槲皮素对多囊卵巢综合征大鼠卵巢颗粒细胞增殖与凋亡的影响研究  

Effect of Quercetin on Proliferation and Apoptosis of Ovarian Granulosa Cells in Rats with Polycystic Ovarian Syndrome

作　　者：江雪娟 陈晓菲 俞佳[1] 王晨晔[1] 胡慧敏[1] 丁彩飞[1] JIANG Xuejuan;CHEN Xiaofei;YU Jia(Zhejiang Hospital of Integrated Traditional Chinese and Western Medicine,Hangzhou 310005,China)

机构地区：[1]浙江省中西医结合医院,杭州310005

出　　处：《浙江中医药大学学报》2022年第10期1055-1062,1069,共9页Journal of Zhejiang Chinese Medical University

基　　金：杭州市卫生科技计划项目(ZD20200016,ZD20210029,OO2019011,OO2019008);浙江省中医药科学研究基金项目(2021ZB210);杭州市红十字会医院院内科研基金项目(HHQN2021005);浙江省自然科学基金项目(LQ19H270005)。

摘　　要：[目的]探究槲皮素(quercetin,Q)对多囊卵巢综合征(polycystic ovarian syndrome,PCOS)大鼠卵巢颗粒细胞增殖与凋亡的影响及其作用机制。[方法]建立PCOS大鼠模型,提取培养卵巢颗粒细胞进行鉴定,并分为PCOS组、PCOS+4-氯-N-环己基-N-(苯基甲基)苯甲酰胺(N-benzyl-4-chloro-N-cyclohexylbenzamide,FZ)组、PCOS+L-Q(槲皮素0.5μg·m L)组、PCOS+H-Q(槲皮素1μg·mL)组、PCOS+H-Q+FZ组、PCOS+盐酸二甲双胍(metformin hydrochloride,MH)组,并取正常颗粒细胞作为对照组。以四甲基偶氮唑盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT]法检测细胞活力,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞上清液中雌二醇(estradiol,E)、孕酮(progesterone,P)、促卵泡生成素(follicle-stimulating hormone,FSH)、黄体生成素(luteinizing hormone,LH)、雄激素(testosterone,T)含量,流式细胞术测定细胞凋亡率,并进行活性氧(reactive oxygen species,ROS)活性检测。实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测细胞中晚期糖基化终末产物受体(receptor of advanced glycation end products,RAGE)、Bax、Bcl-2及炎性介质高迁移率族蛋白B1 (high mobility group protein B1,HMGB1)的mRNA表达情况,免疫印迹法检测细胞中晚期糖基化终末产物/晚期糖基化终末产物受体(advanced glycation end products/receptor of advanced glycation end products,AGEs/RAGE)及细胞外信号调节蛋白激酶/有丝分裂原蛋白激酶(extracellular signal-regulated kinase/mitogen-activated protein kinase,ERK/MAPK)相关蛋白表达情况。[结果]分离的大鼠卵巢颗粒细胞鉴定正常。MTT结果显示,槲皮素在0~1μg·mL时对正常颗粒细胞活力无影响,对于PCOS组细胞,槲皮素作用后各组细胞活力显著升高(P<0.05,P<0.01)。与PCOS组比较,PCOS+FZ组、PCOS+H-Q组、PCOS+H-Q+FZ组和PCOS+MH组细胞中E、LH、T含量以及ROS活性均显著降低(P<0.05,P<0.01),PC[Objective]To investigate the effects of quercetin(Q) on proliferation and apoptosis of ovarian granulosa cells in rats with polycystic ovarian syndrome(PCOS) and its mechanism.[Methods]After PCOS rat model was established,ovarian granulosa cells were extracted and identified.Then the ovarian granulosa cells were divided into PCOS group,PCOS+N-benzyl-4-chloro-N-cyclohexylbenzamide(FZ)group,PCOS+L-Q(quercetin 0.5μg·mL) group,PCOS+H-Q(quercetin 1μg·mL) group,PCOS+H-Q+FZ group,and PCOS+metformin hydrochloride(MH) group,and normal granulosa cells as control group.Cell viability was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT),the contents of estradiol(E),progesterone(P),follicle-stimulating hormone(FSH),luteinizing hormone(LH) and testosterone(T) in cell supernatant were determined by enzyme linked immunosorbent assay(ELISA),and cell apoptosis rate was determined by flow cytometry,and reactive oxygen species(ROS) activity was detected.The m RNA expressions of receptor of advanced glycation end products(RAGE),Bax,Bcl-2 and inflammatory medium high mobility group protein B1(HMGB1) were detected by Real-time quantitative polymerase chain reaction(Real-time qPCR),and the protein expressions of advanced glycation end products/receptor of advanced glycation end products(AGEs/RAGE) and extracellular signal-regulated kinase/mitogen-activated protein kinase(ERK/MAPK) related proteins were detected by Western blot.[Results]The isolated rat ovarian granulosa cells were successfully identified.MTT results showed that quercetin had no effect on viability of normal granulosa cells at 0~1μg·mL,while for PCOS group,the viability of each group was significantly increased(P<0.05,P<0.01).Compared with PCOS group,E,LH and T contents and ROS activity in PCOS+FZ group,PCOS+H-Q group,PCOS+H-Q+FZ group and PCOS+MH group were significantly decreased(P<0.05,P<0.01).In PCOS+H-Q group,PCOS+H-Q+FZ group and PCOS+MH group,the mRNA expression levels of RAGE,HMGB1,Bax and protein expression levels of AGEs an

关 键 词：槲皮素 多囊卵巢综合征 胰岛素抵抗 细胞活力 二甲双胍 细胞增殖 细胞凋亡 AGEs/RAGE 

分 类 号：R331[医药卫生—人体生理学]