Department of Nutrition and Food Hygiene, School of Public Health, Capital Medical University, Beijing 100069, China.
Wanke School of Public Health Tsinghua University, Beijing, China.
Curr Neurovasc Res. 2022;19(1):73-82. doi: 10.2174/1567202619666220406100320.
β-amyloid peptides (Aβ) 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 Aβ, but the specific mechanisms and regulation targets are unclear.
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, Aβ25-35, Gen, and Gen+Aβ25-35 groups.
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.
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.
Gen could alleviate the cerebrovascular cells' oxidative damage induced by Aβ25-35 by regulating the Nrf2 pathway, and Keap1 might be one of the targets of Gen in activating the Nrf2 pathway.
β-淀粉样肽（Aβ）诱导的氧化损伤导致神经退行性疾病的发病机制，脑血管系统更容易受到氧化应激的影响。我们之前的研究表明，染料木黄酮（Gen）可能通过激活核因子 E2 相关因子 2（Nrf2）通路来保护脑血管细胞免受 Aβ诱导的氧化损伤，但具体的机制和调节靶点尚不清楚。
本研究旨在探讨 Gen 对 bEnd.3 细胞 Nrf2 通路的抗氧化作用及其可能的调节靶点。将细胞分为对照组、Aβ25-35 组、Gen 组和 Gen+Aβ25-35 组。
检测细胞活力、丙二醛（MDA）水平、超氧化物歧化酶（SOD）活性和硝基酪氨酸。此外，还检测了 Nrf2 和 Kelch 样 ECH 相关蛋白 1（Keap1）的 mRNA 和/或蛋白表达。然后我们将 Keap1 过表达质粒转染到 bEnd.3 细胞中，检测 Nrf2 通路相关因子的蛋白表达。
结果表明，Gen 可抑制 MDA 和硝基酪氨酸的过度产生，激活 SOD 活性。此外，我们发现 Gen 可以上调 Nrf2 mRNA 和蛋白表达，同时下调 Keap1 蛋白表达。Keap1 过表达质粒研究表明，Gen 预处理上调 Nrf2 蛋白表达可被 Keap1 过表达质粒转染阻断，Nrf2 下游因子也出现同样结果。
Gen 可通过调节 Nrf2 通路减轻 Aβ25-35 诱导的脑血管细胞氧化损伤，Keap1 可能是 Gen 激活 Nrf2 通路的靶点之一。
