ferroptosis-related genes in TMCs during glaucoma pathogenesis. Furthermore, we screened specific small molecule inhibitors based on these ferroptosis-related genes. Our findings aim to pave new avenues for glaucoma prevention, early diagnosis, and therapy targeting trabecular meshwork injury.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments associated with human participants in this study were approved by the Ethics Committee of the Changsha Aier Eye Hospital (No. KYPJ012) and the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal protocols are based on the experimental animal welfare ethics guide drawn up by the Chinese Association for Laboratory Animal Sciences. Animal care and experimental protocol were approved by the Animal Ethical and Welfare Committee of Beijing Gene Line Bioscience Co., Ltd (No. JL-ZXY-20230101).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Data source and preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe searched the Gene Expression Omnibus (GEO) database to obtain the sequencing data of trabecular meshwork (TM) tissues from POAG patients. We used the following key words \u0026ldquo;POAG\u0026rdquo; \u0026ldquo;trabecular meshwork\u0026rdquo; and \u0026ldquo;Home sapiens\u0026rdquo; (organism). Finally, we selected GSE27276 dataset for subsequent analysis, GSE27276 dataset compared genome-wide expression of trabecular meshwork in 13 controls and 15 POAG subjects based on the GPL2507 platform. A total of 19 control TM samples and 17 POAG TM samples were collected. Six controls and one POAG cases had the expression performed from both left and right eyes. One technical replicate was done between two cases. Control TM samples were obtained from donor eyes without a clinical history of glaucoma. POAG TM samples were obtained during trabeculectomy surgery. There were 5 African American and 10 Caucasian POAG patients, 3 African American and 10 Caucasian controls. Their ages ranged from 40 to 94 years[21]. The detailed information can be obtained from GEO database.\u003c/p\u003e\n\u003cp\u003eGEO2R was used to analyze the differentially expressed genes (DEGs) between control and POAG TM samples. |logFC| \u0026gt;1 and adjusted P＜0.05 were used as threshold for identifying DEGs. And logFC \u0026gt;0 represents up-regulated genes; logFC \u0026lt;0 represents down-regulated genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e567 ferroptosis-related genes (FRGs) including ferroptosis driver, marker, suppressor and unclassified regulator were downloaded from FerrDb (http://www.zhounan.org/ferrdb/current/). Venn diagram was used to analyze the overlap between FRGs and DEGs. Heat map showing expression patterns of differentially expressed FRGs between controls and patients with POAG were generated using \u0026ldquo;ggplot2\u0026rdquo; R package. \u0026ldquo;RCircos\u0026rdquo; package was used to display the location of FRGs on chromosomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Screening key genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSearch Tool for the Retrieval of Interacting Genes/Proteins (STRING) online database (http://stringdb.org/) was utilized to construct the protein-protein interaction (PPI) network of differentially expressed FRGs. PPI network helps to construct the interactions among various proteins, which is beneficial for understanding the function and biochemical processes of proteins. It can also reveal disease and drug patterns. Correlation analysis of differentially expressed FRGs expression and visualization were conducted by \u0026ldquo;corrplot\u0026rdquo; R package and \u0026ldquo;ggplot2\u0026rdquo; R package, respectively. Nomograms of multivariable models related to differentially expressed FRGs were generated with \u0026ldquo;rms\u0026rdquo; and \u0026ldquo;rmda\u0026rdquo;R package. Importance score of differentially expressed FRGs was generated by \u0026ldquo;randomForest\u0026rdquo; R package.\u003c/p\u003e\n\u003cp\u003eThe operating characteristic curves (ROC) curves were generated to validate the potential predictive effectiveness of differentially expressed FRGs as biomarkers for POAG through \u0026ldquo;pROC\u0026rdquo; R package.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Functional enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026ldquo;clusterProfiler\u0026rdquo; \u0026ldquo;org.Hs.eg.db\u0026rdquo; \u0026ldquo;enrichplot\u0026rdquo; \u0026ldquo;circlize\u0026rdquo; \u0026ldquo;RColorBrewer\u0026rdquo; \u0026ldquo;dplyr\u0026rdquo; \u0026ldquo;ggpubr\u0026rdquo; \u0026ldquo;ComplexHeatmap\u0026rdquo; and \u0026ldquo;ggplot2\u0026rdquo; R packages were used to perform Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analyses and visualization based on the differentially expressed FRGs. The analysis threshold was used: adjusted P \u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Identification of small molecular compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis-related DEGs were uploaded into the connectivity map (CMap) database (https://clue.io). TOP 10 candidate small molecular compounds and mechanisms of action were discovered by CMap mode-of-action (MoA) analysis and visualized by Hiplot (https://hiplot-academic.com/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Molecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D structure of digoxin was obtained from the PubChem database. The 3D structure of the receptor proteins were obtained from the Protein Data Bank (PDB) or UniProtKB. Then uploaded the structural files of protein receptors and ligand small molecules to the CB-Dock2 website (https://cadd.labshare.cn/cb-dock2). Structure-based blind docking method and Vina\u0026nbsp;score\u0026nbsp;were adopted to predict and evaluate the binding interactions between small molecules (ligands) and target proteins (receptors).