基于TGFβ/Smads信号通路探讨补肾促排卵汤治疗PCOS大鼠卵巢纤维化的分子机制

Discussion on the molecular mechanism of Bushen Cupailuan Decoction in treating ovarian fibrosis in polycystic ovary syndrome rats based on TGF-β/Smads signaling pathway

关键词:  补肾促排卵汤  多囊卵巢综合征  卵巢纤维化  TGFβ  Smads

Key words:  Bushen Cupailuan Decoction  polycystic ovary syndrome  ovarian fibrosis  TGF-β  Smads

摘要:

[目的] 探讨补肾促排卵汤对多囊卵巢综合征（PCOS）模型大鼠的治疗效果，并研究其调节卵巢纤维化TGF-β/Smads信号通路的分子机制。[方法] 将40只SD雌性大鼠随机分为空白组8只及脱氢表雄酮（DHEA）给药组32只，DHEA给药组每日颈部皮下注射DHEA（60 mg/kg），连续给药35 d；将造模成功的大鼠随机分为模型组、补肾促排卵汤低、中、高剂量组（5.58、11.16、22.32 g/kg）。补肾促排卵汤分别用生理盐水配成适当浓度，连续灌胃给药4周，每日1次；同时空白组与模型组大鼠予等量生理盐水进行灌胃。阴道涂片观察大鼠动情周期，HE法观察补肾促排卵汤对各组大鼠卵巢中卵泡发育的影响，天狼猩红染色法观察大鼠卵巢纤维化情况，酶联免疫吸附实验（ELISA）法检测大鼠血清雌二醇（E2）、睾丸酮（T）、黄体生成素（LH）、促卵泡激素（FSH）含量，实时荧光定量聚合酶链反应（RT-qPCR）法检测转化生长因子-β1（TGF-β1）、Smad3、Smad7 mRNA的表达，蛋白免疫印迹（Western Blot）法检测大鼠TGF-β1、p-Smad3、Smad7的蛋白表达。[结果] 与空白组比较，模型组大鼠丧失规律的动情周期，大鼠卵巢中囊状扩张卵泡增多，卵巢纤维化阳性面积明显增多，血清T、LH水平显著升高，同时TGF-β1、p-Smad3蛋白及mRNA表达显著增多，Smad7蛋白及mRNA表达显著减少（P<0.01）。与模型组比较，补肾促排卵汤中、高剂量组可明显改善大鼠的动情周期，减少囊状扩张卵泡的形成，降低卵巢纤维化阳性面积（P<0.01），下调卵巢TGF-β1蛋白及mRNA的表达（P<0.05，P<0.01），显著下调p-Smad3蛋白及mRNA的表达（P<0.01），上调Smad7蛋白及mRNA表达（P<0.05，P<0.01）。[结论] 补肾促排卵汤能有效治疗PCOS模型大鼠，可能是通过调节纤维化TGF-β/Smads信号通路上重要分子TGF-β1、p-Smad3、Smad7的表达从而发挥作用。

Abstract:

[Objective] To investigate the therapeutic effect of Bushen Cupailuan Decoction on polycystic ovary syndrome (PCOS) model rats, and to study the molecular mechanism of regulating TGF-β/Smads signaling pathway in ovarian fibrosis.[Methods] Forty SD female rats were randomly divided into the blank group (8 rats) and the Dehydroepiandrosterone (DHEA) group (32 rats). The DHEA group was injected subcutaneously with DHEA (60 mg/kg) daily for 35 days. The successfully modeled rats were randomly divided into the model group and the low-dose, medium-dose and high-dose groups of Bushen Cupailuan Decoction[5.58, 11.16 and 22.32 g/(kg/d)]. Bushen Cupailuan Decoction was prepared with normal saline at appropriate concentration, and was given continuous intragastric administration for 4 weeks, once a day. At the same time, rats in blank group and model group were given the same amount of normal saline for intragastric administration. The estrous cycle of rats was observed by vaginal smear, the effect of Bushen Cupailuan Decoction on ovarian follicle development was observed by Hematoxylin and eosin (HE) staining, ovarian fibrosis was observed by Sirius red staining. The serum estradiol (E2), testosterone (T) and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were quantified using Enzyme linked immunosorbent assay (ELISA). Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expression of TGF-β1, Smad3, Smad7. Western blot was used to detect the protein expression of TGF-β1, p-smad3 and Smad7 in rats.[Results] Compared with the blank group, the rats in the model group lost regular estrus cycle, partial follicular vesicle-like dilation in the ovaries, increased the positive area of rat ovarian fibrosis, and significantly increased serum T and LH. Meanwhile, the expression of TGF-β1, p-Smad3 protein and mRNA increased significantly, while the expression of Smad7 protein and mRNA decreased significantly (P<0.01). Compared with the model group, the medium and high dose groups of Bushen Cupailuan Decoction could effectively improve the estrus cycle of PCOS rats, reduce the formation of follicle expansion, reduce the positive area of ovarian fibrosis (P<0.01), down-regulate the expression of TGF-β1 protein and mRNA in ovary (P<0.05, P<0.01), and significantly down-regulate the expression of p-Smad7 protein and mRNA (P<0.01).[Conclusion] Bushen Cupailuan Decoction can effectively treat PCOS model rats, it may play a role by regulating the expression of TGF-β1, p-Smad3 and Smad7, which are important molecules in fibrotic TGF-β/Smads signal pathway.