小檗碱干预PCOS胰岛素信号通路关键分子作用机制研究

Polycystic ovary syndrome(PCOS) is a very common and complex endocrine disease that affects 8% to 10% of women in reproductive age life.It is characterized by chronic anovulation,irregular menses and infertility,and symptoms of androgen excess,such as hirsutism and ache.In the long term,it increase the risk of type 2 diabetes,coronary heart disease,hypertensive disease,hyperlipidemia and other metabolic complications. In addition，the role of modern medicine for PCOS is poor.It seriously affects the reproductive function and physical and mental health.Although it’s mechanism entirely remains unclear,hyperinsulinemia and hyperandrogen may be two important foundations of pathophysiology. It is reported that abnormal glucose metabolism and insulin resistance presented in ovaries of patients with PCOS，and excessive androgen synthesis by the ovaries is the main source of hyperandrogenism. Therefore, ovarian dysfunction may be related to the PCOS incidence .In the study ，intended by the detection of abnormal expression and activity of insulin signaling pathway in ovarian granulosa cells of PCOS patients, as well as construction of insulin signaling pathway expression / interference vector and transfected target cells， to investigate the abnormal insulin signaling pathway expression and activity effect on glucose metabolism, hormones and ovarian signaling molecule in PCOS.To prove that abnormality of insulin signaling pathway is a risk factor of PCOS ，and berberine improve abnormal glucose metabolism and hormone levels in ovarian granulosa cells through the influence on insulin signaling pathway and glucose transporter，as well as their phosphorylation and then improve the systemic glucose metabolism abnormalities and hyperandrogenism of PCOS patients, basic experiment and randomized controlled clinical trial about berberine are done. The study will provide evidence of evidence-based medicine for the theoretical mechanism and Clinical application of berberine treatment on PCOS.

多囊卵巢综合征（PCOS）临床表现月经紊乱、不孕、高雄激素等生殖障碍，也表现糖脂代谢和胰岛素抵抗等代谢异常的“双重特征”。我们前期实验发现该“双重特征”也存在于PCOS的卵巢内。文献荟萃分析发现，小檗碱具有降糖、降脂和改善胰岛素抵抗等作用。本项目： （1）体内机制。小檗碱治疗对PCOS患者卵巢细胞胰岛素信号通路关键分子表达的活性调节、胰岛素抵抗/雄激素分泌过多等作用的卵巢内疗效；（2）体外实验。采用基因转染或小RNA干涉技术，运用Wester-Blot， Realtime PCR等方法，探讨胰岛素信号通路关键分子对体外培养卵巢细胞PCOS“双重属性”的关键病理作用以及小檗碱的调控。阐明胰岛素信号传导通路关键分子的活性异常是PCOS卵巢功能“双重属性”偶联的重要病理机制，也是小檗碱对PCOS疗效的重要机制。本项目体现前期基础实验-临床疗效评价和临床基础研究的转化医学要求。

多囊卵巢综合征（polycystic ovary syndrome，PCOS）是常见的女性生殖内分泌代谢异常疾病。PCOS患者在临床上常伴有肥胖、胰岛素抵抗（insulin resistance，IR）、糖代谢异常、血脂异常和高血压等明显的代谢异常表现，远期发生Ⅱ型糖尿病（type 2 diabetes mellitus，T2DM）、心血管疾病（cardiovascular disease，CVD）的风险显著增加。肥胖是导致部分PCOS患者临床症状恶化的重要因素，与IR呈正相关。小檗碱（berberine）又称黄连素，是黄连等中药分离出来的一种异喹啉植物生物碱，具有二甲双胍类似的胰岛素增敏作用，可改善PCOS患者以IR为核心的糖脂代谢异常情况，降低LH水平、抑制雄激素合成及改善排卵情况等。本课题研究小檗碱干预PCOS胰岛素信号通路关键分子作用机制,通过临床基线数据分析，与对照组相比，PCOS患者SBP、BMI、血清LH、LH/FSH、T、FPG、AMH、 FSH水平差异具有统计学意义（P<0.05）。生殖方面可见，PCOS患者可以获得的卵泡数、ICSI成熟卵子数、及正常受精数均显著多于对照组，差异具有统计学意义（P<0.05）。运用多因子检测技术，制作标准曲线。结果显示，PCOS组血清IL-17a 、白介素1受体拮抗剂（IL-1Ra）、IL-6水平显著高于对照组，差异具有统计学意义（P<0.05）。颗粒细胞糖摄取能力检测中，与正常人群获取的颗粒细胞相比，PCOS患者卵巢颗粒细胞在胰岛素作用下葡萄糖的摄取能力显著下降，表现出胰岛素抵抗的病理状态。同时，在小檗碱作用后，细胞内荧光密度显著上升，说明小檗碱显著提高了PCOS患者颗粒细胞的胰岛素敏感性。小檗碱对PCOS患者KGN细胞葡萄糖代谢的调节作用及机制，高胰岛素环境下，IL-6可显著降低胰岛素敏感性，从而进一步降低KGN细胞葡萄糖摄取率。而小檗碱可显著增加胰岛素敏感性，从而显著提高KGN细胞葡萄糖摄取率。C.C降低了小檗碱胰岛素敏感性，从而影响KGN细胞葡萄糖摄取率。提示小檗碱可能是通过激活AMPK信号通路，从而增胰岛素敏感性，提高KGN细胞葡萄糖摄取率。