白藜芦醇调节SIRT1/AMPK通路抑制大鼠多囊卵巢综合征颗粒细胞内自噬的机制研究

Inhibitory effects of resveratrol on autophagy of the granulosa cells in rats with polycystic ovary syndrome through regulating SIRT1/AMPK pathway

目的:探讨植物提取物白藜芦醇对多囊卵巢综合征(polycystic ovary syndrome,PCOS)大鼠模型颗粒细胞内自噬的影响,并探讨其对SIRT1/AMPK通路的调控作用。方法:体外培养PCOS的SD大鼠卵巢颗粒细胞,用不同浓度白藜芦醇(5、10、20、40μmol/L)处理不同时间(24、48、72 h),MTT法观察卵巢颗粒细胞增殖率的变化;电镜观察细胞内自噬水平的变化;Western blot检测细胞内沉默信息调节因子2相关酶1(silent information regulator T1,SIRT1)、单磷酸腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、磷酸化的AMPK(pAMPK)、Beclin1和p62的表达变化,并检测SIRT1特异性抑制剂EX527作用后,细胞内上述蛋白的表达情况。结果:MTT结果显示,白藜芦醇能明显促进颗粒细胞增殖(P<0.05),呈浓度-时间依赖性(P<0.05);电镜结果表明白藜芦醇能降低细胞内的自噬体数量(P<0.05)。Western blot结果显示白藜芦醇可以增强卵巢颗粒细胞内SIRT1和pAMPK的蛋白表达水平(P<0.05),抑制细胞内Beclin1的表达(P<0.05),增加p62的表达;联合SIRT1特异性抑制剂EX527作用后,颗粒细胞内的SIRT1和pAMPK蛋白表达水平明显降低,Beclin1表达明显增加(P<0.05),p62却明显降低(P<0.05)。结论:白藜芦醇能抑制PCOS卵巢颗粒细胞内自噬水平并促进其增殖,这与白藜芦醇激活SITR1/AMPK通路密切相关。

Objective:To investigate the effect of plant extract resveratrol on autophagy of the granulosa cells in rat model with polycystic ovary syndrome and to explore its regulation on SIRT1/AMPK pathway.Methods:Ovarian granulosa cells of Sprague Dawley rats with polycystic ovary syndrome(PCOS)were cultured in vitro and treated with different concentrations of resveratrol(5,10,20,40μmol/L)for different times(24,48,72 h).The proliferation rate of ovarian granulosa cells was observed by MTT method,the changes of intracellular autophagy were observed by electron microscope,and the expression changes of silent information regulator T1(SIRT1),adenosine monophosphate-activated protein kinase(AMPK),phosphorylated AMPK(pAMPK),Beclin1 and p62 were detected by Western blot.Finally,the expression of the above proteins in the cells was detected after the application of EX527,as a specific SIRT1 inhibitor.Results:MTT results showed that resveratrol promoted the proliferation rates of the granulosa cells of PCOS rats(P<0.05),and it was in a concentration-time dependent manner(P<0.05).Electron microscope showed that resveratrol could reduce the number of autophagosome in cells(P<0.05).Western blot results showed that resveratrol could enhance the protein expression levels of SIRT1 and pAMPK in ovarian granulosa cells(P<0.05),inhibit the expression of Beclin1(P<0.05),and increase the expression of p62.After combined with SIRT1 specific inhibitor EX527,the protein expression levels of SIRT1 and pAMPK in granulosa cells decreased significantly,the expression of Beclin1 increased significantly(P<0.05),while p62 decreased significantly(P<0.05).Conclusion:Resveratrol could inhibit the level of autophagy and promote the proliferation of ovarian granulosa cells of the rats with PCOS,which is closely related to the activation of SIRT1/AMPK pathway by resveratrol.