补肾养血活血中药复方通过PI3K/Akt/mTOR信号通路调控细胞自噬促卵泡发育的分子机制研究

Follicular maldevelopment is the main cause of gynecological difficulties such as oligomenorrhea, hypomenorrhea, amenorrhea, infertility and premature ovarian failure. Its incidence continues to rise. The classic method of reinforcing kidney，nourishing and activating blood has been shown to ameliorate such conditions. However, the modern biological mechanism of it has not been clarified. Our finished NSFC (Natural Science Foundation of China) project (NO. 81173289) has elucidated the molecular mechanisms of reinforcing kidney，nourishing and activating blood method from the molecular level involving gene and protein expressions. The results of the experiment have confirmed that the Chinese herbal compound prescription Xinjiaguishenwan(XJGSW) using the method of reinforcing kidney, nourishing and activating blood, can repair the damaged ovarian granulosa cells by activating the PI3K/Akt/mTOR signal pathway. On this basis, this project is at the leading edge of cell autophagy, which is closely related to follicular development and PI3K/Akt/mTOR signal pathways. Our project is to adopt the modern detection technology of cell biology and molecular biology, such as gene chips, and continues to use tripterygium wilfordii to induce ovarian injury in the rat model. In this project, we intend to combine the experiment of animal experiment with isolated cell culture. We will further explore the molecular mechanisms of reinforcing kidney，nourishing and activating blood to promote follicular development. In the present study, we aim to observe the morphology of autophagy using the transmission electron microscopy (TEM), study autophagy correlation factors as well as the relationship between apoptosis and autophagy. The results are expected to elucidate the key issues involved in the reproductive endocrine regulation and follicle growth promotion by traditional Chinese medicine, as well as provide reference for prevention and treatment of reproductive toxicity of Tripterygium wilfordii. Therefore, the present study is of significant scientific meaning and practical application value.

卵泡发育障碍是引起月经稀发、月经过少、闭经、不孕症及卵巢早衰等妇科疑难病的主因，临床呈持续高发态势，补肾养血活血法为治疗此类疾病之经典治法，但其现代科学生物学机制尚未阐明。已结题国家自然科学基金项目（NO.81173289）从蛋白和基因水平初步探明补肾养血活血中药复方新加归肾丸通过激活PI3K/Akt/mTOR信号通路，修复受损的卵巢颗粒细胞的分子机制。在此基础上，本项目紧扣与卵泡发育和PI3K/Akt/mTOR信号通路皆密切相关的细胞自噬这一研究前沿，拟采用基因芯片等细胞及分子生物学现代检测技术，复制雷公藤致卵巢损伤大鼠模型，结合在体动物实验与离体细胞培养，从自噬体的电镜下形态、自噬相关因子表达、自噬及凋亡关系等方面深入探索补肾活血养血法促卵泡发育的分子机制。其成果有望阐明中药调控生殖内分泌与促卵泡发育的关键科学问题，亦可为雷公藤生殖毒性的防治提供参考，具有重要科学意义和实际应用价值。

卵泡发育障碍是引起月经稀发、月经过少、闭经、不孕症及卵巢早衰等妇科疑难病的主因，补肾养血活血法是促卵泡发育的经典治法，其现代科学生物学基础尚未阐明。本项目紧扣与卵泡发育密切相关的细胞氧化应激-自噬-凋亡发病机制和重要细胞器线粒体，采用细胞生物学、分子生物学等现代科技手段，选择雷公藤甲素(triptolide, TP)致卵巢损伤疾病模型，结合在体动物实验与离体细胞培养，深入探索补肾养血活血法促卵泡发育的分子机制。.主要研究结果：.（1）TP不同剂量和时间均可成功建造卵巢功能减退大鼠模型。.(2)新加苁蓉菟丝汤通过激活PI3K/AKT/mTOR信号通路，减少大鼠卵巢中自噬小体和自噬溶酶体的数量，下调自噬相关因子LC3Ⅱ、P53、Beclin1、LAMP2、Cathepsin-D并上调LC3I，抑制大鼠卵巢颗粒细胞自噬,修复TP所致的大鼠卵巢颗粒细胞损伤以促进卵泡发育。.(3)新加从蓉菟丝汤通过激活PI3K/AKT/mTOR信号通路一方面降低颗粒细胞凋亡率、下调凋亡相关分子Cleaved-Caspase-3抑制颗粒细胞凋亡，另一方面减少颗粒细胞自噬小体和自噬溶酶体的数量，下调自噬相关因子LC3II、Beclin1、LAMP2、Cathepsin-D，上调p62抑制颗粒细胞自噬流的过度活化，修复TP所致的大鼠卵巢颗粒细胞损伤。.(4)新加苁蓉菟丝汤可缓解TP所致大鼠卵巢和颗粒细胞氧化应激、线粒体功能障碍，激活PINK1/Parkin介导的线粒体自噬，抑制颗粒细胞凋亡。.(5)新加苁蓉菟丝汤可能通过调控SIRT1/AMPK和PI3K/Akt两条信号通路对抗氧化应激损伤，并恢复卵巢颗粒细胞保护性自噬以促进卵泡发育。.综上所述，补肾养血活血中药复方新加苁蓉菟丝汤通过调控PI3K/Akt/mTOR介导的自噬和凋亡信号通路、SIRT1/AMPK介导的氧化应激信号通路以及PINK1/Parkin介导的线粒体自噬信号通路，能够修复雷公藤甲素(TP)所致的卵巢颗粒细胞损伤，促进卵泡发育和恢复卵巢功能。本项目的研究成果为中医药促卵泡发育以治疗卵巢功能减退类疾病提供了科学依据。