Keap1-C151-dependent activation of Nrf2 by RS9 protects the retinal pigment epithelium cells and retinal macrovascular endothelial cells against phototoxic oxidative injury

Abstract Ultraviolet (UV) radiation (UR) and excessive light exposure induce profound oxidative injury to the neural retina, compromising both the retinal pigment epithelium (RPE) and retinal microvascular endothelial cells (RMECs). This phototoxic damage is an important driver of retinal degenerative diseases. RS9, a highly efficient Nrf2 (nuclear factor erythroid 2-related factor-2) activator developed from bardoxolone methyl (BARD), possesses potent antioxidant properties. This study investigated the comprehensive protective effects of RS9 against phototoxicity in vitro and in vivo. In human ARPE-19 cells, primary murine RPE cells, and hRMECs, nanomolar concentrations of RS9 potently mitigated UVR-induced viability reduction, proliferation inhibition, and apoptosis. RS9 alleviated UVR-triggered reactive oxygen species (ROS) overproduction, mitochondrial depolarization, and oxidative DNA damage in RPE cells and hRMECs. Mechanistically, RS9 caused Nrf2 protein stabilization, nuclear translocation, and the subsequent expression of cytoprotective and antioxidant genes ( HO1 and NQO1 ) in RPE cells and hRMECs. Genetic depletion of Nrf2 (via shRNA or CRISPR/Cas9) abolished RS9-mediated cytoprotection in RPE cells and hRMECs. Conversely, Keap1 depletion mitigated UVR injury but precluded additional RS9-induced benefits in RPE cells and hRMECs. Furthermore, expression of the Keap1-C151S mutant blocked RS9-induced Nrf2 activation and abolished its protective efficacy in both RPE and endothelial cells. In vivo, intravitreal injection of RS9 in a murine light-damage model significantly ameliorated oxidative injury, preserved RPE layer thickness and visual function, and protected retinal vascular integrity by restoring PECAM-1 expression. Collectively, these findings demonstrate that RS9 effectively engages the Keap1-Nrf2 signaling cascade to provide robust, comprehensive protection to both the outer and inner blood-retinal barriers against phototoxic oxidative injury.