黄芪多糖调控Sirt1/FoxO1通路抑制多囊卵巢综合征大鼠颗粒细胞自噬的研究

Astragalus polysaccharide regulates the Sirt1/FoxO1 pathway and inhibits granulosa cell autophagy in polycystic ovary syndrome rats

目的探究黄芪多糖(APS)调控沉默信息调节因子1/叉头状转录因子O1(Sirt1/FoxO1)通路对多囊卵巢综合征(PCOS)大鼠颗粒细胞自噬的影响。方法大鼠中分离卵巢颗粒细胞并经促卵泡刺激素受体(FSHR)免疫荧光鉴定,且CCK8检测APS对卵巢颗粒细胞增殖的影响。10-5mol/L丙酸睾丸酮、10μmol/L C2-神经酰胺作用大鼠卵巢颗粒细胞24 h诱导PCOS大鼠颗粒细胞自噬模型,CCK8检测细胞增殖情况;透射电镜观察自噬体情况;免疫印迹法检测细胞中Sirt1、Fox O1、微管相关蛋白1轻链3(LC3)蛋白表达情况。结果免疫荧光检测显示,FSHR蛋白在大鼠卵巢颗粒细胞中的平均阳性表达量93.18%>90%,可进行接下来实验。0、100、200、400μg/mL APS处理正常卵巢颗粒细胞,在贴壁0、24 h比较,细胞OD450差异均无统计学意义(P>0.05)。与正常组相比,模型组细胞核附近出现大量自噬小体,双层、单层膜包裹胞质形成封闭、圆形结构,部分自噬小体内膜溶解,自噬小体周围可见单层膜结构包裹着一些被降解的胞浆、类似自噬小体结构;100μg/mL APS组与模型组细胞结构类似,随着APS剂量的升高,自噬小体数量减少,在400μg/m L APS组时细胞正常。正常组、模型组、100、200、400μg/mL APS组在细胞贴壁0 h时,细胞OD450差异无统计学意义(P>0.05)。细胞贴壁24 h,与正常组相比,模型组细胞OD450降低(P<0.05),细胞中Sirt1、Fox O1、LC3Ⅱ/LC3Ⅰ蛋白水平升高(P<0.05);与模型组相比,100、200、400μg/mL APS组细胞OD450升高(P<0.05),细胞中Sirt1、Fox O1、LC3Ⅱ/LC3Ⅰ蛋白水平降低(P<0.05)。结论APS可以抑制PCOS大鼠颗粒细胞自噬,可能是通过抑制自噬通路Sirt1/FoxO1实现的。

Objective To investigate the effect of astragalus polysaccharides(APS)on autophagy of granulosa cells in polycystic ovary syndrome(PCOS)rats by regulating the silent mating type information regulation 2 homolog 1(Sirt1)/forkhead box transcription factor O1(FoxO1)pathway.Methods Ovarian granulosa cells were isolated from rats and identified by follicle stimulating hormone receptor(FSHR)immunofluorescence.CCK8 was used to assess the effect of APS on the proliferation of ovarian granulosa cells.Ovarian granulosa cells were treated with 1×10^(-5) mol/L testosterone propionate and 10μmol/L C2 ceramide for 24 h to induce the autophagy model of PCOS rat granulosa cells.CCK8 was used to measure cell proliferation.Autophagy was observed by transmission electron microscope.Protein expression of Sirt1,FoxO1,and microtubule-associated protein 1 light chain 3(LC3)was detected by Western blot.Results Immunofluorescence showed that average positive expression of FSHR protein in rat ovarian granulosa cells was 93.18%>90%.In 0,100,200,and 400μg/mL APS-treated normal ovarian granulosa cells,there was no significant difference in OD450 after adherence at 0 or 24 h(P>0.05).Compared with the normal group,a large number of autophagy bodies appeared near the nucleus of the model group,and double and single layer membranes wrapped the cytoplasm to form a closed and round structure.Some of the autophagy body inner membrane had dissolved and there was a single membrane structure around the autophagosome,which contained some degraded cytoplasm similar to the autophagosome structure.The cell structure of the 100μg/mL APS group was similar to that of the model group.With the increase of APS dosage,the number of autophagy bodies decreased and the cells were normal in the 400μg/mL APS group.There was no significant difference in OD450 between normal,model,and 100,200,and 400μg/mL APS groups at 0 h of cell adhesion(P>0.05).After 24 h of cell adhesion,compared with the normal group,OD450 was lower in the model group(P<0.05)and the protein levels of Sirt1,FoxO1,and LC3 II/LC3 I were higher(P<0.05).Compared with the model group,OD450 was higher in 100,200,and 400μg/mL APS groups(P<0.05)and the protein levels of Sirt1,FoxO1,and LC3 II/LC3 I were lower(P<0.05).Conclusions APS inhibits autophagy of granulosa cells in PCOS rats,which may be mediated by inhibiting the autophagy pathway Sirt1/FoxO1.