基于Kisspeptin/GPR54系统探讨七子益肾理冲汤对多囊卵巢综合征模型大鼠卵泡发育的影响

Effect of Qizi Yishen Lichong Decoction(七子益肾理冲汤)on Follicular Development of Polycystic Ovary Syndrome Model Rats based on Kisspeptin/GPR54 System

目的探讨七子益肾理冲汤对多囊卵巢综合征(PCOS)卵泡发育的影响及可能作用机制。方法将17只动情规律雌性SD大鼠随机分为空白组5只、模型组6只、七子益肾理冲汤组6只。模型组及七子益肾理冲汤组大鼠每日予来曲唑-羧甲基纤维素混悬液1 mg/(kg·d)连续灌胃21天构建PCOS大鼠模型。第22天开始,七子益肾理冲汤组以七子益肾理冲汤34.02 g/(kg·d)灌胃,每日1剂,空白组、模型组以10 ml/(kg·d)无菌饮用水灌胃。15天后比较各组大鼠体质量、卵巢系数,体视显微镜观察各组大鼠卵巢外观,HE染色观察各组大鼠卵巢组织形态学改变,放射免疫法检测各组大鼠血清激素卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、睾酮(T)、孕激素(P)水平、抗苗勒管激素(AMH)、抑制素B(INHB)水平,Western blot法检测下丘脑及卵巢Kisspeptin(Kp)、G蛋白偶联受体54(GPR54)、细胞外信号调节激酶1/2(ERK1/2)蛋白表达水平。结果各组大鼠的体质量、卵巢系数比较,差异均无统计学意义(P>0.05)。与空白组比较,模型组大鼠卵巢外观较苍白,囊状扩张卵泡及闭锁卵泡增加,卵泡颗粒细胞层变薄;血清FSH、LH、AMH水平,下丘脑及卵巢Kp、GPR54、ERK1/2蛋白表达显著升高(P<0.05或P<0.01),血清E2、P水平显著降低(P<0.01)。与模型组比较,七子益肾理冲汤组大鼠卵巢外观较红润,囊状扩张卵泡减少,卵泡颗粒细胞层增加,出现近成熟卵泡,可见少量黄体;大鼠血清FSH、LH、AMH水平,下丘脑、卵巢Kp、GPR54蛋白表达显著下降,下丘脑ERK1/2蛋白表达显著下降(P<0.05或P<0.01)。结论七子益肾理冲汤可以改善PCOS模型大鼠卵泡发育,其机制可能是通过激活Kp/GPR54系统,调节下游ERK信号通路,进而调控下丘脑-垂体-卵巢轴,改善激素水平,发挥调节卵巢功能、促进卵泡发育的作用。

Objective To explore the effect and the potential mechanism of Qizi Yishen Lichong Decoction(七子益肾理冲汤)on follicular development of polycystic ovary syndrome(PCOS).Methods Seventeen female SD rats with regular estrus were randomly divided into blank group(n=5),model group(n=6)and Qizi group(n=6).Rats in the model group and Qizi group were given 1 mg/(kg·d)of letrozole-carboxymethylcellulose suspension by gavage for 21 days to establish the model of PCOS.Since the 22nd day,Qizi group was given 34.02 g/(kg·d)of Qizi Yishen Lichong Decoction,one dose daily,while the blank group and model group were given 10 ml/(kg·d)of sterile drinking water by gavage.After 15 days,the rat weight and ovarian coefficient were compared.The appearance of the ovaries was observed with a stereo microscope.Histomorphological Changes of ovary in all groups of rats were observed by HE staining.The levels of serum follicle stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2),testosterone(T),progesterone(P),anti-Müllerian hormone(AMH),and inhibin B(INHB)were detected by radioimmunoassay.The expression levels of kisspeptin(Kp),G protein-coupled receptor 54(GPR54)and extracellular signal-regulated kinase 1/2(ERK1/2)in hypothalamus and ovary were detected by Western blot.Results There was no significant difference in weight and ovarian coefficient of rats between the groups(P>0.05).Compared to thoese in the blank group,the ovaries of rats in the model group were paler in appearance,with cystic dilatation,increased follicles and atretic follicles,and thinning follicular granulosa cell layer;the serum FSH,LH,AMH levels,as well as the expression of Kp,GPR54,and ERK1/2 in hypothalamus and ovary increased(P<0.01),while the serum E2 and P levels decreased(P<0.01).Compared to those in the the model group,the ovarian surface of rats in the Qizi group was more ruddy in appearance,with decreased number of cystic follicles and increased follicular granulosa cell layer,and near-mature follicles were found with a small amount of corpus luteum;the serum FSH,LH and AMH levels,and the expression of Kp and GPR54 in the hypothalamus and ovary significantly decreased,as well as the ERK1/2 in the hypothalamus(P<0.05 or P<0.01).Conclusion Qizi Yishen Lichong Decoction can improve the follicular development of PCOS,and its mechanism may be related to the regulation of the downstream ERK signaling pathway by activating the Kp/GPR54 system,which further regulates the hypothalamus-pituitary-ovary axis and improves the sex hormones levels,playing the role in regulating ovarian function and promoting follicular development.