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Cell culture and treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary human trabecular meshwork cells (HTMCs) were isolated from corneal rims of volunteers without glaucoma who donated their cornea. After penetrating keratoplasty, TM tissue strips containing pigment were carefully dissected free. HTMCs from TM tissues were cultured in DMEM/F12 medium with 10% fetal bovine serum and 1% penicillin and streptomycin. And they were placed in a humidified incubator at 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3% Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was purchased from Sigma Aldrich. When the cell density reached approximately 70%, HTMCs were pre-treated with digoxin for 24h followed by 1.13mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 1 h. Finally, all cells were collected and the relevant indicators were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Quantitative Real-time PCR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted and purified from HTMCs with Trizol reagent (Transgen #ER501), and RNA was reverse-transcribed to cDNA using reverse transcriptase kit (Transgen #AU341). qPCR was performed to realize gene amplification and quantification in the help of SYBR Green qPCR Super Mix (Transgen #AQ601).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Cell viability and cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHTMCs were stimulated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to induce cell injury. Lactate dehydrogenase (LDH) assay was released from injured cells. HTMCs were seeded in 96-well plates and stimulated by corresponding intervention. The cell supernatant was collected and measured by LDH cytotoxicity assay kit (Beyotime) at the absorbance of 490nm and 600nm according to the manufacturer\u0026rsquo;s instructions. The cells were incubated with the CCK-8 (APExBIO) solution for 2 h. Then the cell viability was detected by measuring the optical density at 450 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. Western-blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHTMCs proteins were extracted using 98% radio immunoprecipitation assay (RIPA) lysis buffer, 1% protease inhibitor and 1% phosphatase inhibitor. Lysates were centrifuged at 15,000 rpm for 15 min, and sediment was removed. Protein expression (50 \u0026micro;g/lane) was detected by SDS PAGE (12\u0026ndash;20% gel). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and then incubated by blocking with PBST containing 5% skim milk blocking buffer at room temperature for 2 h. Subsequently, the immunoblots were washed and incubated with antibodies at 4\u0026deg;C for 24 h. These primary antibodies includes GAPDH (HUABIO, EM1901-57, 1:1000), Hemoglobin \u0026alpha; (HBA1) (Zen-bioscience, R25675, 1:1000), Glut3 (SLC2A3) (Santa Cruz, sc-74399, 1:1000), SCD (Zen-bioscience, R25675, 1:1000), and \u0026alpha;-SMA (Abcam, ab5694, 1:1000). Then, the membranes were washed and incubated with appropriate secondary antibody (HUABIO, HA1006 and HA1001) at room temperature for 2 h. Bands of the proteins were visualized with enhanced chemiluminescence luminescence (ECL) reagents and scanned with Bio-Rad Imager.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11. Animal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e6 healthy male Brown Norway rats aged 6 weeks were adapted to feeding schedules for 2 weeks. From 1 week before surgery to 4 weeks after the surgery, IOP of the rats was measured 6 times a day using Icare rebound tonometer. The average value was taken as the preoperative IOP. Intravenous injection of hypertonic saline on episcleral veins in right eyes were used as the chronic ocular hypertension (COH) group, while left eyes as control group. The specific protocol was described in previous studies[22]. After maintaining stable IOP for 4 weeks, rats were sacrificed. Aqueous humor and corneoscleral limbus were collected for ELISA and histological staining detection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e12. Digoxin administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese experiments were conducted in two consecutive steps. Firstly, right eyes of 10 healthy male Brown Norway rats were induced by intravenous injection of hypertonic saline to construct COH model as described above. Left eyes were used as control. The second step was performed 4 weeks after COH modeling. These successfully created COH rat models with stable elevated IOP were randomly assigned for two groups : digoxin