中成药

目的 探讨补肾助排汤对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞凋亡的影响。方法 SD大鼠皮下注射60 mg/kg脱氢表雄酮(DHEA)21 d构建PCOS模型，将造模成功的大鼠随机分为模型组、优思悦组(0.01 g/kg)和中药(补肾助排汤)低、中、高剂量组(8.82、17.64、35.28 g/kg),每组10只，另以10只皮下注射大豆油的大鼠为空白组。各组灌胃给药28 d后，观察大鼠一般状况、动情周期及卵巢的形态学变化；ELISA法检测血清睾酮水平；HE染色观察卵巢组织病理学；TUNEL染色检测卵巢颗粒细胞凋亡率；透射电镜观察卵巢组织线粒体超微结构及自噬变化；RT-qPCR和Western blot法检测卵巢组织线粒体自噬信号通路和卵巢颗粒细胞凋亡相关mRNA及蛋白表达。结果 与空白组比较，模型组大鼠失去规律动情周期；卵巢组织中出现大量的小卵泡，并呈现多囊样改变；血清睾酮水平升高(P<0.01);卵泡颗粒细胞凋亡率升高(P<0.01);杆状线粒体减少，肿胀的线粒体逐渐增多，且伴有线粒体膜损伤、线粒体嵴断裂，同时自噬现象也随之增多；卵巢组织Bax、Caspase-3、Beclin1、LC3B、PINK1、Parkin mRNA及蛋白表达升高(P<0.01),P62、Bcl-2 mRNA及蛋白表达降低(P<0.01)。与模型组比较，中药各剂量组和优思悦组大鼠行为表现出现不同程度的改善；卵巢组织中小卵泡数量减少；血清睾酮水平降低(P<0.01);卵泡颗粒细胞凋亡率降低(P<0.01);线粒体结构损伤程度减轻，自噬现象减少；中药低、中、高剂量组和优思悦组卵巢组织Bax、Caspase-3、Beclin1、LC3B、PINK1、Parkin mRNA及蛋白表达降低(P<0.05,P<0.01),P62、Bcl-2 mRNA及蛋白表达升高(P<0.05,P<0.01)。结论 补肾助排汤能够改善PCOS大鼠卵巢颗粒细胞凋亡，其作用与调控PINK1/Parkin信号通路介导的线粒体自噬相关。

AIM To explore the effect of Bushen Zhupai Decoction on the apoptosis of ovarian granulosa cells in rats with polycystic ovary syndrome(PCOS).METHODS PCOS models were established by subcutaneous injection of 60 mg/kg dehydroepiandrosterone(DHEA) in SD rats for 21 days.The successfully modeled rats were randomly divided into the model group, the Yousiyue [drospirenone and ethinylestradiol tablets(Ⅱ)] group(0.01 g/kg), and the low-, medium-, and high-dose groups of traditional Chinese medicine(Bushen Zhupai Decoction, 8.82, 17.64, 35.28 g/kg), with 10 rats in each group. Additionally, 10 rats subcutaneously injected with soybean oil were taken as the blank group. After 28 days of intragastric administration in each group, the general condition, estrous cycle and morphological changes of the ovaries of the rats were observed. Serum testosterone levels were detected by ELISA. HE staining was used to observethe histopathology of ovarian tissue. The apoptosis rate of ovarian granulosa cells was detected by TUNEL. The ultrastructure of mitochondria and the changes of autophagy in ovarian tissue were observed by transmission electron microscopy. The mRNA and protein expressions related to mitochondrial autophagy signaling pathways and apoptosis of ovarian granulosa cells inovarian tissues were detected by RT-qPCR and Western blot.RESULTS Compared with the blank group, the rats in the model group lost their regular estrous cycles. A large number of small follicles appear in the ovarian tissue and show polycystic changes. The serum testosterone level increased(P<0.01). The apoptosis rate of follicular granulosa cells increased(P<0.01). The number of rod-shaped mitochondria decreased, while the number of swollen mitochondria gradually increased, accompanied by mitochondrial membrane damage and mitochondrial crest rupture. Meanwhile, the phenomenon of autophagy also increased. The mRNA and protein expressions of Bax, Caspase-3, Beclin1, LC3B, PINK1 and Parkin in ovarian tissue increased(P<0.01), while the mRNA and protein expressions of P62 and Bcl-2 decreased(P<0.01). Compared with the model group, the behavioral performance of rats in each dose group of TCM and the Yousiyue group improved to varying degrees. The number of small follicles in ovarian tissue decreased. The serum testosterone level decreased(P<0.01). The apoptosis rate of follicular granulosa cells decreased(P<0.01). The degree of mitochondrial structural damage was alleviated and the phenomenon of autophagy was reduced. The mRNA and protein expressions of Bax, Caspase-3, Beclin1, LC3B, PINK1 and Parkin in the ovarian tissues of the low, medium and high-dose groups of TCM and the Yousiyue group decreased(P<0.05, P<0.01), while the mRNA and protein expressions of P62 and Bcl-2 increased(P<0.05, P<0.01).CONCLUSION The Bushen Zhupai Decoction can improve the apoptosis of ovarian granulosa cells in PCOS rats, and its effect is related to the regulation of mitochondrial autophagy mediated by the PINK1/Parkin signaling pathway.