益母草碱调节RhoA/ROCK信号通路对多囊卵巢综合征大鼠卵巢颗粒细胞自噬和凋亡的影响

Impacts of leonurine on autophagy and apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome by regulating the RhoA/ROCK signaling pathway

目的 探讨益母草碱(Leo)调节RhoA/ROCK信号通路对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞(GCs)自噬和凋亡的影响。方法 将SD大鼠随机分为正常组(NC组)和模型组(PCOS组),分离卵巢GCs,H-E染色观察其形态;FSHR免疫荧光鉴定PCOS大鼠卵巢GCs。不同浓度的Leo处理PCOS大鼠卵巢GCs,CCK-8法检测细胞增殖并筛选最佳药物浓度。将PCOS大鼠GCs随机分为模型组(PCOS组)、益母草碱低剂量组(Leo-L组)、益母草碱中剂量组(Leo-M组)、益母草碱高剂量组(Leo-H组)、益母草碱高剂量组+RhoA/ROCK激活剂组(Leo+LPA组),流式细胞术检测细胞凋亡率;透射电子显微镜检测细胞自噬;Western blot检测Bax、Cleaved-Caspase-3、Bcl-2、Beclin1、LC3-Ⅱ、LC3-Ⅰ、RhoA、ROCK1、ROCK2蛋白表达。结果 与NC组比较,PCOS组可见大小不等的囊性卵泡,且卵巢GCs数增多(P<0.05);PCOS大鼠FSHR阳性率达92.13%,即成功分离卵巢GCs;Leo处理PCOS卵巢GCs后,细胞凋亡率、Bax、Cleaved-Caspase-3、Beclin1、LC3-Ⅱ/LC3-Ⅰ、RhoA、ROCK1、ROCK2蛋白表达及MDA含量显著降低,细胞OD值、Bcl-2蛋白表达及GSH、SOD、GSH-PX含量显著增加,且呈浓度依赖性(P<0.05);LPA逆转了Leo对PCOS大鼠卵巢GCs凋亡和自噬的抑制作用。结论 Leo通过抑制RhoA/ROCK信号通路抑制PCOS大鼠中GCs凋亡和自噬。

Objective To investigate the impacts of leonurine(Leo)on autophagy and apoptosis of ovarian granulosa cells(GCs)in polycystic ovary syndrome(PCOS)rats by regulating the RhoA/ROCK signaling pathway.Methods SD rats were randomly grouped into normal group(NC group)and model group(PCOS group),ovarian GCs were isolated,and its morphology was observed by H-E staining.FSHR immunofluorescence to dectect ovarian GCs of PCOS rat.After treating ovarian GCs with different concentrations of Leo,CCK-8 method was used to detect the cell proliferation,and the optimal drug concentration was screened in PCOS rat.Ovarian granulosa cells of PCOS rat were randomly grouped into a PCOS group,a low-dose leonurinegroup(Leo-L),a medium-dose leonurinegroup(Leo-M),a high-dose leonurinegroup(Leo-H),and a high-dose leonurine+RhoA/ROCK activator group(Leo+LPA).Flow cytometry was applied to detect the cell apoptosis.Transmission electron microscopy was applied to detect the autophagy.Western blot was applied to detect the expression of Bax,Cleaved-Caspase-3,Bcl-2,Beclin1,LC3-II,LC3-I,RhoA,ROCK1,and ROCK2 proteins.Results Compared with the NC group,cystic follicles of different sizes were observed in PCOS group,and the number of ovarian GCs was increased(P<0.05).The positive rate of FSHR in PCOS rats was 92.13%,indicating ovarian granulosa cells were successfully isolated,after treating ovarian GCs with Leo in PCOS rat,the apoptosis rate,the protein expression of Bax,Cleaved-Caspase-3,Beclin1,LC3-II/LC3-I,RhoA,ROCK1,ROCK2,and MDA content were obviously reduced,the cell OD value,Bcl-2 expression,and GSH,SOD,GSH-PX contents were obviously increased,and in a concentration-dependent manner(P<0.05).The inhibitory effect of apoptosis and autophagy for Leo on ovarian GCs were reversed by LPA in PCOS rats.Conclusion Leo inhibits apoptosis and autophagy of GCs in PCOS rats by inhibiting the RhoA/ROCK signaling pathway.