补肾促排卵汤通过FSH-FOXO1信号轴和表观遗传修饰调控DOR小鼠氧化应激的作用机制

Decline in ovarian reserve(DOR) in elderly women is a major problem in infertility diagnosis and treatment. Study confirms that DOR is induced by oxidative stress bacause of the imbalance of ROS and antioxidant in ovarian. Professor Xia Guicheng applied the Tonifying kidney and Inducing Ovulation Decoction for the treatment of Decline in ovarian reserve (DOR), the clinical effect was significant. Previous studies confirmed that Tonifying kidney and Inducing Ovulation Decoction can effectively protect oxidative stress injury. Oxidative stress can induce granulosa cell apoptosisby FSH-FOXO1 signal axis. Epigenetic modification can regulate the antioxidant enzyme systems to clear ROS. In this study, We used the mouse model of DOR to study the effect of Tonifying kidney and Inducing Ovulation Decoction and its active ingredients on the methylation mechanism of histone H3K4 to to regulate antioxidant enzyme activity by CFP1 and mechanism of granulosa cell survival in mice by FSH-PKA-PI3K-AKT-Foxo1 signal axis in the process of oocyte maturation. We also want to explore the mechanism of DNA demethylation to activate gene expression by TET enzyme mediated by CRL4 complex during fertilization and early embryonic development by means of RNA-seq, gene transfection, RNAi, et al. The purpose of the subject is to explore the mechanism of Tonifying kidney and Inducing Ovulation Decoction regulating oxidative stress in DOR mice and to provide experimental basis for the prevention and treatment of premature ovarian failure, improving ovarian reserve function, improving the quality of oocytes and embryos.

高龄女性卵巢储备功能低下（DOR）是不孕症诊疗一大难题。研究证实DOR是卵巢内活性氧（ROS）与抗氧化剂失衡导致氧化应激损伤所致。国医大师夏桂成教授运用补肾促排卵汤治疗DOR，临床疗效显著。前期研究证实补肾促排卵汤可有效保护氧化应激损伤，氧化应激可通过FSH-FOXO1信号轴诱导颗粒细胞凋亡，表观遗传修饰调控抗氧化酶系统影响ROS清除。本项目借助小鼠DOR模型，运用RNA-seq，基因转染、RNAi等技术手段，研究补肾促排卵汤及有效成分在卵母细胞成熟过程中通过CFP1介导组蛋白H3K4甲基化调控抗氧化酶活性和通过FSH-FOXO1信号轴促进颗粒细胞存活，受精及早期胚胎发育过程中通过CRL4复合体激活TET酶介导DNA去甲基化激活基因表达，全面阐释补肾促排卵汤调控DOR小鼠氧化应激的作用机制，为补肾促排卵汤防治卵巢早衰、改善卵巢储备功能、提高卵子和胚胎质量提供科学依据。

本课题通过研究补肾促排卵汤在卵母细胞成熟过程中通过CFP1介导组蛋白H3K4甲基化调控抗氧化酶活性，通过FSH-PKA-PI3K-AKT-FOXO1信号轴促进小鼠颗粒细胞存活，在受精及早期胚胎发育过程中通过CRL4激活TET酶介导 DNA去甲基化激活基因表达，全面深入探讨补肾促排卵汤治疗卵巢储备功能下降的作用机制。本课题通过网络药理学和UHPLC-QE-MS筛选中药复方与疾病靶点、关键通路及蛋白，对临床DOR患者颗粒细胞进行5mc和5hmc甲基化测序，筛选出DNA甲基化和去甲基化过程差异位点邻近基因通路；通过体内实验检测DOR小鼠生殖激素及氧化应激相关指标ROS、SOD、GSH-Px、MDA，卵巢组织形态，卵巢内8-OHdG、4-HNE、NTY及KISS1的表达，卵巢颗粒细胞凋亡相关基因Bax、Bak、Bcl-xl、Bcl-2及Bim mRNA表达及FOXO1、FOXO3a、KISS1、P53、P-AKT、PI3K及FSHr蛋白表达差异，研究补肾促排卵汤通过FSH-PKA-PI3K-AKT-FOXO 信号轴促进小鼠颗粒细胞存活机制；通过建立氧化应激导致DOR小鼠模型及注射MLN4924抑制小鼠卵巢CRL4蛋白表达，比较各组小鼠获卵数、生殖激素及早期胚胎中CFP1蛋白定位及表达、卵母细胞中TET1/2/3、受精卵全能性基因、颗粒细胞排卵相关基因mRNA表达及卵巢组织CRL4、H3K4me3、CFP1蛋白表达差异，证明补肾促排卵汤在受精及早期胚胎发育过程中通过CRL4激活TET酶介导DNA去甲基化激活基因表达；研究IVM期间补肾促排卵汤中丹参有效成分RA对猪卵母细胞成熟及孤雌激活后体外胚胎发育的影响，探索中药单体对猪胚胎的体外发育能力影响。研究结果表明补肾促排卵汤能改善DOR氧化应激状态，通过FSH-PKA-PI3K-AKT-FOXO1信号轴促进小鼠颗粒细胞存活而改善DOR，在受精及早期胚胎发育过程中能通过CRL4 激活TET酶介导DNA去甲基化激活基因表达；RA提高猪体外受精卵裂率、囊胚形成率，改善猪卵母细胞和卵丘细胞内活性氧水平，提高猪卵母细胞内游离硫醇水平。本课题全面深入阐释补肾促排卵汤及其有效成分调控DOR小鼠氧化应激的作用机制，为补肾促排卵汤防治卵巢早衰、改善卵巢储备功能、提高卵子和胚胎质量提供实验依据，为传统中医更好地与ART技术结合治疗不孕症奠定基础。