淫羊藿苷调控PI3K/Akt/mTOR通路对早发性卵巢功能不全大鼠卵巢结构及功能的保护作用及机制

Protective effect and mechanism of icariin regulating PI3K/Akt/mTOR pathway on ovarian structure and function in rats with early-onset ovarian insufficiency

目的探讨淫羊藿苷(ICA)对早发性卵巢功能不全(POI)大鼠卵巢结构、功能的影响及可能作用机制。方法从55只SPF级雌性SD大鼠中随机选取10只动情周期正常的大鼠作为对照组,剩余大鼠经腹腔注射环磷酰胺制备POI模型,成模大鼠随机分为模型组、ICA低(15 mg·kg^(-1))、中(30 mg·kg^(-1))、高(60 mg·kg^(-1))剂量组,对照组及模型组大鼠每日灌胃等量生理盐水,连续28 d。计算各组大鼠卵巢湿重和卵巢指数;HE染色观察卵巢组织结构;TUNEL染色检测颗粒细胞凋亡率;ELISA法检测血清促卵泡激素(FSH)、雌二醇(E2)、抗苗勒管激素(AMH)、丙二醛(MDA)及超氧化物歧化酶(SOD)含量;RT-qPCR技术检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)、核糖体S6蛋白激酶1(S6k1)mRNA相对表达量;Western blot法检测PI3K、Akt、mTOR和S6k1蛋白活性水平及B细胞淋巴瘤/白血病-2基因(Bcl2)、Bcl2相关X蛋白(Bax)和Cleaved Caspase-3蛋白相对表达情况。结果成功诱导POI大鼠模型;与对照组比较,模型组及ICA低、中、高剂量组大鼠卵巢湿重、卵巢指数、成熟卵泡和黄体数、血清E2、AMH及SOD水平降低,闭锁卵泡数、颗粒细胞凋亡率、血清FSH、MDA水平升高,卵巢见有不同程度萎缩,卵泡结构、形态见有不同程度退化,另卵巢组织中Bax、Cleaved Caspase-3蛋白相对表达量升高,Bcl2蛋白相对表达量降低,PI3K、Akt、mTOR、S6k1 mRNA及其蛋白活性水平降低(P<0.05);与模型组比较,ICA低、中、高剂量组大鼠上述指标及因子水平均逆转(P<0.05);ICA作用效果呈剂量依赖性(P<0.05)。结论ICA可提高POI大鼠血清E2水平,降低FSH水平,改善POI大鼠卵巢结构及卵巢储备功能,其作用机制可能与激活PI3K/Akt/mTOR通路有关。

Objective To investigate the effect of icariin(ICA)on ovarian structure and function in rats with early-onset ovarian insufficiency(POI)and its possible mechanism.Methods Ten SD female rats with normal estrous cycle were randomly selected as the control group.The remaining rats were injected intraperitoneally with cyclophosphamide to prepare the POI model.The model rats were randomly divided into model group,ICA low(15 mg·kg^(-1)),medium(30 mg·kg^(-1))and high(60 mg·kg^(-1))dose groups.The rats in the control group and model group were gavaged with the same amount of normal saline every day for 28 days.The wet weight and ovarian index of rats in each group were calculated.The histological structure of ovary was observed by HE staining.TUNEL staining was used to detect the apoptosis rate of granulosa cells.The levels of serum follicle stimulating hormone(FSH),estradiol(E2),anti mullerian hormone(AMH),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.RT-qPCR was used to detect the relative expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),mammalian rapamycin target protein(mTOR)and ribosomal S6 protein kinase 1(S6K1).The activity levels of PI3K,Akt,mTOR and S6K1 proteins and the relative expression of B-cell lymphoma/leukemia-2 gene(Bcl2),Bcl2 related X protein(Bax)and cleaved caspase-3 protein were detected by Western blot.Results POI rat model was successfully induced.Compared with the control group,the wet weight of ovaries,ovarian index,number of mature follicles and corpus luteum,serum E2,AMH and SOD levels in the model group and low,medium and high dose ICA groups decreased,the number of atresia follicles,apoptosis rate of granulosa cells,serum FSH and MDA levels increased,ovaries shrank in varying degrees,follicular structure and morphology degenerated in varying degrees,and the relative expression of Bax and cleaved caspase-3 protein in ovarian tissue increased,the relative expression of Bcl2 protein decreased,and the PI3K,Akt,mTOR,S6K1 mRNA and activities of protein decreased(P<0.05).Compared with the model group,the above indexes and factor levels of rats in low,medium and high dose ICA groups were reversed(P<0.05).The effect of ICA was dose-dependent(P<0.05).Conclusion ICA could increase serum E2 level,reduce FSH level,improve ovarian structure and ovarian reserve function in POI rats.Its mechanism may be related to the activation of PI3K/Akt/mTOR pathway.