促卵合剂调控AMPK/mTOR/HIF-1/VEGF通路改善卵泡发育不良大鼠氧化应激及凋亡的作用机制

Mechanism of Culuan Heji in Regulating AMPK/mTOR/HIF-1/VEGF Pathway to Improve Oxidative Stress and Apoptosis in Rats with Follicular Maldevelopment

目的:探讨促卵合剂通过调控腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白/缺氧诱导因子-1/血管内皮生长因子(AMPK/mTOR/HIF-1/VEGF)通路改善卵泡发育不良(FM)大鼠氧化应激及凋亡的作用机制。方法:将48只雌性SD大鼠随机分为正常组8只,造模组40只,造模组大鼠连续灌胃雷公藤多苷混悬液(75 mg·kg^(-1))30 d进行造模,造模后随机分为模型组、克罗米芬组(4.5 mg·kg^(-1))、促卵合剂低、中、高剂量组(8.1、16.2、32.4 g·kg^(-1)),每组8只。相应剂量药物给药,正常组及模型组灌胃等体积灭菌水,连续干预14 d。巴氏染色检测各组大鼠动情周期变化;苏木素-伊红(HE)染色观察卵巢组织病理学及卵泡计数;酶联免疫吸附测定法(ELISA)检测血清促卵泡激素(FSH)、黄体生成素(LH)及雌二醇(E2)含量;化学荧光法检测卵巢组织中活性氧(ROS)、超氧化物歧化酶(SOD)水平;原位末端标记法(TUNEL)检测卵巢颗粒细胞凋亡率;免疫组化法检测卵巢组织B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、Bcl-2蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测卵巢组织AMPK/mTOR/HIF-1/VEGF mRNA及蛋白表达。结果:与正常组比较,模型组大鼠动情周期紊乱,次级、成熟卵泡减少,闭锁卵泡增多,血清FSH及LH含量,卵巢组织ROS含量、Bax表达、卵巢颗粒细胞凋亡率、AMPK mRNA及蛋白表达均显著升高(P<0.01),E2含量、SOD活性、Bcl-2表达、mTOR、HIF-1、VEGF mRNA及蛋白表达均显著降低(P<0.01);与模型组比较,克罗米芬组及促卵合剂高、中剂量组次级卵泡和成熟卵泡增加,闭锁卵泡显著减少,血清FSH及LH含量、卵巢组织ROS含量、Bax表达、细胞凋亡、AMPK mRNA及蛋白表达降低(P<0.05,P<0.01),E2含量、SOD活性、Bcl-2、mTOR、HIF-1、VEGF mRNA及蛋白表达均升高(P<0.05,P<0.01)。结论:促卵合剂可能通过调控AMPK/mTOR/HIF-1/VEGF通路来抑制氧化应激减少颗粒细胞凋亡,促进卵泡正常发育成熟,减少卵泡闭锁,保护卵巢功能,进而对FM发挥治疗作用。

Objective:To explore the mechanism of Culuan Heji in improving oxidative stress and apoptosis in rats with follicular maldevelopment(FM)by regulating the adenylate activated protein kinase/mammalian target protein of rapamycin/hypoxia-inducing-factor-1/vascular endothelial growth factor(AMPK/mTOR/HIF-1/VEGF)pathway.Method:A total of 48 female SD rats were randomly divided into a normal group(eight rats)and a modeling group(40 rats).The rats in the modeling group were given continuous instillation of tripterygium wirelli polyside suspension(75 mg·kg^(-1))for 30 days for modeling.After modeling,the rats were randomly divided into model group,clomiphene group(4.5 mg·kg^(-1)),and low-,medium-,and high-dose groups of Culuan Heji(8.1,16.2,32.4 g·kg^(-1)),with eight rats in each group.The groups were administered the corresponding drugs,and the normal group and the model group were injected with the same volume of sterilized water.The intervention lasted for 14 days.The changes in the estrous cycle of rats in each group were detected by Pap staining.Ovarian histopathology and follicle count were observed by hematoxylineosin(HE)staining.Serum follicle-stimulating hormone(FSH),luteinizing hormone(LH),and estradiol(E2)were determined by enzyme-related immunosorbent assay(ELISA).The contents of reactive oxygen species(ROS)and superoxide dismutase(SOD)in ovarian tissue were detected by chemical fluorescence.The apoptosis rate of ovarian granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).The expression of B-cell lymphoma-2 protein(Bcl-2)associated X protein(Bax)and Bcl-2 in ovarian tissue was detected by immunohistochemistry.Real-time fluorescence quantitative PCR(Real-time PCR)and Western blot were used to detect AMPK/mTOR/HIF-1/VEGF mRNA and protein expressions in ovarian tissue.Result:Compared with that in the normal group,the estrous cycle of rats in the model group was disturbed,and secondary and mature follicles were decreased.Atretic follicles were increased,and FSH and LH contents,ROS content,Bax expression,apoptosis rate of ovarian granulosa cells,and AMPK mRNA and protein expressions were significantly increased(P<0.01).E2 content,SOD content,Bcl-2 expression,mTOR,HIF-1,and VEGF mRNA and protein expressions were significantly decreased(P<0.01).Compared with those in the model group,secondary follicles and mature follicles increased in the clomiphene group and medium-and high-dose groups of Culuan Heji.Atretic follicles significantly decreased,and FSH and LH contents,ROS content,Bax expression,apoptosis,and AMPK mRNA and protein expressions decreased(P<0.05,P<0.01).E2 content,SOD content,Bcl-2 expression,mTOR,HIF-1,and VEGF mRNA and protein expressions were all increased(P<0.05,P<0.01).Conclusion:Culuan Heji may inhibit oxidative stress by regulating the AMPK/mTOR/HIF-1/VEGF pathway to reduce granulosa cell apoptosis,promote normal follicle development and maturation,reduce follicle atresia,protect ovarian function,and thus play a therapeutic role in FM.