白藜芦醇调节Akt/mTOR通路对大鼠多囊卵巢综合征颗粒细胞自噬的影响机制研究

Effects of resveratrol on granulosa cell autophagy by regulating Akt/mTOR pathway in rats with polycystic ovarian syndrome

目的:探讨白藜芦醇对多囊卵巢综合征大鼠模型颗粒细胞自噬的影响,并探讨其机制。方法:40只雌性SD大鼠幼鼠随机分为2组,即多囊卵巢综合征模型组(n=30)和模型对照组(n=10)。将经免疫细胞化学法鉴定证实的多囊卵巢综合征(polycystic ovarian syndrome,PCOS)颗粒细胞进行体外培养。用不同浓度的白藜芦醇处理,CCK-8观察细胞的增殖率变化情况;电镜观察颗粒细胞内自噬水平的变化;Western blot检测细胞内自噬相关蛋白轻链3Ⅰ(light chain 3Ⅰ,LC3Ⅰ)、自噬相关蛋白轻链3Ⅱ(light chain 3Ⅱ,LC3Ⅱ)、Beclin1的表达,以及总(磷酸化)蛋白激酶B[total(phosphorated)protein kinase B,T(p)-Akt]、总(磷酸化)雷帕霉素靶蛋白[total(phosphorated)mammalian target of rapamyoin,T(p)-mTOR]的变化,并检测Akt抑制剂LY294002作用后,细胞内上述蛋白的表达情况。结果:免疫细胞化学结果显示,与模型对照组比较,模型组卵巢颗粒细胞中卵泡刺激素(follicle-stimulating hormone,FSH)表达明显增强。这提示模型构建成功且分离成功。CCK-8结果表明,白藜芦醇能够提高颗粒细胞的增殖率,有浓度-时间依赖性;并降低细胞内的自噬水平;而Western blot结果显示,白藜芦醇可以减低细胞内LC3Ⅰ、LC3Ⅱ和Beclin1的表达,提高p-Akt和p-mTOR的蛋白表达;LY249002作用后,细胞内的LC3Ⅰ、LC3Ⅱ和Beclin1表达明显增加,同时p-Akt和p-mTOR的表达则明显降低。结论:白藜芦醇能够明显降低多囊卵巢综合征卵巢颗粒细胞内自噬水平并影响细胞的增殖率,其机制可能与白藜芦醇上调Akt/mTOR通路密切相关。

Objective:To investigate the effect of the plant extract resveratrol on autophagy of granulosa cells in the rats with polycystic ovarian syndrome(PCOS)and to explore its mechanism of action.Methods:Forty female SD(Sprague Dawley)rats were randomized into two groups:PCOS model group(n=30)and model control group(n=10).PCOS granulosa cells identified and confirmed by immunocytochemistry were cultured in vitro,and dealt with different concentrations of resveratrol,and the changes of cell proliferation rate were observed by CCK-8.Electron microscope was used for the changes of autophagy level in granular cells.Western blot was used to detect the expression of light chain 3Ⅰ(LC3Ⅰ),light chain 3Ⅱ(LC3Ⅱ),Beclin1,total(phosphorylated)protein kinase B[T(p)-Akt],and total(phosphorylated)mammalian target of rapamyoin[T(p)-mTOR],and the expression of these proteins in cells after the action of Akt inhibitor LY294002 was detected.Results:Compared with the model control group,the immunocytochemical results showed that the expression of follicle-stimulating hormone(FSH)in ovarian granulosa cells in the model group was significantly enhanced,which indicated that the model was successfully constructed and isolated.The CCK-8 results showed that resveratrol could improve the proliferation rate of granulosa cells,with concentration-time dependence.The Western blot results showed that resveratrol could decrease the expression of LC3Ⅰ,LC3Ⅱand Beclin1,but increase the protein expression of p-Akt and p-m TOR.After LY249002 treatment,the expression of LC3 I,LC3Ⅱand Beclin1 increased significantly,while the expression of p-Akt and p-m TOR decreased significantly.Conclusion:Resveratrol can significantly reduce the autophagy level of ovarian granulosa cells in PCOS and affect the cell proliferation rate,and its mechanism may be closely related to the upregulation of Akt/mTOR.