电针刺通过Nrf2介导的抗氧化通路改善卵巢储备功能的机制研究

Diminished ovarian reserve (DOR) leads to significantly decreases on the number and quality of oocytes, which resulted worse embryo implantation rate and increased abortion rate. What’s more，fewer eggs were obtained after ovulation induction of DOR patients, and the available of embryos for implantation was limited, which led to the outcome of assisted reproductive was always less than ideal..Our previous studies have found that electroacupuncture can improve ovarian function and pregnancy outcome in DOR patients, and knock down the antioxidant molecule Nrf2 leads to a decrease in oocyte quality in normal mouse oocytes. Literature studies have shown that imbalance between DNA damage and repair system was closely correlated with ovarian dysfunction, which could be regulated by Nrf2 pathway. Therefore, we proposed that the Nrf2 antioxidant pathway may mediate the improvement of the ovarian function and the outcome of assisted outcome in DOR by regulating the process of DNA damage and repair. .To this end, this study intends to 1) establish an electroacupuncture intervened DOR rat model, detect Nrf2 pathway and the expression of DNA damage and repair related molecule, and evaluate oocyte quality; 2) regulate the level of Nrf2 in oocyte, and determine the effects on DNA damage and repair molecules, oocyte quality and reproductive outcomes; 3) verify the importance of this mechanism in the improvement of ovarian function in DOR patient whom treated with electroacupuncture. Those findings are of particular importance for elucidating the mechanism of electroacupuncture treatment of DOR and introducing the national medical EA into assisted reproductive technology.

卵巢储备功能下降（DOR）患者卵母细胞数量及质量显著下降，导致胚胎着床率降低、流产率增加；诱导排卵后获卵数少、可利用胚胎少导致助孕结局始终达不到理想状态。前期研究发现，电针刺可改善DOR患者卵巢功能和生殖结局；小鼠卵母细胞敲低抗氧化分子Nrf2导致卵母细胞质量下降。文献显示DNA损伤修复失衡参与卵巢功能减退并受Nrf2通路的调节。由此提出本研究假说：Nrf2抗氧化通路可能通过调节DNA损伤修复过程介导电针刺改善DOR卵巢功能及助孕结局。为此，本研究拟1)构建电针刺干预DOR大鼠模型，检测卵巢及卵母细胞中Nrf2通路、DNA损伤修复水平并评估卵母细胞质量；2)调节卵母细胞Nrf2表达，明确对DNA损伤修复过程、卵母细胞质量、生殖结局的影响；3)验证该机制在电针刺改善临床DOR患者卵巢功能及生殖结局中的重要价值。本研究对阐明电针刺治疗DOR的机制、将电针刺技术引入人类辅助生殖技术具有深远意义。

目的:观察卵巢储备功能下降（DOR）大鼠及患者模型的相关表型，探讨电针治疗的疗效及内在机制。.方法:8周龄SD雌性大鼠随机分为对照组(CTL)、DOR组和电针+DOR组(EAD)。采用生理盐水灌胃建立CTL模型，DOR组采用雷公藤多苷片悬液75 mg/ (kg.d)灌胃建立CTL模型，连续14 d。根据选择肾俞穴、气海穴、关元穴，给予DOR模型大鼠相应的电针方案干预，每日1次，连续2周，建立EAD模型。记录各组大鼠体重变化趋势，观察各组间是否有显著差异。自灌胃第8天起，行阴道涂片观察发情周期的变化。采用ELISA法测定血清性激素和抗苗勒氏管激素(AMH)水平。评价卵巢组织的组织学变化及排卵诱导、细胞凋亡和DNA损伤/修复相关蛋白、氧化和抗氧化指标、Nrf2水平。ART周期患者经卵巢逆转诊断为正常卵巢早衰，POF患者采用EA治疗。采集血清、卵泡液和颗粒细胞，检测上述指标，阐明EA治疗价值和机制。.结果:EA可有效改善DOR症状。三组大鼠体重变化趋势基本一致，DOR组大鼠卵巢体积、卵巢/体重比、卵巢组织总卵泡数均明显降低，DOR模型大鼠闭锁卵泡数明显增加，而EA治疗组大鼠明显改善。EA处理通过调控Nrf2介导的抗氧化途径降低细胞凋亡和DNA损伤相关蛋白的表达水平。临床数据还表明，EA治疗可以通过调节卵巢中的Nrf2水平来改善氧化应激，从而增加ART周期中DOR患者的卵母细胞回收数量和MII率。.结论:卵巢早衰导致卵巢功能障碍，卵母细胞储备和质量下降，卵泡闭锁增加，卵巢细胞凋亡增加，导致卵巢氧化和抗氧化状态失衡，DNA损伤修复失衡参与卵巢功能障碍，并受EA治疗介导的Nrf2通路调控。本研究对阐明EA在DOR治疗中的作用机制，并将其引入ART治疗具有重要意义。