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  

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

作　　者：白俊 赵晓鹏 王海静 王丽君 张辉 李楠 陈梅 贺丰杰 吴克明[2] BAI Jun;ZHAO Xiaopeng;WANG Haijing;WANG Lijun;ZHANG Hui;LI Nan;CHEN Mei;HE Fengjie;WU Keming(Affiliated Hospital of Shaanxi University of Chinese Medicine,Xianyang 712000,China)

机构地区：[1]陕西中医药大学附属医院,陕西咸阳712000 [2]成都中医药大学附属医院,四川成都610075

出　　处：《陕西中医》2023年第5期547-551,共5页Shaanxi Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81873334);陕西省自然科学基础研究项目(2023-JC-QN-0899);陕西省教育厅专项科研计划项目(20JK0596);陕西省咸阳市科技计划项目(2021ZDYF-SF-0033);陕西中医药大学附属医院院级课题(2020QN002);全国名老中医药专家传承工作室建设项目。

摘　　要：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(E_(2))、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E_(2)含量显著降低,中药各剂量组E_(2)含量均上升(均P<0.05),且中药高剂量组与果纳芬组E_(2)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:补肾活血中药复方新加归肾丸可能是通过激活PI3K/AKT/mTOR通路,下调下游自噬效应分子Beclin 1及LC3的表达并减少p62的消耗来修复OGCs的自噬损伤。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 E_(2) 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:E_(2) content 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 E_(2) content 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/LC3Ⅰwere 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.

关 键 词：补肾活血法 卵巢颗粒细胞 自噬 雷公藤 PI3K/AKT/mTOR通路 卵泡发育障碍 

分 类 号：R711.75[医药卫生—妇产科学]