Ranitidine protects Müller cells against ferroptosis in diabetic retinopathy by regulating the AKT1/GSK3β pathway

PURPOSE: This study was designed to investigate the primary targets and possible mechanisms of ranitidine (Ra) against diabetic retinopathy (DR). METHODS: Single-cell sequencing technology and the SPIED3 platform were employed to characterize key genes in retinal Müller cells (RMCs) of diabetic mice and identify potential small-molecule compounds separately. The effects of small-molecule compounds on the cell viability and proliferative capacity of mouse retinal Müller cells (rMC-1) cultured in high-glucose (HG) were evaluated using the cell counting kit-8 (cck-8) and 5-ethyl-2-deoxyuridine (Edu) assay. Glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), and Fe2+ were identified as indicators of ferroptosis. Then, network pharmacology was used to predict specific targets for Ra. Western blotting was used to identify ferroptosis-related proteins, including glutathione peroxidase 4 (GPX4), cystine/glutamate transporter (xCT), serine/threonine-protein kinase AKT1, and glycogen synthase kinase-3β (GSK3β). RESULTS: The predicted results suggested that the potential mechanism of RMCs damage in diabetic mice is associated with ferroptosis. The cck-8 results indicated Ra played a regulatory role in HG-induced rMC-1 by enhancing cell viability. Besides, Edu results showed that Ra promoted the proliferation of rMC-1 cells. Network pharmacological analyses predicted a potential mechanism of Ra effect in HG-induced rMC-1, mainly associated with the AKT1 and GSK3β genes. Phenotypically, Ra elevated intracellular GSH levels, while reducing MDA, Fe²⁺, and ROS concentrations. Mechanistically, Ra increased xCT and GPX4 expression through the promotion of AKT1/GSK3β phosphorylation, thereby alleviating ferroptosis in HG-induced rMC-1 cells. CONCLUSIONS: The study highlighted that the mechanism of DR is closely associated with ferroptosis and demonstrated that Ra inhibits HG-induced ferroptosis of rMC-1 cells by regulating the AKT1/GSK3β signaling pathway, thereby providing a theoretical basis for using Ra in managing DR.