桂枝茯苓丸对PCOS模型小鼠卵巢颗粒细胞自噬的影响  

Effect of Guizhi Fulingwan on Autophagy of Ovarian Granulosa Cells in Mice with Polycystic Ovary Syndrome

作　　者：胡小丹 朱鸿秋[2] 朱影 刘敏[1] 陈海燕[1] HU Xiao-dan;ZHU Hong-qiu;ZHU Ying;LIU Min;CHEN Hai-yan(Chengdu University of Traditional Chinese Medicine(TCM),Chengdu 610072,China;School of Medical and Life Sciences,Chengdu University of TCM,Chengdu 610072,China)

机构地区：[1]成都中医药大学,成都610072 [2]成都中医药大学医学与生命科学院,成都610072

出　　处：《中国实验方剂学杂志》2021年第15期1-7,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81873335);四川省科学技术厅基金项目(2020YJ0376);成都中医药大学医学与生命科学院医院专项基金(2020yky05)。

摘　　要：目的:探讨桂枝茯苓丸对多囊卵巢综合征(PCOS)模型小鼠颗粒细胞自噬的影响。方法:20只SD小鼠中随机选取10只建立PCOS小鼠模型,并提取小鼠卵巢颗粒细胞进行体外培养。设置正常小鼠颗粒细胞为空白组,PCOS小鼠颗粒细胞为模型组,模型组颗粒细胞分别给予10%体积的17.6,35.1,70.2 mg·kg^(-1)桂枝茯苓丸含药血清,10%体积的25 mg·kg^(-1)二甲双胍含药血清作为培养基培养颗粒细胞,空白组颗粒细胞用10%体积的空白血清培养72 h。造模期间,测量小鼠体质量变化;造模结束后用显微镜观察小鼠卵巢形态,检测空腹血糖;使用放射免疫法检测空腹胰岛素水平和睾酮水平;细胞增殖与活性检测-8(CCK-8)检测卵巢颗粒细胞增殖情况,找到对于细胞活力无明显影响的最高含药血清剂量,用于后续实验;流式细胞仪检测颗粒细胞自噬情况;蛋白免疫印迹法(Western blot)检测细胞内微管相关蛋白1轻链3Ⅰ(LC3Ⅰ),LC3Ⅱ,Beclin1,p62蛋白的表达。结果:与空白组比较,模型组小鼠体质量增加、空腹胰岛素及空腹血糖水平增加、睾酮水平增加(P<0.05,P<0.01),表明PCOS小鼠建模成功。与空白组比较,经10%桂枝茯苓丸含药血清培养基孵育后,各组细胞自噬率均有所下降(P<0.05);Western blot检测结果表明,与空白组比较,模型组中Beclin1,LC3Ⅱ/Ⅰ的表达水平明显升高,p62的蛋白表达水平明显降低(P<0.05,P<0.01);与模型组比较,桂枝茯苓丸中、高剂量组中Beclin1,LC3Ⅱ/Ⅰ的表达水平明显下降,p62水平明显升高(P<0.05,P<0.01)。结论:桂枝茯苓丸可以通过下调Beclin1,LC3Ⅱ/Ⅰ蛋白的表达,抑制卵巢颗粒细胞的自噬水平。Objective:To investigate the effect of Guizhi Fulingwan on autophagy of ovarian granulosa cells in mice with polycystic ovary syndrome(PCOS).Method:Twenty SD mice were randomized into a normal group(n=10)and a PCOS model group(n=10),followed by PCOS modeling and in vitro culture of extracted ovarian granulosa cells.The ovarian granulosa cells of normal mice were classified into the control group and treated with 10%blank serum while those of PCOS mice into the experimental groups and with 10%Guizhi Fulingwan-containing serum at different concentrations(17.6,35.1,70.2 mg·kg^(-1))and 10%metformincontaining serum(25 mg·kg^(-1)),respectively,for 72 h.During the modeling,the changes in mouse body weight were measured.After modeling,the ovarian morphology was observed by microscopy,and the fasting blood glucose(FBG)was measured by Roche glucometer.Following the detection of fasting insulin(FI)and testosterone(T)levels by radioimmunoassay,the proliferation of ovarian granulosa cells was determined using cell counting kit-8(CCK-8)to figure out the maximal dose of drug-containing serum that did not obviously affect the cell viability for subsequent assay.The autophagy of ovarian granulosa cells was examined by flow cytometry,and the protein expression levels of intracellular microtubule-associated protein 1 light chain 3Ⅰ(LC3Ⅰ),LC3Ⅱ,Beclin1,and p62 were assayed by Western blott.Result:Compared with the blank group,the model group showed increased body weight and elevated FI,FBG,and T levels(P<0.05,P<0.01),indicating the successful modeling of PCOS mice.Flow cytometric assay proved that the incubation with 10%Guizhi Fulingwan serum-containing medium resulted in a decline of autophagy(P<0.05).As demonstrated by Western blot assay results,the protein expression levels of Beclin1 and LC3Ⅱ/Ⅰin the model group increased significantly as compared with those of the blank group,whereas the expression level of p62 decreased significantly(P<0.05,P<0.01).Compared with the model group,the medium-and high-dose Guizhi Fuling

关 键 词：桂枝茯苓丸 颗粒细胞 自噬 多囊卵巢综合征(PCOS) 

分 类 号：R2-0[医药卫生—中医学] R22