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二甲双胍改善多囊卵巢综合征并激活雌性生殖干细胞

王春红^1,2, 王强强^1,2, 苏亚珊¹, 孙亚群¹, 孙苗¹, 刘心蕊¹, 马会明¹, 李广永³, 杜小利², 何瑞^1,2,*

¹宁夏医科大学基础医学院，生育力保持教育部重点实验室，银川 750004；²宁夏医科大学中医学院，回医药现代化教育部重点实验室，银川 750004；³宁夏医科大学总医院泌尿外科，银川 750004

摘要

多囊卵巢综合征(polycystic ovary syndrome, PCOS)是生育年龄妇女常见的一种复杂的内分泌及代谢异常所致的疾病，是女性不孕的重要因素。二甲双胍是临床应用最广泛的经典口服降糖药物，近年来被应用于PCOS治疗，但其作用机制尚不明确。本研究通过建立小鼠PCOS模型，论证二甲双胍对PCOS的改善作用及其机制。4~5周龄雌性C57BL/6J小鼠连续21天给予来曲唑(每天灌胃1 mg/kg)联合高脂饮食，建立PCOS模型。成模后灌胃给予二甲双胍(每天200 mg/kg)，1个月后取材，期间检测体重并进行糖耐量试验。取材后行苏木素-伊红(H&E)染色检测卵巢病理改变；ELISA法测定血清中抗缪勒氏管激素(anti-Mullerian hormone, AMH)、卵泡刺激素(follicle-stimulating hormone, FSH)、黄体生成素(luteinizing hormone, LH)、雌二醇(E2)、睾酮等激素水平；免疫组织化学及免疫荧光染色法检测雌性生殖干细胞(female germline stem cells, FGSCs)标志物DDX4/MVH及PCNA表达及定位；Western blot检测小鼠卵巢中DDX4/MVH、PCNA、cyclin D2以及AMPK/mTOR通路蛋白。结果显示，PCOS小鼠在接受二甲双胍治疗后，体重逐渐恢复正常，糖耐量试验显示糖耐量明显改善，血清E2水平升高，AMH、LH、睾酮水平及LH/FSH比值下降，卵巢多囊病变减轻，闭锁卵泡减少，且具有增殖性的FGSCs数目明显增多，卵巢组织PCNA、cyclin D2等增殖相关蛋白增加，p-mTOR、p-AMPK等蛋白明显上调。以上结果提示，二甲双胍在改善PCOS高雄激素血症、糖耐量异常及卵巢多囊病变的同时，还可以促进FGSCs增殖并激活其功能，其作用可能与AMPK、mTOR磷酸化有关。这些发现为二甲双胍治疗PCOS以及改善卵泡发育提供了新的科学依据和方向。

关键词： 二甲双胍; 多囊卵巢综合征; 雌性生殖干细胞; AMPK/mTOR

Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice

WANG Chun-Hong^1,2, WANG Qiang-Qiang^1,2, SU Ya-Shan¹, SUN Ya-Qun¹, SUN Miao¹, LIU Xin-Rui¹, MA Hui-Ming¹, LI Guang-Yong³, DU Xiao-Li², HE Rui^1,2,*

¹宁夏医科大学基础医学院，生育力保持教育部重点实验室，银川 750004；²College of Traditional Chinese Medicine, Key Laboratory of Hui Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan 750004, China；³Urology Department of General Hospital, Ningxia Medical University, Yinchuan 750004, China

Abstract

Polycystic ovary syndrome (PCOS) is a common disease caused by complex endocrine and metabolic abnormalities in women of childbearing age. Metformin is the most widely used oral hypoglycemic drug in clinic. In recent years, metformin has been used in the treatment of PCOS, but its mechanism is not clear. In this study, we aimed to investigate the effect of metformin on PCOS and its mechanism through PCOS mouse model. Female C57BL/6J mice aged 4–5 weeks were intragastrically given letrozole (1 mg/kg daily) combined with a high-fat diet (HFD) for 21 days to establish the PCOS model. After modeling, metformin (200 mg/kg daily) was intragastrically administered. One month later, the body weight and oral glucose tolerance test (OGTT) were measured. Hematoxylin eosin (H&E) staining was used to detect the pathological changes of ovary. The serum levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2 and testosterone (T) were measured by ELISA. The expression of DDX4/MVH was detected by immunohistochemistry. DDX4/MVH and PCNA were co-labeled by immunofluorescence. The protein levels of DDX4/MVH, PCNA, cyclin D2, AMPK and mTOR were detected by Western blot. The results showed that after metformin treatment, the body weights of PCOS mice were gradually returned to normal, glucose tolerance was significantly improved, serum E2 levels were increased, while AMH, LH, T levels and LH/FSH ratio were decreased. Ovarian polycystic lesions were reduced with reduced atresia follicles. Furthermore, the number of proliferative female germline stem cells (FGSCs) and levels of proliferation related proteins (PCNA, cyclin D2) were significantly increased, and the p-mTOR and p-AMPK levels were markedly up-regulated. These results suggest that metformin treatment not only improves hyperandrogenemia, glucose intolerance and polycystic ovarian lesions in PCOS, but also activates the function of FGSCs. The underlying mechanism may be related to the phosphorylation of AMPK and mTOR. These findings provide new evidence to use metformin in the treatment of PCOS and follicular development disorder.

Key words: metformin; polycystic ovary syndrome; female germline stem cells; AMPK/mTOR

收稿日期：　　录用日期：

通讯作者：何瑞　　E-mail: ruihe515@163.com

DOI: 10.13294/j.aps.2021.0090

引用本文：

王春红, 王强强, 苏亚珊, 孙亚群, 孙苗, 刘心蕊, 马会明, 李广永, 杜小利, 何瑞. 二甲双胍改善多囊卵巢综合征并激活雌性生殖干细胞[J]. 生理学报 2022; 74 (3): 370-380.

WANG Chun-Hong, WANG Qiang-Qiang, SU Ya-Shan, SUN Ya-Qun, SUN Miao, LIU Xin-Rui, MA Hui-Ming, LI Guang-Yong, DU Xiao-Li, HE Rui. Metformin improves polycystic ovary syndrome and activates female germline stem cells in mice. Acta Physiol Sin 2022; 74 (3): 370-380 (in Chinese with English abstract).