Keap1 as Target of Genistein on Nrf2 Signaling Pathway Antagonizing A beta induced Oxidative Damage of Cerebrovascular Endothelial Cells

Background: beta-amyloid peptides (A beta) induced oxidative damage contributes to the pathogenesis of neurodegenerative diseases, and the cerebrovascular system is more vulnerable to oxidative stress. Our earlier study showed a clue that Genistein (Gen) might activate the Nf-E2 related factor 2 (Nrf2) pathway to protect cerebrovascular cells from oxidative damage induced by A beta, but the specific mechanisms and regulation targets are unclear. Objective: In this study, the anti-oxidative effects and the possible targets of Gen on regulating the Nrf2 pathway in bEnd.3 cells were investigated. Cells were divided into control, A beta 25-35, Gen, and Gen+A beta 25 -35 groups. Methods: Cell viability, levels of malondialdehyde (MDA), Superoxide Dismutase (SOD) activity, and nitrotyrosine were evaluated. Moreover, mRNA and/or protein expressions of Nrf2 and kelchlike ECH-associated protein 1 (Keap1) were measured. Then we transfected Keap1 over-expressed plasmid into bEnd.3 cells and measured the protein expressions of Nrf2 pathway related factors. Results: Data showed that Gen could inhibit the over-production of MDA and nitrotyrosine and activate SOD activity. Furthermore, we discovered that Gen could up-regulate Nrf2 mRNA and protein expression while down-regulating Keap1 protein expression. The Keap1 over-expressed plasmid study revealed that the up-regulation of Nrf2 protein expression induced by Gen pretreatment could be blocked by transfection of Keap1 over-expressed plasmid, and the same results were observed in Nrf2 downstream factors. Conclusion: Gen could alleviate the cerebrovascular cells' oxidative damage induced by A beta 25-35 by regulating the Nrf2 pathway, and Keap1 might be one of the targets of Gen in activating the Nrf2 pathway.