Proteomic insights into nepetin-mediated protection against oxidative stress in ARPE-19 cells

Oxidative stress is one of the crucial factors associated with the pathogenesis of age-related macular degeneration (AMD), particularly the degeneration of retinal pigment epithelium (RPE) cells. In this study, we identified nepetin, a natural flavonoid compound, as a potential inhibitor of hydrogen peroxide (H₂O₂)-induced ARPE-19 cell death. Pretreatment of nepetin significantly reduced intracellular generation of reactive oxygen species (ROS). Quantitative proteomics was applied to explore the underlying molecular response, revealing that 77 proteins were up-regulated, and 198 proteins were down-regulated significantly after nepetin treatment. Gene ontology (GO) analysis and the protein-protein interaction (PPI) network analysis showed that heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1), Sequestosome 1 (SQSTM1)/p62, and glucose-regulated protein 78 (GRP78) were associated with nepetin-mediated antioxidative responses. Western blotting confirmed the altered expression of these key proteins, with HO-1, p62, and GRP78 being upregulated and KEAP1 being downregulated. Immunofluorescence further showed nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, suggesting the involvement of Nrf2-related antioxidant signaling in nepetin-treated ARPE-19 cells. Although direct causal interactions were not established, the proteomic and bioinformatic analyses provide correlative and suggestive evidence that nepetin modulates key proteins within the oxidative stress response network. Based on our previous research and the current study, nepetin exhibits both anti-inflammatory and antioxidative properties in RPE cells, and may have potential implications for the prophylaxis and treatment of AMD, particularly dry AMD.