基于“以方测证”理论从AMPK/mTOR/HIF-1/VEGF通路探讨雷公藤多苷诱导卵泡发育不良大鼠模型的中医证候属性

Determination of Traditional Chinese Medicine Syndrome of Rat Model of Follicular Dysplasia Induced by Tripterygium Glycosides Based on Prescriptions via AMPK/mTOR/HIF-1/VEGF Pathway

目的:采用雷公藤多苷诱导卵泡发育不良大鼠模型,基于“以方测证”理论从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)信号通路探索该模型的证候属性。方法:将48只具有规律动情周期大鼠随机分为正常组(n=8)和造模组(n=40),造模组大鼠连续灌胃雷公藤多苷混悬液(75 mL·kg^(-1))30 d,造模后将其分为模型组、四物汤组(3.69 g·kg^(-1))、右归饮组(3.11 g·kg^(-1))、左归饮组(7.29 g·kg^(-1))和归肾丸组(10.35 g·kg^(-1)),每组8只。相应药物干预14 d。巴氏染色检测各组大鼠动情周期变化;体视镜观察卵巢组织形态;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)测定血清促卵泡激素(FSH)、黄体生成素(LH)及雌二醇(E2)含量;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,FSH、LH、AMPK mRNA及蛋白表达显著升高(P<0.01),E2含量,mTOR、HIF-1、VEGF mRNA及蛋白表达显著降低(P<0.01);与模型组比较,归肾丸组次级、成熟卵泡增多,闭锁卵泡减少,FSH、LH含量、AMPK mRNA表达及蛋白表达显著降低(P<0.01),E2含量、mTOR、HIF-1、VEGF mRNA表达及蛋白表达显著升高(P<0.01)。与归肾丸组比较,四物汤组、右归饮组、左归饮组成熟卵泡数量显著减少,闭锁卵泡数量显著增多(P<0.01);四物汤组、右归饮组、左归饮组LH显著升高(P<0.01),FSH明显升高(P<0.05),E2明显降低(P<0.05);右归饮组AMPK蛋白表达明显升高(P<0.05),mTOR和HIF-1 mRNA表达显著降低(P<0.01),VEGF蛋白及mRNA表达显著降低(P<0.01);四物汤组mTOR和HIF-1 mRNA,VEGF蛋白及mRNA表达明显降低(P<0.05);左归饮组mTOR mRNA和VEGF蛋白及mRNA表达明显降低(P<0.05)。结论:归肾丸可以抑制AMPK/mTOR/HIF-1/VEGF通路来改善卵泡发育不良大鼠模型的卵巢功能、促进卵泡成熟,且总体疗效优于左归饮、右归饮和四物汤,表明该模型的证候更符合肾精亏虚证。

Objective:To determine the syndrome of a rat model of follicular dysplasia induced by Tripterygium glycosides based on prescriptions and investigate the mechanism of traditional Chinese medicine intervention via the adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/hypoxia-inducible factor-1(HIF-1)/vascular endothelial growth factor(VEGF)pathway.Method:Forty-eight rats with regular estrous cycles were randomly assigned into a normal group(n=8)and a modeling group(n=40).The rats in the modeling group were administrated with Tripterygium glycoside suspension(75 mL·kg^(-1))by gavage for 30 days.The modeled rats were assigned into model,Siwutang(3.69 g·kg^(-1)),Youguiyin(3.11 g·kg^(-1)),Zuoguiyin(7.29 g·kg^(-1)),and Guishenwan(10.35 g·kg^(-1))groups,with 8 rats in each group.The drug intervention lasted for 14 days.The changes of estrous cycle were detected by Pap staining,and a stereoscope was used to observe the morphology of the ovarian tissue.Hematoxylin-eosin staining was employed to observe the pathological changes and follicle count in the ovarian tissue.Enzyme-related immunosorbent assay(ELISA)was used to measure the levels of follicle-stimulating hormone(FSH),luteinizing hormone(LH),and estradiol(E2)in the serum.Real-time fluorescence quantitative polymerase chain reaction and Western blot were employed to determine the mRNA and protein levels,respectively,of AMPK,mTOR,HIF-1,and VEGF in the ovarian tissue.Result:Compared with the normal group,the model group had a disordered estrous cycle,reduced secondary and mature follicles,increased atretic follicles,elevated FSH and LH levels,lowered E2 level,up-regulated mRNA and protein levels of AMPK,and downregulated mRNA and protein levels of mTOR,HIF-1,and VEGF(P<0.01).Compared with the model group,Guishenwan increased secondary and mature follicles,decreased atretic follicles,lowered the FSH and LH levels,elevated the E2 level,down-regulated the mRNA and protein levels of AMPK,and up-regulated the mRNA and protein levels of mTOR,HIF-1,and VEGF(P<0.01).Compared with Guishenwan group,Siwutang,Youguiyin,and Zuoguiyin decreased mature follicles,increased atretic follicles(P<0.01),elevated the LH(P<0.01)and FSH(P<0.05)levels,and lowered the E2 level(P<0.05).In addition,Youguiyin upregulated the protein level of AMPK(P<0.05)and down-regulated the mRNA levels of mTOR and HIF-1(P<0.01)as well as the mRNA and protein levels of VEGF(P<0.01).Siwutang down-regulated the mRNA levels of mTOR and HIF-1 as well as the mRNA and protein levels of VEGF(P<0.05).Zuoguiyin down-regulated the mRNA level of mTOR and the protein and mRNA levels of VEGF(P<0.05).Conclusion:Guishenwan may improve the ovarian function and promote follicle maturation in a rat model of follicular dysplasia by inhibiting the AMPK/mTOR/HIF-1/VEGF pathway,with the therapeutic effect superior to Zuoguiyin,Youguiyin,and Siwutang.It was hypothesized that this model presented the syndrome of kidney-essence deficiency.