补肾活血法调控PI3K/AKT/mTOR通路修复卵巢颗粒细胞自噬损伤机制研究

目的:观察补肾活血法调控磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/mTOR)通路对体外培养大鼠卵巢颗粒细胞(OGCs)自噬损伤的影响。方法:将原代培养的大鼠卵巢颗粒细胞分为六组：空白对照组、模型组、中药低剂量组、中药中剂量组、中药高剂量组、果纳芬组。采用ELISA法检测各组中雌二醇(E₂)、孕酮(P)分泌量；Western blot法和免疫荧光法分别检测各组OGCs中PI3K、AKT、mTOR及自噬关键分子酵母Atg6同系物1(Beclin 1)、微管相关蛋1轻链3(LC3)、自噬受体蛋白p62的蛋白表达。结果:模型组E₂含量显著降低，中药各剂量组E₂含量均上升(均P<0.05),且中药高剂量组与果纳芬组E₂含量相当；各组对P的含量均没有影响(均P>0.05)。中药各剂量组及果纳芬组p-PI3K、p-AKT、p-mTOR水平和p62的表达上调，Beclin 1和LC3Ⅱ/LC3Ⅰ的表达下调，且中药高剂量组效果优于果纳芬组(均P<0.05)。结论:补肾...

Research on the mechanism of repairing autophagic damage in ovarian granulosa cells by regulating PI3K/AKT/mTOR pathway by tonifying kidney and activating blood

Objective:To observe the effect of PI3K/AKT/mTOR pathway regulation by kidney tonic and blood activation method on autophagic injury in cultured rat ovarian granulosa cells in vitro.Methods:Primary cultured rat ovarian granulosa cells were divided into 6 groups:blank control group,model group,Chinese medicine low-dose group,Chinese medicine medium-dose group,Chinese medicine high-dose group and Gonal-F group.The E₂ and P content and the expression of PI3K,AKT,mTOR,Beclin 1,LC3 and p62 proteins in each group of OGCs.were detected by WB method and immunofluorescence method,respectively.Results:E₂content were detected by was significantly decreased in the model group and increased in all dose groups of Chinese medicine(all P<0.05),and the E₂content in the high dose group of Chinese medicine was comparable to that in the Gonal-F group.There was no effect of each group and on the content of P(P>0.05).The expression of p-PI3K,p-AKT,p-mTOR levels and p62 were up-regulated in each dose group of Chinese medicine and Gonal-F group,and the expression of Beclin 1 and LC3Ⅱ/LC3Ⅰ were down-regulated,and the effect of high-dose group of Chinese medicine was better than that of western medicine Gonal-F group(all P<0.05).Conclusion:The results suggest that the kidney tonic and blood activating Chinese herbal medicine compound Xinjia Guishen Wan may repair autophagic damage in ovarian granulosa cells by activating the PI3K/AKT/mTOR pathway,down-regulating the expression of downstream autophagic effector molecules Beclin 1 and LC3 and reducing the depletion of p62.