College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China.
Biomed Res Int. 2022 Aug 18;2022:8562795. doi: 10.1155/2022/8562795. eCollection 2022.
Nrf2 plays a key role in the antioxidant system, and many antioxidants can activate the Nrf2/ARE signaling pathway and alleviate oxidative stress. However, the underlying mechanisms of antioxidants, such as proanthocyanidin- (PC-) induced Nrf2 activation, remain poorly understood. In this study, PC was used on MODE-K cells at different concentrations (0, 1, 2.5, and 5 g/mL) and different times (0, 3, 6, 12, and 24 h); then, immunoprecipitation, immunofluorescence, and Western blotting were performed to test Nrf2, Bach1, Keap1, HO-1, and NQO1 protein expressions in MODE-K cells. Results showed that PC increased Nrf2, HO-1, and NQO1 protein expressions, decreased Keap1 and Bach1 protein expressions, and enhanced ARE gene activity. PC also decreased the ubiquitinated degradation of the Nrf2 protein, increased Nrf2 protein stability, and increased Nrf2 protein expression by inhibiting Keap1-dependent Nrf2 protein degradation, promoted Nrf2 entry into the nucleus, competed with Bach1, and activated ARE elements, which in turn initiated the Nrf2/ARE signaling pathway. Thus, we conclude that PC activates the Nrf2/ARE signaling pathway in intestinal epithelial cells by inhibiting the ubiquitinated degradation of Nrf2, increasing Nrf2 protein stability and expression, and then regulating key antioxidant enzymes such as HO-1 and NQO1 to initiate cytoprotective effects.
Nrf2 在抗氧化系统中发挥关键作用，许多抗氧化剂可以激活 Nrf2/ARE 信号通路并减轻氧化应激。然而，抗氧化剂的潜在机制，如原花青素（PC-）诱导的 Nrf2 激活，仍知之甚少。在这项研究中，使用不同浓度（0、1、2.5 和 5 g/mL）和不同时间（0、3、6、12 和 24 h）的 PC 处理 MODE-K 细胞；然后，进行免疫沉淀、免疫荧光和 Western blot 以检测 MODE-K 细胞中的 Nrf2、Bach1、Keap1、HO-1 和 NQO1 蛋白表达。结果表明，PC 增加了 Nrf2、HO-1 和 NQO1 蛋白表达，降低了 Keap1 和 Bach1 蛋白表达，并增强了 ARE 基因活性。PC 还通过抑制 Keap1 依赖性 Nrf2 蛋白降解，减少了 Nrf2 蛋白的泛素化降解，增加了 Nrf2 蛋白的稳定性，并增加了 Nrf2 蛋白的表达，促进了 Nrf2 进入细胞核，与 Bach1 竞争并激活 ARE 元件，从而启动了 Nrf2/ARE 信号通路。因此，我们得出结论，PC 通过抑制 Nrf2 的泛素化降解、增加 Nrf2 蛋白稳定性和表达，然后调节关键的抗氧化酶，如 HO-1 和 NQO1，来激活肠上皮细胞中的 Nrf2/ARE 信号通路，从而引发细胞保护作用。