administration group and vehicle group. Digoxin and vehicle were prescribed to drip rats\u0026rsquo; eyes twice a day for 4 weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e13. Histological staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter sacrifice, the corneoscleral limbus tissues obtained from rats were fixed in 4% paraformaldehyde and then embedded in paraffin wax. The sections were stained with hematoxylin and eosin (H\u0026amp;E). For tissue immunofluorescence, the sections were incubated overnight with HBA1, SCD, SLC2A3, \u0026alpha;-SMA and Collagen I (COL-1) (Affinity, AF7001) antibody at 4 ℃. The sections were subsequently incubated with goat anti rabbit IgG (H+L)-Cy3 secondary antibody for 1 h at room temperature followed by DAPI staining for 5min. For immunohistochemistry (IHC), after antigen-repairing, the sections were incubated overnight with 4-Hydroxynonenal (4-HNE) antibody (BIOSS, bs-6313R) at 4 ℃. The sections were subsequently incubated with polymer-HRP goat anti rabbit secondary antibody for 40 min at room temperature followed by DBA staining for 10min. Images were obtained with a scan platform.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e14. Detection of MDA, GPX4, GSH and iron ion levels in aqueous humor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA (Jianglai biology, Shanghai, JL-11466 for human, JL13297 for rat) and glutathione peroxidase 4 (GPX4) (Jianglai biology, Shanghai, JL46163 for human, JL48671 for rat) levels in aqueous humor were detected using corresponding species ELISA kit according to the instructions. Glutathione (GSH) (Jianglai biology, Shanghai, JL-T0906) and iron ion (Jianglai biology, Shanghai, JL-T1116) levels were detected using corresponding biochemical\u0026nbsp;kits. GSH reacts with DTNB to form a complex with a characteristic absorption peak at 412nm. Its absorbance is directly proportional to the GSH content. Iron dissociates from the complex in an acidic medium, is then reduced to divalent iron by a reducing agent, and reacts with ferrous zine to form purplish red compound. This colored substance has a characteristic absorption peak at 562nm, which can be used to calculate the iron content The optical density was measured by microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e15. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were analyzed with stats [4.2.1], car [3.1-0] packages of R software (4.2.1) and GraphPad Prism software. All data were given as means \u0026plusmn; SEM. MDA and GPX4 levels in aqueous humor of rats were analyzed using paired sample t test. Student\u0026rsquo;s t-test (satisfied normality test and homogeneity of variance), Welch t\u0026apos; test (satisfied normality test and not satisfied homogeneity of variance) or Wilcoxon rank sum test (not satisfied normality test, nonparametric test) were used to compare two groups. One-way ANOVA was used to compare four groups. \u003cem\u003eP\u003c/em\u003e-values \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Enhanced ferroptosis levels in POAG patients and chronic ocular hypertension rat model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential role of ferroptosis in POAG, we conducted an analysis of ferroptosis indicators in the aqueous humor of POAG patients and control individuals. Our findings revealed that, compared to the control group, patients with POAG exhibited elevated levels of iron ions and MDA in their aqueous humor, which are indicative of elevated lipid peroxidation (Figure 1A, B). Conversely, the expression levels of GPX4 and GSH, key antioxidants that counteract ferroptosis, were notably lower in the POAG group compared to the control group (Figure 1C, D). To further elucidate the role of ferroptosis in glaucoma, we developed a COH rat model by injecting hypertonic saline into the episcleral vein of the right eye, while using the left eye as a control. After one week of modeling, the IOP remained stable and elevated for more than four weeks (Figure 1E). Consistent with our findings in POAG patients, the MDA content in the aqueous humor of the COH eyes was increased, while the GPX4 content was decreased compared to the control eyes (Figure 1F, G). H\u0026amp;E staining displayed the morphological structure of the trabecular meshwork in both control and COH eyes (Figure 1H). Furthermore, IHC and immunofluorescence detection revealed that the lipid peroxidation marker 4-HNE was significantly overexpressed in the COH eyes. These results suggest that the imbalance in redox homeostasis, characterized by increased levels of iron ions and MDA, and decreased levels of GPX4 and GSH, indicates a potential mechanism through which ferroptosis may contribute to glaucoma pathogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Ferroptosis-related genes patterns in POAG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 194 DEGs were identified in TM tissue between POAG and control samples, with 14 of these genes being associated with ferroptosis (Figure 2A). A heatmap was utilized to visualize the expression patterns of these 14 FRGs, which included 5 ferroptosis drivers, 5 ferroptosis suppressors, and 4 unclassified genes (Figure 2B). Notably, most