Astaxanthin attenuates H2O2-induced oxidative injury in R28 retinal precursor cells via the Keap1-Nrf2/HO-1 signaling pathway

Objective To investigate the protective effects of astaxanthin (AST) against H 2 O 2 -induced oxidative injury in R28 retinal precursor cells using network pharmacology and in vitro experiments, and to evaluate the involvement of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway. Methods Network pharmacology was used to identify shared targets of AST and glaucoma, construct a protein-protein interaction (PPI) network, and perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and molecular docking. An oxidative stress model was established in rat R28 retinal precursor cells using hydrogen peroxide (H 2 O 2 ). Cells were assigned to control, H 2 O 2 , AST + H 2 O 2 , AST + H 2 O 2 + ML385, ML385-alone, and H 2 O 2 + ML385 groups; an AST-alone group was included in selected experiments. Cell viability, reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG), protein carbonyls, mitochondrial membrane potential, Nrf2 nuclear translocation, inflammation-related genes and proteins, apoptosis-related proteins, and expression of Keap1-Nrf2/HO-1/NQO1 pathway molecules were assessed. Reverse validation was performed using the Nrf2 inhibitor ML385. Results Network pharmacology indicated that the mechanisms by which AST may act in glaucoma involve oxidative stress, inflammation, and apoptosis. H 2 O 2 treatment reduced R28 cell viability, increased ROS, MDA, 8-OHdG, and protein carbonyl levels, decreased SOD activity, and induced inflammation, loss of mitochondrial membrane potential, and an imbalance in apoptosis-related proteins. AST treatment reversed these changes, downregulated Keap1, upregulated Nrf2, HO-1, and NQO1, and promoted Nrf2 nuclear translocation. ML385 partially attenuated the antioxidant, anti-inflammatory, and anti-apoptotic effects of AST. Conclusion AST may alleviate H 2 O 2 -induced oxidative injury, inflammation, and apoptosis in R28 cells by activating the Keap1-Nrf2/HO-1 antioxidant pathway. These findings provide experimental evidence for the potential protective role of AST in glaucoma-related oxidative stress, although its use as an adjunctive neuroprotective strategy for glaucoma requires further validation in animal models that more closely recapitulate the clinical pathology.