护阳养坤方通过RNA-m6A甲基化动态修饰调控卵巢颗粒细胞死亡线粒体通路的机制研究

Premature ovarian insufficiency, with little effective treatment so far, affects women’s physical and mental health seriously. Stress and environmental factors damage the ovarian function, which still is an unclear mechanism. RNA m6A methylation is a hot topic in epigenetics, it participates in gene modification and expression, as well as cell fate determination under the influence of those factors. Apoptosis caused by knockdown of m6A transferase is closely related to P53 pathway. Mitochondria is the center regulator of apoptosis in ovarian granulosa cells, while P53-mediated Bcl-2 family protein is its "switch". Based on the reproductive aging theory of "the end of Tian gui belongs to the Tai-Yin ", our team created "Hu-yang-Yang-kun decoction" by referencing the classic Danggu Buxue decoction. Hu-yang-Yang-kun decoction has shown effectiveness in clinical and basic research, it can regulate mitochondria Pathway MAPK/P53/Bcl-2, and inhibit ovarian granulosa cell apoptosis. In addition, we first found the correlation between RNA m6A and reproductive aging. Therefore, targeting the mitochondrial pathway of cell death and its key-point P53， we will use m6A-seq and RNA-seq technology in both disease and gene modification models to reveal the anti-ovarian aging mechanism of Chinese Herb decoction form an epigenetic perspective.

早发性卵巢功能不全严重影响女性心身健康，尚缺乏有效治疗手段。应激和环境等因素损伤卵巢功能，但机制不清。RNAm6A甲基化是表观遗传学新晋热点，受应激环境因素影响修饰基因表达，决定细胞命运。m6A转移酶敲低所致的细胞凋亡与P53信号密切相关。线粒体是卵巢颗粒细胞凋亡调控中心，P53介导Bcl-2家族蛋白则是其“开关”。本团队基于“天癸已绝，乃属太阴经也”的生殖衰老理论，创制以经典名方当归补血汤为基础的“护阳养坤方”，在临床和基础研究中显示有效性，可调控MAPK/P53/Bcl-2家族蛋白抑制卵巢颗粒细胞凋亡，此外我们首次检测RNAm6A甲基化与生殖衰老有关。故本项目以P53为纽带，以细胞死亡线粒体通路为靶点，通过疾病、基因修饰模型，运用m6A-seq、RNA-seq测序技术，创新性的从表观遗传学角度，探讨m6A甲基化修饰介导生殖衰老以及护阳养坤方抗衰老机制，有重大理论和应用价值。

早发性卵巢功能不全（POI）在临床缺乏有效治疗，既往临床研究表明中药护阳养坤方治疗POI有药效。m6ARNA修饰是近年来表观遗传学方向的研究热点，m6ARNA修饰可能参与到生殖衰老疾病的发生机制，为了阐述其在POI发病机制及护阳养坤方作用相关的分子机制，本研究通过动物、细胞实验进行研究探索。本研究基于前期研究基础，建立VCD诱导POI大鼠模型，并构建了VCD诱导的人卵巢颗粒细胞COV434细胞凋亡模型，首先验证了护阳养坤方的药效表型，大鼠实验结果提示护阳养坤方可以有效保护POI大鼠卵巢体积、可促使发育卵泡中成熟卵泡数量增加、促使血清AMH上升、一定程度恢复血清FSH、E2的周期性变化趋势；细胞实验结果提示护阳养坤方可以对抗细胞凋亡、保护细胞增殖。为探索其m6A RNA修饰作用机制，通过Dotblot、LC-MS/MS和或比色法对卵巢组织及COV434细胞的m6A水平进行检测，结果提示：VCD诱导下POI大鼠卵巢m6A RNA修饰水平的下降，而护阳养坤方干预下POI大鼠卵巢整体m6A RNA修饰增加，可能与FTO、ALKBH5等催化酶的相对变化有关。通过Western Blot法筛选JNK/Nrf2/P53/BCL-2信号通路上的差异靶标有P53、(p)JNK、BAK、PUMA和BAX蛋白，然后通过MeRIP-qPCR实验验证目的靶标，结果表明m6A-Jnk和m6A-Tp53转录本是POI发病P53/BCL-2线粒体凋亡信号上的重要靶点，护阳养坤方通过调控m6A-Tp53转录本可逆修饰抑制P53介导的BCL-2线粒体凋亡，从而发挥抗细胞凋亡作用保护卵巢功能。此外项目还通过MeRIP-seq测序筛选出了m6A 修饰的差异表达基因主要为：SAC,Bub1,Espl1,Mapk15等；KEGG pathway富集提示VCD-POI疾病模型及中药护阳养坤方干预的发生机制与卵母细胞减数分裂：微管蛋白聚合、调节动粒-微管连接的蛋白及染色体分离等密切相关；本项目通过疾病、基因修饰模型，运用MeRIP-seq测序技术，创新性的从表观遗传学角度，探讨m6A RNA甲基化修饰介导生殖衰老以及护阳养坤方抗衰老机制。通过MeRIP-seq筛选出护阳养坤方通过m6A甲基化酶调控的卵巢颗粒细胞凋亡的核心通路及相关基因，为后续研究提供了理论基础和方向，有重大理论和应用价值。