A dual Keap1 and p47phox inhibitor Ginsenoside Rb1 ameliorates high glucose/ox-LDL-induced endothelial cell injury and atherosclerosis

Oxidative stress is a vital contributor to the development and progression of diabetes-accelerated atherosclerosis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a well-known molecule that participates in cellular defense against oxidative stress. Utilizing luciferase reporter assay from 379 Natural Products, we reported here that Ginsenoside Rb1 played a dual role in inhibiting Kelch-like ECH-associated protein 1 (Keap1) and p47^phox luciferase reporter activities. In endothelial cells (ECs), Rb1 pretreatment enhanced cell viability, reduced oxidative stress, inflammation, endothelial-mesenchymal transition (EndMT), and Apoptosis, as well as ameliorated mitochondrial quality following oxidized low-density lipoprotein (ox-LDL) plus high glucose (HG) challenge. Rb1 directly bound to Keap1 and promoted its ubiquitination and proteasomal degradation dependent on lysine residues (K108, K323, and K551) by recruiting the E3 Ligase synovial Apoptosis inhibitor 1 (SYVN1), leading to Nrf2 dissociation from Keap1, Nrf2 nuclear translocation, Nrf2/PGC-1α complex formation. We further identified that Rb1 could bind to p47^phox and reduce its phosphorylation and membrane translocation, thereby disrupting the assembly of the NOX2 complex. Importantly, Rb1-mediated preservation of cytoplasmic p47^phox stabilized and contributed to Nrf2 activation. Additionally, we revealed that Rb1 reduced aortic atherosclerotic plaque formation along with reductions in oxidative stress and inflammatory response in streptozotocin (STZ)-induced ApoE^-/- mice, but not in ApoE^-/- mice with deficiency of Nrf2 and PGC-1α. Collectively, we demonstrated that Rb1, which directly targeted Keap1 and p47^phox in ECs, may be an attractive candidate for the treatment of atherosclerosis in diabetes.