of these genes were down-regulated, with only FADS2, SCD, and HBA1 showing up-regulation. The chromosomal locations of these FRGs were depicted in Figure 2C. GO and KEGG enrichment analysis revealed that these 14 DEGs were significantly enriched in processes related to oxygen levels, glucocorticoid response, one-carbon compound transport, gas transport, water transport, and iron ion binding (Figure 2D, E). To further investigate the interactions among these FRGs, a PPI network was constructed (Figure 3A). Within this network, SLC2A3 emerged as a hub, participating in the majority of connections. However, HBA1 did not display known PPIs with other genes in the network. Despite this, correlation analysis demonstrated that HBA1 exhibited a strong expression correlation with the other 13 genes (Figure 3B). Gene importance scoring was performed, and it was found that HBA1, SLC1A5, and FADS2 (with importance scores of\u0026nbsp;\u0026ge;2) might play critical roles in POAG (Figure 3C). In a nomogram, HBA1 also demonstrated excellent predictive performance for POAG (Figure 3D). Additionally, Receiver ROC curves were established to assess the potential diagnostic efficiency of the 14 hub genes as biomarkers for POAG (Figure 3E). HBA1 showed exceptional ability to distinguish POAG patients from controls, with an Area Under the Curve (AUC) of 0.997. The other 13 hub genes also exhibited favorable accuracy as biomarkers for POAG. These findings collectively reveal a ferroptosis-related gene signature in TM tissues of POAG patients, highlighting the potential importance of these genes in the pathogenesis of the disease..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Verification of hub genes expression levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe developed an oxidative stress model in HTMCs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which is commonly employed as a cellular model for POAG. To stimulate the HTMCs, various concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (ranging from 0 to 20mM, including 0.1, 0.5, 1, 2, 5, and 10mM) were applied for a duration of 1 hour. Cell viability was assessed using the CCK-8 kit, revealing that the median effective concentration (EC50) was 1.13mM, as depicted in Figure 4A. Consequently, a concentration of 1mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was selected for subsequent experimental procedures. Subsequently, the expression levels of 14 hub genes were quantified through qPCR. Compared to the control group, three hub genes (LCN2, HILPDA, and HBA1) exhibited increased expression, while five hub genes (SLC2A3, SCD, FADS2, DDIT4, and PROM2) showed decreased expression in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs (Figure 4B~O). Notably, the aberrant expressions of HBA1, DDIT4, PROM2, and SLC2A3 align with the sequencing results. However, no significant differences were observed in the expression levels of six hub genes (AQP3, AQP5, GDF15, SLC1A5, ZFP36, and DUSP1) between the control and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated groups. These findings highlight the pivotal role of HBA1-mediated ferroptosis in the pathogenesis of POAG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Potential small-molecule compounds screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify potential small-molecule compounds for the treatment of POAG, we uploaded ferroptosis-related DEGs to the Connectivity Map small-molecule drug database for annotation of chemical compounds and mechanisms of action. The top 10 potential small-molecule drugs identified were LDN-193189, BX-795, everolimus, ingenol, XMD-892, cycloheximide, digoxin, proscillaridin, calmidazolium, and digitoxin. Additionally, eight mechanisms of action (MOA) were identified: serine/threonine kinase inhibitor, I\u0026kappa;B kinase (IKK) inhibitor, mammalian target of rapamycin (mTOR) inhibitor, protein kinase C (PKC) activator, mitogen-activated protein (MAP) kinase inhibitor, protein synthesis inhibitor, calcium channel blocker, and ATPase inhibitor, as shown in Figure 5A. These results suggest that digoxin and other ATPase inhibitors may exhibit therapeutic potential in POAG. Furthermore, we conducted molecular docking between digoxin and proteins encoded by eight differentially expressed hub genes in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs (Figure 5B). Based on structure-based blind docking, the Vina score results indicated that digoxin is more likely to regulate ferroptosis in HTMCs by targeting HBA1, SCD, and SLC2A3 (Vina score \u0026lt; -10), as detailed in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 CurPocket between digoxin and corresponding target in CB-dock2\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eLigand\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVina score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCavitym volume(A\u0026sup3;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCenter (x, y, z)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDocking size (x, y, z)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDDIT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"8\"\u003e\n \u003cp\u003eDigoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-2, 9, -9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFADS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-1, 7, -1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHBA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67, 66, 61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHILPDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0, -1, -3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSLC2A3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1814\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-22, -5, -11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePROM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-55, 2, 38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24, 65, 45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLCN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-9.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41, 84, 45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33, 33, 33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\n\u003cp\u003e\u003cstrong\u003e5. Digoxin protect HTMCs from ferroptosis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the potential therapeutic benefits of digoxin in treating HTMCs ferroptosis, various concentrations of digoxin were utilized to treat HTMCs exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Notably, the enhanced LDH release into the supernatant, a hallmark of cell death induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, was inhibited by 0.1 nM and 1 nM digoxin (Figure 6A). Moreover, digoxin reversed the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced increases in MDA and iron ions, as well as the decrease in GPX4 levels in the cell culture medium (Figure 6B, C, D). While statistical analysis did not reveal significant effects of digoxin on GSH content, the trends in GSH level changes were consistent with expectations (Figure 6E). Western blot analysis further demonstrated that digoxin reversed the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced downregulation of SLC2A3 and SCD, as well as the upregulation of HBA1 (Figure 6F). Additionally, digoxin suppressed the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced upregulation of \u0026alpha;-SMA, which is indicative of extensive ECM remodeling. These findings provide evidence that digoxin may protect HTMCs from ferroptosis by targeting SLC2A3, SCD, and HBA1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. IOP lowering effect of digoxin for POAG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effect of digoxin on IOP reduction in POAG in vivo, COH rat model was established as previously described, with IOP stabilized for a month. Subsequently, COH rats received twice-daily topical administrations of either 1 mM digoxin or vehicle for an additional month (Figure 7A). As anticipated, digoxin significantly decreased IOP in COH eyes compared to the vehicle-treated group (Figure 7B). Furthermore, digoxin mitigated the elevation of MDA levels in the aqueous humor of COH eyes (Figure 7C) and enhanced the reduced GPX4 levels in the same (Figure 7D). Histological staining of the eyeballs revealed that digoxin inhibited the COH-induced upregulation of 4-HNE in the TM tissue (Figure 8). Additionally, digoxin reversed the COH-induced downregulation of SLC2A3 and SCD, as well as the upregulation of HBA1, while also inhibiting COH-induced ECM remodeling, as indicated by \u0026alpha;-SMA and COL-1 expression. These findings suggest that digoxin reduces IOP by inhibiting ferroptosis in the trabecular meshwork.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we conducted an exhaustive analysis of the clinical manifestations of ferroptosis patterns in Primary Open-Angle Glaucoma (POAG). Despite accumulating evidence pointing to ferroptosis as a pivotal factor in glaucoma pathogenesis, the precise mechanism underlying ferroptosis-induced trabecular meshwork injury remains elusive. Our GO and KEGG analysis revealed that ferroptosis is triggered and executed through hypoxia, iron ion binding, and carbon metabolism pathways, aligning with prior research findings. Notably, we observed significant enrichment in the responses to glucocorticoid and water transport, which are congruent with the reported pathogenic mechanisms of POAG. Our study unveiled several genes that mediate ferroptosis in POAG, including SLC2A3, HBA1, and SCD. Notably, SLC2A3 has been recognized as a crucial biomarker of ferroptosis in various diseases [23, 24]. It primarily functions in glucose transmembrane transport and may contribute to ferroptosis by modulating glycolysis and immune infiltration[25]. Abhishek\u0026apos;s research highlighted the 12p13.3 CNV locus overlapping the SLC2A3 gene as a vital determinant for IOP control[26]. Furthermore, Xuan\u0026apos;s study demonstrated that inhibiting FADS2 disrupts lipid peroxidation, leads to mitochondrial dysfunction, and triggers ferroptosis by directly downregulating GPX4[27]. Additionally, HBA1 regulates ferroptosis through GSH-related pathways and influences the transport of p-pyruvate and p-serine[28]. Intriguingly, HbA1c levels exhibit a positive correlation with IOP in patients with non-proliferative diabetic retinopathy[29]. These findings provide valuable insights into the complex interplay between ferroptosis and POAG, suggesting potential therapeutic targets for future interventions.