基于PPARG1介导线粒体调控的䃼肾填精法改善高雄性PCOS不孕IVF-ET妊娠结局和子代表型的作用机制

Based on the theories of “Shen governing reproduction" and "Shen as the congenital source", the traditional Chinese medicine therapeutic principle of Bu-Shen-Tian-Jing is important for the treatment of infertility in traditional Chinese medicine. The applicant found that Bu-Shen-Tian-Jing can significantly improve the pregnancy outcomes and subtype of the offpring of hyperandrogenism polycystic ovary syndrome (PCOS) patients undergoing in vitro fertilization embryo transfer (IVF-ET) by regulating peroxisome proliferator-activated receptor gamma 1 (PPARG1). PPARG is an important target for improving mitochondrial function. Mitochondria are significantly associated with follicular development, fertilization and embryo development, which provides new ideas for revealing the epigenetic mechanism of Bu-Shen-Tian-Jing. This project aims to explore whether Bu-Shen-Tian-Jing can improve the impaired mitochondrial functions and activities of PCOS patients by regulating PPARG1 methylation levels of ovarian granulosa cells and oocytes, and ultimately improve the pregnancy outcomes of IVF-ET through pyrosequencing, targeted metabolomics and ChIP methods; to investigate the effects of Bu-Shen-Tian-Jing on phenotype of the PCOS offspring, including reproductive function, glucose metabolism and nerve functions, and the epigenetic mechanisms through pyrosequencing, transmission electron microscopy, mice IVF-ET technologies. The present study is of great significance to reveal the biological basis of the theories of “Shen governing reproduction" and "Shen as the congenital source" and has certain academic value for enriching and developing reproductive theories of traditional Chinese medicine.

基于“肾主生殖”和“肾为先天之本”理论，补肾填精法是中医治疗不孕的重要治则。申请人发现补肾填精法通过调控PPARG1显著改善了行体外受精-胚胎移植（IVF-ET）的高雄性多囊卵巢综合征（PCOS）不孕患者的妊娠结局和子代表型。PPARG是改善线粒体功能的重要靶点，而线粒体与卵泡发育、受精和胚胎发育密切相关，这为揭示补肾填精法的表观遗传学机制提供了新的思路。本项目拟应用焦磷酸测序、靶向代谢组学、ChIP等技术研究补肾填精法是否通过调控卵巢颗粒细胞和卵母细胞中PPARG1的甲基化水平而拯救PCOS受损的线粒体功能活性，最终改善IVF-ET妊娠结局；通过焦磷酸测序、透射电镜、小鼠IVF-ET等技术探讨补肾填精法对PCOS子代生殖功能、糖代谢、神经功能等表型的影响及其表观遗传学机制。这对揭示中医“肾主生殖”和“肾为先天之本”理论的生物学基础具有重要意义，对丰富和发展中医生殖理论具有一定的学术价值。

多囊卵巢综合征（PCOS）是引起不孕的常见妇科内分泌疾病，具有高发性、异质性、难治性和终身性的特点。任何单一的化学药物在治疗PCOS方面都存在其局限性。PCOS对子代的远期健康会产生严重的负面影响。这些事实表明：改善高雄性PCOS不孕患者的妊娠结局并关注这一群体的子代健康具有重要的临床意义和科学价值。中医经典理论强调肾与生殖的关系最为密切。肾藏精，主生殖，为先天之本，是生长发育与生殖之根本。补肾填精法是临床治疗PCOS不孕的常见治则，疗效确切。本课题首先发现补肾填精方显著提高了行IVF-ET的PCOS患者的累计临床妊娠率、活产率和足月产率。进一步的临床随访的队列研究发现：补肾填精法对高雄性PCOS患者的1-3岁龄女性子代的神经发育起到了一定的改善作用。通过进一步采用Mito-SOX标记细胞线粒体超氧化物，发现补肾填精方可通过上调SIRT3表达进而抑制PA诱导的线粒体ROS产生。基于高雄性多囊卵巢综合征模型大鼠的研究发现：补肾填精方干预PCOS模型大鼠可改善动情周期、葡萄糖耐量、卵巢形态和卵泡发育；同时可改善PCOS模型大鼠卵巢颗粒细胞的线粒体氧化应激水平，抑制p38 MAPK激活，促进PI3K/AKT信号通路的激活，从而有利于PCOS卵泡发育。进一步研究发现：补肾填精方显著改善了PCOS组雌性子代的水迷宫目标停留时间（5周龄：P=0.039）和海马齿状回区树突棘密度（5周龄P=0.003，13周龄P=0.000）；改善了PCOS组雌性子代海马神经元和突触的超微结构；显著回调了PCOS组雌性子代海马齿状回GABRB1表达水平（5周龄：P=0.016；13周龄：P=0.014）。研究成果对揭示中医“肾主生殖”和“肾为先天之本”理论的生物学基础具有重要意义，对丰富和发展中医生殖理论具有一定的学术价值。