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目的探究白藜芦醇对经典抗氧化信号通路Keap1-Nrf2-ARE中关键蛋白Keap1的调控作用与机制。方法培养MDA-MB-231细胞利用不同浓度的白藜芦醇(5μmol/L和20μmol/L)处理24和48h,Western blot方法检测细胞内Keap1蛋白和其调控转录因子Nrf2的表达变化。结合泛素-蛋白酶体抑制剂MG-132和溶酶体酶抑制剂氯喹的应用,进一步研究白藜芦醇降解Keap1蛋白的可能途径。结果白藜芦醇明显降低MDA-MB-231细胞内Keap1蛋白的表达水平,具有浓度和时间依赖的倾向性;加入MG-132和氯喹并不影响白藜芦醇的作用。结论白藜芦醇能够明显促进Keap1蛋白降解,进而激活Keap1-Nrf2-ARE信号通路,增强细胞抗氧化应激的能力;而导致Keap1蛋白降解的途径有可能不依赖于泛素-蛋白酶体和溶酶体通路,详细机制仍有待进一步研究。
Objective To investigate the regulatory effect and mechanism of resveratrol on the key protein Keap1 in the classical antioxidant signaling pathway Keap1-Nrf2-ARE. Methods MDA-MB-231 cells were cultured in different concentrations of resveratrol (5μmol / L and 20μmol / L) for 24 and 48 hours, and the expression of Keap1 protein and its regulatory transcription factor Nrf2 were detected by Western blot. Based on the application of ubiquitin-proteasome inhibitor MG-132 and lysosomal enzyme inhibitor chloroquine, the possible mechanism of resveratrol degradation of Keap1 protein was further studied. Results Resveratrol significantly reduced the expression of Keap1 protein in MDA-MB-231 cells in a time-and concentration-dependent manner. The addition of MG-132 and chloroquine did not affect the effect of resveratrol. Conclusion Resveratrol can significantly promote the degradation of Keap1 protein and activate Keap1-Nrf2-ARE signaling pathway, which enhances the ability of cells to resist oxidative stress. However, the mechanism of Keap1 degradation may not depend on ubiquitin-proteasome and lysosome Enzyme pathway, the detailed mechanism remains to be further studied.