\u003c/p\u003e\n\u003cp\u003eHBA1 stands out as a distinctive gene in our study, displaying a notable lack of direct interactions while exhibiting a robust correlation in expression with other key genes. This gene demonstrated the highest importance score and significant diagnostic potential for POAG. Remarkably, HBA1 is the sole gene that consistently appeared in sequencing results from TM tissues, PCR results from HTMCs, and histological sections of a COH rat model. Despite these promising findings, the link between these hub genes, ferroptosis, and the underlying pathophysiological mechanisms of POAG remains unclear due to a lack of sufficient evidence. Our data strongly indicate that these hub genes, including HBA1, hold great promise for diagnosing POAG. However, further clinical and basic research is imperative to validate their pivotal role in the disease process. By delving deeper into the mechanisms by which these genes influence ferroptosis and POAG pathogenesis, we may uncover novel therapeutic targets and improve our understanding of this blinding condition.\u003c/p\u003e\n\u003cp\u003eFurthermore, potential small-molecule compounds for the treatment of POAG have been identified, offering a theoretical foundation for the development of therapeutic strategies targeting TM injury. Currently, the effective treatment of glaucoma faces numerous challenges, with most anti-glaucoma medications focusing on lowering IOP by either increasing aqueous humor outflow or reducing its production through the uveoscleral outflow pathway. Traditional anti-glaucoma medications suffer from issues such as limited potency, poor adherence to long-term use, ocular surface damage, surgical failure, and other adverse reactions[30, 31]. Prostaglandin analogs targeting extracellular matrix remodeling to increase aqueous humor outflow through uveoscleral pathway are currently the most widely used IOP lowering drugs[32]. Another relatively new type of anti-glaucoma medication, Rho kinase inhibitor, reduces IOP by directly targeting traditional outflow pathway and simultaneously relaxing trabecular meshwork cells[33]. Since the drainage of aqueous humor through the TM channel accounts for 50% to 75% of the total outflow, developing medications that target TM injury to promote increased aqueous humor outflow is currently the most promising approach for glaucoma treatment. In this study, we screened eight pharmacological and genetic inhibitors of ferroptosis as potential anti-glaucoma medications. Notably, three of these inhibitors are Na+,K+-ATPase inhibitors: digoxin, proscillaridin, and digitoxin. Previous studies have shown that iron overload increases Na,K-ATPase activity in erythrocyte plasma membranes[34]. Na,K-ATPase can interact with Parkin which prevents Parkin translocating to the mitochondria and inactivates mitophagy, leading to ferroptosis[35]. On the other hand, Na,K-ATPase also interacts with SLC7A11 and inhibits ferroptosis in Parkinson\u0026apos;s disease. So the relationships between Na,K-ATPase and ferroptosis are complicated and varies from different cell types and disease. Our results suggested a therapeutic role of digoxin in POAG. Consistent with our study, Adriana found that various digoxin derivatives exhibited great IOP lowering effects in animal ocular hypertension model[36]. It is supposed that the mechanism may be associated with the ability of digoxin to inhibit the production of aqueous humor in the ciliary body. In this study, we discovered the role of digoxin in TM protection. However, narrow therapeutic window and corneal endothelial toxicity limits digoxin application in ocular disease[37]. IKK inhibitor BX-795 has the potential to protect RGCs in glaucoma, while no research has shown its correlation with IOP[38]. Protein synthesis inhibitor cycloheximide was reported to reverse glucocorticoid-induced responsiveness in HTMCs, such as up-regulated MYOC[39, 40]. Several calcium channel blockers, such as verapamil and diltiazem, can significantly reduce IOP and improve ophthalmic blood flow[41, 42]. These novel small-molecule compounds are expected to be anticipated options for novel therapeutic strategies of glaucoma. While it still requires more basic experiments and clinical cohorts to further validate the therapeutic effect of these medications.\u003c/p\u003e\n\u003cp\u003eThis study provides clarity on the pivotal role of ferroptosis-related gene signatures in the underlying mechanisms of POAG. It presents a novel viewpoint regarding the clinical manifestations and ramifications of ferroptosis in glaucoma, thereby establishing a robust theoretical framework for the prevention and early identification of this condition. Additionally, the potential for developing tailored small molecule drugs targeting ferroptosis-related pathways holds promise for groundbreaking therapeutic approaches to glaucoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China [grant numbers 82401262 to Xiaoyu Zhou, 82471079 to Xuanchu Duan]; Hunan Engineering Research Center for Glaucoma with Artificial Intelligence in Diagnosis and Application of New Materials [grant numbers 2023TP2225 to Xuanchu Duan]; the Natural Science Foundation of Hunan Province, China [grant numbers 2023JJ40004 to Xiaoyu Zhou, 2023JJ70014 to Xuanchu Duan]; the Natural Science Foundation of Changsha [grant numbers kq2403205 to Jiahao Xu]; and the Science and Technology Foundation of Aier Eye Hospital Group, China [grant numbers AMF2406D02 to JiahaoXu].