Targeting KEAP1/NRF2 interaction with oleuropein ameliorates atherosclerosis by inhibiting macrophage ferroptosis

Atherosclerosis (AS) is a chronic inflammatory arterial disease. Oleuropein (OL), extracted from olive leaves, has demonstrated broad cardioprotective properties. However, the effects of OL on AS remain to be fully elucidated. ox-LDL incubated macrophages were used to imitate macrophage damage within plaques in vitro. Recombinant adeno-associated virus serotype 9 (AAV9) encoding a short hairpin RNA targeting NRF2 (AAV9-shNRF2) and AAV-KEAP1-R451S were administered to OL-treated ApoE^-/- mice. The molecular mechanisms were explored through immunoprecipitation and Chip-qPCR, seahorse assays, and proteomics analysis. OL administration significantly attenuated the progression of AS and enhanced plaque stability, as evidenced by reduced plaque area and lipid deposition, along with increased collagen content. Proteomics revealed that OL suppressed ferroptosis by upregulating GPX4/xCT level, improving mitochondrial function, alleviating oxidative stress and suppressing lipid peroxidation in macrophages treated by ox-LDL and atherosclerotic plaques. Moreover, OL enhanced NRF2 activation and nuclear translocation, while NRF2 inhibition or knockdown abolished the protective effect of OL on AS and macrophage ferroptosis. Mechanistically, molecular dynamics analysis suggested that OL may bind to the Arg415 site of KEAP1 competitively, promoting the separation and nuclear translocation of NRF2 from KEAP1. Additionally, transfection with the Arg415 mutant plasmid (KEAP1-R415S) abolished the antioxidative and anti-ferroptosis effects of OL in macrophages induced by ox-LDL. Moreover, the protective effects of OL against atherosclerosis and macrophage ferroptosis were significantly attenuated in AAV-KEAP1-R415S mutant ApoE^-/- mice. OL attenuated AS progression and macrophage ferroptosis by facilitating NRF2 nuclear translocation and activation via binding to the Arg415 residue of KEAP1 competitively. These findings identified a novel insight into potential therapeutic strategies for the treatment of AS.