桂枝茯苓丸调节PI3K/Akt/mTOR通路对PCOS-IR大鼠排卵障碍的影响  

Effect of Guizhi Fulingwan on Ovulation Dysfunction in PCOS-IR Rats by Regulating PI3K/Akt/mTOR Pathway

作　　者：刘敏[1] 朱鸿秋 李印 朱影 胡小丹 LIU Min;ZHU Hong-qiu;LI Yin;ZHU Ying;HU Xiao-dan(Chengdu University of Traditional Chinese Medicine(TCM),Chengdu 610075,China;College of Medicine and Life Sciences,Chengdu University of TCM,Chengdu 610041,China)

机构地区：[1]成都中医药大学,成都610075 [2]成都中医药大学医学与生命科学学院,成都610041

出　　处：《中国实验方剂学杂志》2021年第6期7-14,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81873335);四川省科技厅面上项目(2020YJ0376);成都中医药大学科技发展基金医院专项重点项目(2017-EL-13)。

摘　　要：目的:探讨桂枝茯苓丸对来曲唑联合高脂乳剂导致的多囊卵巢综合征伴胰岛素抵抗(PCOS-IR)大鼠排卵障碍的影响。方法:将72只雌性SD大鼠随机分为空白组、模型组、二甲双胍组和桂枝茯苓丸低、中、高剂量组,每组12只。除空白组外,其余大鼠每日予来曲唑0.001 g·kg^(-1)结合高脂乳剂15 mL·kg^(-1)连续灌胃30 d建立PCOS-IR大鼠模型。桂枝茯苓丸低、中、高剂量组每日分别灌服桂枝茯苓丸混悬液0.31,0.62,1.24 g·kg^(-1),二甲双胍组每日灌服二甲双胍混悬液0.27 g·kg^(-1),空白组和模型组每日灌服12 mL·kg^(-1)生理盐水,共干预30 d。苏木素-伊红(HE)染色观察卵巢组织病理形态学变化;酶联免疫吸附测定(ELISA)检测血清促卵泡生成素(FSH),促黄体生成素(LH),睾酮(T),空腹胰岛素(FINS)水平,计算LH/FSH,胰岛素抵抗指数(HOMA-IR)值;蛋白免疫印迹法(Western blot)检测大鼠卵巢组织中磷脂酰肌醇-3激酶/蛋白激酶B/雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路相关分子及自噬关键分子酵母Atg6同系物(Beclin-1),自噬相关基因5(Atg5),微管相关蛋白轻链3(LC3)Ⅱ蛋白表达水平;免疫组化(IHC)检测Beclin-1,LC3Ⅱ蛋白表达。结果:与空白组比较,模型组大鼠卵巢中各级卵泡、卵泡颗粒细胞层厚度也减少,黄体数量明显减少,而卵巢白膜厚度增加,闭锁卵泡及囊状扩张卵泡数量明显增多;血清T,LH,LH/FSH,FINS,HOMA-IR显著升高(P<0.01);卵巢组织中磷酸化(p)-PI3K,p-Akt,p-mTOR蛋白表达均明显降低(P<0.05,P<0.01);自噬相关蛋白LC3Ⅱ与Beclin-1的相对表达量明显升高(P<0.05,P<0.01)。与模型组比较,桂枝茯苓丸低、中、高剂量组及二甲双胍组囊状扩张卵泡数、闭锁卵泡数减少,卵泡颗粒细胞层厚度增加,可见到各级卵泡分布及较多黄体;桂枝茯苓丸低、中、高剂量组血清T,LH,LH/FSH,FINS,HOMA-IR明显降低(P<0.05,P<0.01),二甲双胍组血清FINS,HOMA-IR显著降低(P<0.01);各给药组p-PI3K,Objective:To investigate the effect of Guizhi Fulingwan on ovulation dysfunction in rats with polycystic ovary syndrome with insulin resistance(PCOS-IR)induced by letrazole combined with high fat emulsion.Method:A total of 72 female SD rats were randomly divided into control group,model group,metformin group and Guizhi Fulingwan low,medium and high dose groups,with 12 rats in each group.Except for control group,rats were given letrozole 0.001 g·kg^(-1) combined with high-fat emulsion 15 mL·kg^(-1) for 21 consecutive days to establish model of PCOS-IR.Guizhi Fulingwan low,medium and high-dose groups were administrated with Guizhi Fulingwan 0.31,0.62,1.24 g·kg^(-1) respectively,metformin group was administrated with metformin 0.27 g·kg^(-1),control group and model group were administrated with 12 mL·kg^(-1) of normal saline daily for 30 days.Hematoxylin-eosin(HE)staining was used to observe ovarian tissue pathology morphology,and enzyme-linked immunoassay method(ELISA)was used to detect serum follicle stimulating hormone(FSH),luteinizing hormone(LH),testosterone(T),fasting insulin(FINS)level,and LH/FSH and insulin resistance index(HOMA-IR)were calculated.Western blot was used to detect the expression levels of autophagy key molecular Atg6 yeast homologue(Beclin-1),autophagy related gene 5(Atg5),microtubule associated protein light chain 3(LC3)Ⅱproteins in the phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein(PI3 K/Akt/mTOR)signaling pathway and autophagy related indicators in rat ovarian tissue.Beclin-1 and LC3Ⅱprotein expressions were detected by immunohistochemistry(IHC).Result:Compared with control group,the thickness of follicles and follicular granulosa cells in the ovary of the model group also decreased,and the number of corpus luteum significantly decreased,while the white membrane thickness of the ovary increased,and the number of atresia follicles and cystic dilatation follicles increased significantly.Serum T,LH,LH/FSH,FINS,FINS,HOMA-IR were significantly increased(P<0.01).

关 键 词：桂枝茯苓丸 多囊卵巢综合征 排卵障碍 胰岛素抵抗 磷脂酰肌醇-3激酶/蛋白激酶B/雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路 自噬 

分 类 号：R2-0[医药卫生—中医学] R289