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoyu Zhou conducted study design, experiments, data analysis, and manuscript writing. Xinyue Zhang and Ping Wu provided advice and helped to conduct animal experiments. Jiahao Xu conducted qPCR experiments. Xuanchu Duan revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStein JD, Khawaja AP, Weizer JS (2021) Glaucoma in Adults-Screening, Diagnosis, and Management: A Review. JAMA 325(2):164\u0026ndash;174\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuffault J, Labbe A, Hamard P, Brignole-Baudouin F, Baudouin C (2020) The trabecular meshwork: Structure, function and clinical implications. A review of the literature. J Fr Ophtalmol 43(7):e217\u0026ndash;e230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaceres-Velez PR, Hui F, Hercus J, Bui B, Jusuf PR (2022) Restoring the oxidative balance in age-related diseases - An approach in glaucoma. 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Redox Biol 57:102509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Allingham RR, Qin X, Layfield D, Dellinger AE, Gibson J, Wheeler J, Ashley-Koch AE, Stamer WD, Hauser MA (2013) Gene expression profile in human trabecular meshwork from patients with primary open-angle glaucoma. Invest Ophthalmol Vis Sci 54(9):6382\u0026ndash;6389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrison JC, Moore CG, Deppmeier LM, Gold BG, Meshul CK, Johnson EC (1997) A rat model of chronic pressure-induced optic nerve damage. Exp Eye Res 64(1):85\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Li C, Qin Y, Zhang G, Zhao B, Wang Z, Huang Y, Yang Y (2021) A Novel Prognostic Model Based on Ferroptosis-Related Gene Signature for Bladder Cancer. 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Eur J Pharmacol 739:96\u0026ndash;105\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Primary open-angle glaucoma, Ferroptosis, Small-molecule drugs","lastPublishedDoi":"10.21203/rs.3.rs-5869894/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5869894/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecent studies have found that ferroptosis may be involved in the process of trabecular meshwork injury in glaucoma. This study aims to reveal ferroptosis-related gene signature in primary open-angle glaucoma (POAG) and identify small molecule drugs\u0026nbsp;as new direction of\u0026nbsp;therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis-related indicators in POAG patients and chronic ocular hypertension (COH) rats were detected by ELISA kits. The dataset\u0026nbsp;(GSE27276) from\u0026nbsp;GEO database and ferroptosis-related genes from\u0026nbsp;FerrDb were\u0026nbsp;downloaded for analysis. Small molecule drugs targeting ferroptosis-related signature components were predicted via CMap database and CB-Dock2. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced human trabecular meshwork cells (HTMCs) oxidative stress model was constructed to validate the expression of hub genes and efficacy of drugs. Digoxin was made into eye drops to verify its intraocular pressure (IOP) lowering effect in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis levels were enhanced in POAG patients and COH eyes of rats. A total of 14 ferroptosis-related differentially expressed genes were identified. PPI analysis and in vitro experiments showed HBA1, SLC2A3 and SCD played an important role in ferroptosis-mediated POAG. CMap and molecular docking indicated that ATPase inhibitors digoxin might be considered as potential therapeutic drugs for POAG. Digoxin administration alleviated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HTMCs ferroptosis and lowered IOP of COH eyes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study clarified the ferroptosis-related gene signature in the pathogenesis of POAG, which provide a theoretical basis for the prevention and early diagnosis of POAG. Translation of specific small molecule drugs will propose new ideas for therapy of POAG.\u003c/p\u003e","manuscriptTitle":"Ferroptosis-mediated primary open-angle glaucoma: Insight from gene signature and identification of potential small-molecule drugs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-22 08:23:30","doi":"10.21203/rs.3.rs-5869894/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1fd87ff9-5e07-4f3e-a0da-7fb81e604725","owner":[],"postedDate":"January 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-24T09:54:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-22 08:23:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5869894","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5869894","identity":"rs-5869894","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}