滋阴补阳序贯法联合西药对卵巢储备功能下降排卵障碍性不孕大鼠TGF-β1/Smads信号通路的影响

Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve

目的:探讨滋阴补阳序贯法联合西药对卵巢储备功能下降(diminished ovarian reserve,DOR)排卵障碍性不孕大鼠的TGF-β1/Smads信号通路的影响。方法:SD雌性大鼠40只,随机分为空白组、模型组、西药组(人工周期+促排卵)、滋阴补阳序贯组、联合用药组共5组,每组8只。用雷公藤多苷片50 mg/(kg.d)灌胃连续2周,建立DOR大鼠模型。空白组、模型组用生理盐水灌胃,连续10日;西药组予人工周期加促排卵治疗;滋阴补阳序贯组在动情前期予滋阴方,动情后期予补阳方灌胃;联合用药组在西药组的基础上动情前期予滋阴方,动情后期予补阳方。HE染色观察大鼠卵巢组织形态变化,免疫组织化学检测大鼠卵巢转化生长因子β1受体(transforming growth factorβ1 receptor,TGF-β1R)的水平,Western印迹检测大鼠卵巢Smad2,Smad3,Smad7蛋白的表达。结果:与空白组比较,DOR模型组大鼠卵巢黄体数、成熟卵泡数、生长卵泡数显著减少,闭锁卵泡数升高(P<0.01);TGF-β1R表达升高,Smad2和Smad3蛋白水平显著降低,Smad7蛋白显著升高(P<0.01)。与模型组比较,各治疗组黄体数、成熟卵泡数、生长卵泡数增多,闭锁卵泡数减少,Smad2和Smad3蛋白水平升高,Smad7蛋白水平降低(P<0.05或P<0.01)。联合用药组与西药组比较,黄体数及生长卵泡数增加,TGF-β1R表达升高,Smad2和Smad3蛋白水平升高,Smad7蛋白水平降低(P<0.05或P<0.01)。结论:滋阴补阳序贯法联合西药能够上调DOR大鼠卵巢TGF-β1R,Smad2,Smad3表达,降低Smad7表达,改善DOR大鼠卵巢储备功能。

Objective： To explore the influence for combination of nourishing yin and tonifying yang sequential therapy （NYTYST） with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve （DOR） based on TGF-β1/Smads signaling pathway.Methods： A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group （n=8 in each group）. The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy （HRT） and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor （TGF-β1R） in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.Results： Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group （P〈0.01）; the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly （P〈0.01）. Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group （P〈0.05 or P〈0.01）. The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group （P〈0.05）. Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group （P〈0.05 or P〈0.01）.Conclusion： NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.