Mitophagy dysfunction is a critical contributor to retinal pigment epithelial (RPE) cell damage during the progression of retinal degenerative diseases, including age-related macular degeneration (AMD). In this study, we investigated the effects of paeoniflorin (PF) on mitophagy in RPE cells, with a particular focus on the CUL3/LKB1/AMPK/ULK1 signaling pathway. ARPE-19 cells were treated with different concentrations of PF to evaluate cytotoxicity, and its protective effects were further examined in H₂O₂-induced oxidative stress models in vitro and in sodium iodate (NaIO₃)-induced RPE injury models in vivo. Protein levels of CUL3, apoptosis-related factors, mitophagy markers, and components of the LKB1/AMPK/ULK1 pathway were assessed by western blotting, and mitophagy was visualized using MitoTracker labeling. Cycloheximide (CHX) and coimmunoprecipitation (Co-IP) assays were performed to analyze the interaction between CUL3 and LKB1. PF treatment enhanced mitophagy in H₂O₂-stimulated ARPE-19 cells, whereas Parkin knockdown markedly attenuated this effect. In oxidatively damaged cells, PF promoted AMPK and ULK1 phosphorylation, increased mitophagy-associated protein expression, and alleviated mitochondrial dysfunction; these protective effects were abolished by pharmacological inhibition of AMPK or ULK1. In addition, CUL3 overexpression significantly attenuated PF-induced mitophagy activation and reduced PF-associated phosphorylation of LKB1, AMPK, and ULK1. Mechanistically, PF downregulated CUL3 expression, while CUL3 promoted the ubiquitination and degradation of LKB1. Silencing CUL3 induced mitophagy in H₂O₂-treated cells, whereas concurrent knockdown of CUL3 and LKB1 abolished this effect. In vivo, PF mitigated RPE cell loss, enhanced mitophagy, and activated the CUL3/LKB1/AMPK/ULK1 signaling pathway in the retinal tissues of NaIO₃-induced mice. Collectively, these findings indicate that PF protects against RPE injury in an NaIO₃-induced AMD-like model by downregulating CUL3 expression and activating LKB1/AMPK/ULK1-mediated mitophagy.
