Purpose: Diabetic retinopathy (DR) is a common and serious eye complication in patients with diabetes. With DR progresses, destruction of tight junctions (TJs) in RPE cells leads to irreversible visual impairment. Gypenoside XLIX (Gyp XLIX) is a dammarane-type glycoside, which can suppress inflammation and oxidative stress. This study aimed to investigate and verify the mechanism underlying the regulatory effects of Gyp XLIX in the treatment of DR.

Materials and methods: We combined bioinformatics and network pharmacology to identify core targets of Gyp XLIX in DR treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed to elucidate the biological functions of the composite targets. The binding between Gyp XLIX and the core targets was verified with molecular docking. Furthermore, mice with diabetes mellitus (DM) and high glucose (HG)-stimulated ARPE-19 cells were treated with Gyp XLIX. Its impact on TJ function in RPE cells and ferroptosis was evaluated via western blotting, immunofluorescence staining, and assays for iron content, lipid peroxidation, and Glutathione (GSH) levels. Prostaglandin-endoperoxide synthase 2 (PTGS2) was overexpressed to investigate the mechanism underlying the therapeutic effect of Gyp XLIX on DR.

Results: A total of 66 common targets of Gyp XLIX and DR were identified. Among these targets, ALB, VEGFA, JUN, ESR1, PTGS2, STAT3, MMP9, HSP90AA1, BCL2L1 and AR were identified through protein–protein interaction (PPI) network analysis. Enrichment analysis revealed the involvement of the core genes in various biological processes. Molecular docking showed that Gyp XLIX could stably bind to the core targets except JUN and AR. Western blotting and immunofluorescence staining revealed that Gyp XLIX alleviated TJs disruption in RPE cells. In addition, iron, lipid peroxidation and GSH assays revealed that Gyp XLIX inhibited ferroptosis in both mice with DM and HG-stimulated ARPE-19 cells. Overexpression of PTGS2 partially reversed the protective effects of Gyp XLIX, indicating that Gyp XLIX alleviated DR by targeting PTGS2.

Conclusions: This study revealed Gyp XLIX inhibited ferroptosis and prevented the disruption of TJ function in RPE cells by targeting PTGS2, thereby exerting protective effects in DR.