补肾健脾方通过“SIRT3-线粒体-活性氧”调控途径改善老龄小鼠卵子质量的机制研究

Poor oocytes quality is a main concern for decreased fertility of advanced age women with infertility, currently there is no effective treatment available. Past research had found that damages to mitochondrial function caused by oxidative stress is one of the main mechanism that causes decreased oocyte quality in advanced age women with infertility. Our research had shown that Bu Shen Jian Pi formula can improve oocytes and embryo qualities of advanced age women with infertility, the effective component of this formula can improve mitochondrial function in the embryo, regulate the antioxidative enzymic activity and decrease reactive oxygen species level. Combining the newest researches that found SIRT3, which came from the family of proteins Silent Information Regulators Two, can improve oocyte quality of advanced age women with infertility through regulating mitochondrial function and the antioxidative enzymic activity and this particular family of protein has thus became the new target of research on. Our study will first observe the effect of Bu Shen Jian Pi formula on oocyte quality of naturally aged C57BL/6J mice in vivo, and attempt to explain its mechanism on a molecular level; then further this study by using an oocyte in vitro maturation system to investigate the effect of Bu Shen Jian Pi formula serum in regards to SIRT3 gene and its downstream protein expression level on the process of oocytes in vitro maturation of advanced age mice. Using "SIRT3 – Mitochondrial - Reactive oxygen species" regulation mechanism to demonstrate the effect of Bu Shen Jian Pi formula on improving oocyte quality, thus provides scientific evidence for the clinical efficacy of Bu Shen Jian Pi formula.

卵子质量低下是导致高龄不孕症妇女生育能力下降的主要问题，目前缺乏有效的治疗措施。既往的研究证实氧化应激所致的线粒体功能损害是高龄不孕症妇女卵子质量下降的重要机制之一。而我们的前期研究发现中药补肾健脾方能够改善高龄不孕症妇女的卵子及胚胎质量，其有效成分具有改善胚胎内部线粒体功能，调节抗氧化酶的活性，降低活性氧水平的作用。结合最新的研究发现沉默信息调节因子2的同源蛋白质SIRT3可通过调节线粒体功能及抗氧化酶的活性来调控高龄不孕症妇女的卵子质量。基于此，本课题拟采用自然衰老C57BL/6J小鼠作为研究对象，首先通过体内实验观察补肾健脾方对老龄小鼠卵子质量及线粒体功能的影响；进一步采用小鼠窦卵泡的体外成熟培养体系，观察补肾健脾方含药血清对老龄小鼠卵母细胞成熟过程中“SIRT3-线粒体-活性氧”调控机制中关键分子及蛋白的影响，阐明补肾健脾方改善卵子质量的作用机制。

【研究背景】：卵子质量低下是导致高龄不孕症妇女生育能力下降的主要问题，目前缺乏有效的治疗措施。既往的研究证实氧化应激所致的线粒体功能损害是高龄不孕症妇女卵子质量下降的重要机制之一。而我们的前期研究发现中药补肾健脾方能够改善高龄不孕症妇女的卵子及胚胎质量，其有效成分具有改善胚胎内部线粒体功能，调节抗氧化酶的活性，降低活性氧水平的作用。结合最新的研究发现沉默信息调节因子2的同源蛋白质SIRT3可通过调节线粒体功能及抗氧化酶的活性来调控高龄不孕症妇女的卵子质量。因此推测，补肾健脾中药可能是通过调控“SIRT3-线粒体-活性氧”通路中的关键分子及蛋白来发挥改善卵子及胚胎质量的作用。.【研究内容】：体内实验观察补肾健脾方对自然衰老小鼠的卵子及胚胎质量的影响；采用次级卵泡体外培养体系，观察氧化应激状态下小鼠卵泡的体外发育情况。围绕“Sirt3-线粒体-活性氧”通路，观察健脾方有效成分槲皮素对氧化应激状态下小鼠卵泡体外成熟的影响。.【主要成果】：1.补肾健脾方调控老龄小鼠胚胎质量的关键环节与其减少了老龄过程中窦前卵泡的损失，保存了小鼠的生育能力相关；2.补肾健脾方君药菟丝子有效成分槲皮素具有保护氧化应激状态下小鼠次级卵泡体外发育的作用；3.槲皮素通过调控“Sirt3-线粒体-活性氧”通路中的关键分子及蛋白来发挥保护氧化应激状态下小鼠窦前卵泡发育的作用。.【关键数据】：五月龄和六月龄小鼠采用补肾健脾方灌胃后，卵巢中窦前卵泡的计数较对照组明显增多；槲皮素可以改善氧化应激状态下次级卵泡发育迟缓的问题，使卵泡发育的成熟度接近正常；槲皮素能够促进氧化应激状态下卵泡的发育是通过上调Sirt3的表达，提高线粒体的功能及抗氧化酶的活性，抑制凋亡基因的表达，降低活性氧的水平来发挥作用。.【科学意义】：首次明确了补肾健脾方改善卵子及胚胎质量可能与其减少了老龄过程中窦前卵泡的损失相关，其有效成分槲皮素通过调控“Sirt3-线粒体-活性氧”通路中的相关蛋白，改善了老龄过程中的氧化应激环境，从而保存了卵巢功能；这为补肾健脾方保护卵子及胚胎质量提供科学依据，为中药保存生育能力提供理论指导。