基于TGF-β_(1)/Smads信号通路探讨当归芍药散对卵巢储备功能低下模型大鼠的影响

Effect of Danggui Shaoyaosan on Diminished Ovarian Reserve in Model Rats:An Exploration Based on TGF-β_(1)/Smads Signaling Pathway

目的:观察当归芍药散对雷公藤多苷片联合应激所致的卵巢储备功能下降(DOR)模型大鼠卵巢储备功能的改善作用,并探讨转化生长因子-β_(1)(TGF-β_(1))/Smads信号通路在其中的作用机制。方法:选取性周期正常的雌性SD大鼠48只,随机分为正常组8只和模型制备组40只。模型制备组以灌胃雷公藤多苷片(50 mg·kg^(-1))联合随机应激法建立模型,时间15 d,造模成功后将其随机分为模型组、当归芍药散低、中、高剂量组(3.96,7.92,15.84 g·kg^(-1))及戊酸雌二醇组(0.09 mg·kg^(-1))各8只,各组在继续给予应激的同时,分别用生理盐水,低、中、高剂量当归芍药散及戊酸雌二醇进行相应剂量灌胃干预,连续15 d。各组大鼠每日观察动情周期,干预结束后处死大鼠,计算大鼠卵巢脏器指数,苏木素-伊红(HE)染色观察大鼠卵巢组织病理变化,免疫组化法(IHC)检测大鼠卵巢组织中TGF-β_(1),转化生长因子-β_(1)受体(TGF-β_(1)R)蛋白表达水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠卵巢组织中Smad2,Smad3,Smad7 mRNA表达水平。结果:与正常组比较,模型组大鼠动情周期紊乱率明显升高(P<0.05),卵巢指数明显下降(P<0.01),卵巢组织中TGF-β_(1),TGF-β_(1)R蛋白及Smad2,Smad3 mRNA表达水平降低(P<0.01),Smad7 mRNA表达水平上升(P<0.01);与模型组比较,中药3组与戊酸雌二醇组大鼠动情周期均有不同程度改善(P<0.05),卵巢指数均有不同程度回升,其中以中、高剂量组最为明显(P<0.05,P<0.01),卵巢组织中TGF-β_(1),TGF-β_(1)R蛋白及Smad2,Smad3 mRNA水平均有不同程度升高(P<0.01),Smad7 mRNA表达水平均下降(P<0.01),其中TGF-β_(1),TGF-β_(1)R蛋白表达改善程度以中剂量组和戊酸雌二醇组最为明显。结论:当归芍药散能够明显改善DOR大鼠卵巢储备功能,且作用机制与调控TGF-β_(1)/Smads信号通路密切相关。

Objective:To observe the improving effect of Danggui Shaoyaosan on diminished ovarian reserve(DOR)in rats triggered by Tripterygia wilfordii polyglycoside tablet combined with stress,and to explore the role of transforming growth factor-β_(1)(TGF-β_(1))/Smads signaling pathway in such improvement.Method:Forty-eight female SD rats with normal sexual cycle were selected and randomly divided into a normal group(n=8)and a modeling group(n=40),and the ones in the modeling group were given Tripterygium wilfordii polyglycoside tablets(50 mg·kg^(-1))combined with random stress for 15 d.After successful modeling,the rats were randomized into the model group,low-,medium-,and high-dose(3.96,7.92,15.84 g·kg^(-1))Danggui Shaoyaosan groups,and estradiol valerate group(0.09 mg·kg^(-1)),with eight in each group.Under the premise of stress exposure,they were separately gavaged with the normal saline,low-,medium-and high-dose Danggui Shaoyaosan,and estradiol valerate for 15 successive days.The estrous cycle of rats in each group was observed daily.After intervention,the rats were sacrificed and the ovarian visceral index was calculated.The pathological changes in ovarian tissues were observed by hematoxylin eosin(HE)staining.The protein expression levels of TGF-β_(1) and TGF-β_(1) receptor(TGF-β_(1)R)in the ovarian tissues of rats were measured by immunohistochemistry(IHC),and the mRNA expression levels of Smad2,Smad3,and Smad7 in the ovarian tissues by real-time polymerase chain reaction(Real-time PCR).Result:Compared with the normal group,the model group exhibited disordered estrus cycle(P<0.05),reduced visceral index(P<0.01),and down-regulated TGF-β_(1) and TGF-β_(1)R protein and Smad2 and Smad3 mRNA expression in the ovarian tissues(P<0.01),and upregulated Smad7 mRNA expression(P<0.01).Compared with the model group,Danggui Shaoyaosan at the low,medium,and high doses and estradiol valerate improved the estrus cycle of rats to varying degrees(P<0.05)and increased the visceral index,with better effects observed in the medium-group and high-dose Danggui Shaoyaosan groups(P<0.05,P<0.01).Besides,the protein expression levels of TGF-β_(1) and TGF-β_(1)R and the mRNA expression levels of Smad2 and Smad3 in the ovarian tissues were elevated to varying degrees(P<0.01),and the Smad7 mRNA expression declined(P<0.01).The improvements in TGF-β_(1) and TGF-β_(1)R protein expression of the medium-dose Danggui Shaoyaosan group and estradiol valerate group were more obvious.Conclusion:Danggui Shaoyaosan significantly improves ovarian reserve in DOR rats,which is closely related to the regulation of TGF-β_(1)/Smads signaling pathway.