基于PGC-1α-TFAM-ROS途径探讨六味地黄丸调控POF小鼠卵巢颗粒细胞线粒体功能的机制

Ovarian premature aging (POF) is the important cause of female infertility,ovarian granulosa cell apoptosis can induced POF follicular atresia. Liuwei dihuang pill is the basis for the treatment of POF, curative effect, but the mechanism is unknown. The study found that liuwei dihuang pill can promote follicular granulosa cell proliferation and resistance and another role is oxidative stress, our initial success establish POF mice model, and confirmed that the antioxidant therapy of granulosa cells have a protective effect. Oxidative stress caused by a reactive oxygen species (ROS) is a key link in the process of trigger apoptosis, PGC-1αcan control mitochondrial transcription factors (TFAM) which can reverse regulation of mitochondrial dysfunction, reduce ROS generation. So whether the liuwei dihuang pill by PGC-1α-TFAM-ROS ways to improve the function of mitochondria, break the vicious cycle mitochondrial-ROS , inhibiting cell appotosis, so as to achieve the purpose of treatment of POF? To mitochondrial function as the breakthrough point, this study intends to use animal models + cell culture method, using scanning electron microscopy , laser confocal, RT-PCR,immunohistochemistry and Western blot techniques, from molecular Angle discusses the mechanism of mitochondrial function of liuwei dihuang pill possible regulation POF ovarian granular cells in mice,clarify the mechanism of the traditional Chinese medicine treatment.

卵巢早衰（POF）是导致女性不孕的重要原因。卵巢颗粒细胞凋亡会引起卵泡闭锁诱导POF的发生。六味地黄丸是治疗POF的基础方，疗效确切，但机制不明。研究发现六味地黄丸有促卵巢颗粒细胞增殖、卵泡发育及抗氧化应激的作用，我们前期成功建立POF小鼠模型，并证实抗氧化剂治疗对颗粒细胞有保护作用。活性氧（ROS）所致的氧化应激是触发细胞凋亡的关键环节，PGC-1α可调控线粒体转录因子（TFAM）从而逆转线粒体功能障碍、减少ROS产生。那么六味地黄丸是否通过PGC-1α-TFAM-ROS途径改善线粒体功能，打破线粒体-ROS恶性循环，抑制细胞凋亡，从而达到治疗POF的目的？本研究以线粒体功能为切入点，拟采用动物模型+细胞培养方法，运用扫描电镜、激光共聚焦、RT- PCR、免疫组化、Western blot等技术，从分子角度探讨六味地黄丸调控POF小鼠卵巢颗粒细胞线粒体功能的机制，阐明中药治疗的作用机理。

卵巢早衰（POF）是指卵巢功能衰竭所导致40岁之前出现原发或继发性闭经、雌激素缺乏、促性腺激素水平升高，是女性不孕的重要原因之一，卵巢颗粒细胞凋亡会引起卵泡闭锁诱导POF的发生。六味地黄丸是治疗POF的基础方，疗效确切，但机制不明。我们运用六味地黄丸干预自然衰老大鼠发现六味地黄丸可能通过抑制活性氧-细胞凋亡的途径在一定程度上改善卵巢功能，且六味地黄丸可以有效干预环磷酰胺联合白消安引起的小鼠卵巢功能减退，改善卵巢的内分泌功能，提高小鼠血清激素水平，促使卵泡发育，并降低化疗药物生殖毒副作用。进一步探索六味地黄丸的作用机理发现六味地黄丸可能通过PGC-1α-TFAM-ROS途径在一定程度上改善卵巢早衰小鼠卵巢颗粒细胞线粒体的功能，改善颗粒细胞凋亡情况，从而改善卵巢早衰小鼠卵巢功能。运用蛋白组学分析POF模型小鼠、中药干预后小鼠及空白小鼠卵巢蛋白，发现与免疫炎症反应相关蛋白有较明显的变化，提示卵巢功能与免疫炎症反应之间的关系。制备人颗粒细胞损伤模型，六味地黄丸含药血清干预，细胞层面再次验证六味地黄丸可能通过PGC-1α-TFAM-ROS途径改善卵巢功能。本课题阐明了六味地黄丸治疗POF的分子机制，为其治疗POF有效性提供实验依据，希冀为作用机制研究提供新思路，为其临床推广应用和进一步的开发提供研究基础。