基于Nrf2/ARE通路探讨针灸抗氧应激改善卵巢功能的miRNA作用机制

Oxidative stress is a key factor that contributes to ovarian disorders such as menopausal syndrome, diminished ovarian reserve, and premature ovarian failure. Therefore, the maintenance of rebalance between oxidative and anti-oxidative state is vital to protect ovarian reserve function and delay decline of ovarian function. In addition, it is also a novel breakthrough point to study the underlying mechanisms by which acupuncture protect ovarian reserve function. Our previous study had confirmed that anti-oxidative stress of acupuncture and moxibustion was one of the key therapeutic effect mechanisms. In this project, we tried to observe the differential protective effects of acupuncture and moxibustion on ovarian reserve function in animal model. Furthermore, we utilized the Nrf2 knock-out mice and perform the gene chip, RT-PCR, IHC, and ELISA tests. Moreover, we searched the characteristic miRNAs of ovary by which acupuncture and moxibustion exerts its protective effects on ovarian reserve function. Finally, we wanted to investigate the vital role of oxidative/antioxidative balance mediated by Nrf2 signaling pathway in effects of acupuncture on protection of the ovarian reserve function and delay of ovarian aging. Our research would shed light on research of mechanisms of acupuncture and moxibustion in preventing and treating menopausal syndrome, ovarian reserve decline, and premature ovarian failure. Ultimately, our findings would provide scientific experimental evidences in terms of the clinical application of acupuncture and moxibustion and its curative effect in improving ovarian function, and delaying ovarian aging.

氧化应激是导致绝经综合征、卵巢储备功能减退、卵巢早衰等卵巢功能下降疾病的重要因素，维持氧化/抗氧化再平衡是保护卵巢储备功能，延缓卵巢功能下降的关键，也是针灸处理保护卵巢储备功能机制研究的新切入点。课题组前期研究已证实针灸抗氧应激是针灸治病的重要机理之一。本课题拟通过动物模型观察针、灸处理对卵巢储备功能整体保护效应差异的基础上，采用Nrf2基因敲除小鼠，运用基因芯片、RT-PCR、免疫组化和ELISA等技术，寻找针灸抗氧应激保护卵巢储备功能效应中卵巢组织的特征性miRNA，探讨Nrf2信号通路介导的氧化/抗氧化稳态平衡在针灸保护卵巢储备功能，延缓卵巢衰老中的关键作用，为针灸防治绝经综合征、卵巢储备功能减退、卵巢早衰的机制研究提供新思路，为推广针灸改善卵巢功能，延缓卵巢衰老的临床应用和提高临床疗效提供科学的实验依据。

背景：氧化应激是导致卵巢功能下降的重要因素，与抗氧化密切相关的Nrf2/ARE信号通路是机体抵抗伤害性刺激的防御性转导通路。而针灸具有抗氧应激效应，是针灸治病的重要机理之一。维持氧化/抗氧化平衡是保护卵巢储备功能，延缓卵巢功能下降的关键，也是针灸处理保护卵巢储备功能机制研究的新切入点。.研究方法及结果：课题将SD大鼠、ICR小鼠随机分为空白组、模型组、针刺组、艾灸组和雌激素组；Nrf2+/+、Nrf2-/-小鼠随机分为模型组、针刺组和艾灸组。模型组、针刺组、艾灸组和雌激素组用雷公藤多甙片悬浮液制备卵巢储备功能减退模型，空白组用生理盐水灌胃。针刺组和艾灸组穴位选用“关元”、“中脘”穴和双侧“肾俞”穴，在造模开始的同时，分别每日干预针刺或艾灸1次，连续干预14d。结果发现，针刺、艾灸均能改善雷公藤多苷片所致的动情周期、血清性激素水平、卵巢组织形态学等，发挥对卵巢储备功能整体保护效应，且针刺和艾灸两种干预方法对卵巢功能减退模型发挥的保护效应有所不同；针刺、艾灸均能调节卵巢的氧化应激状态，降低卵泡周围颗粒细胞的凋亡，改善炎性水平，从而阻抑大鼠的卵巢储备功能减退，其可能与通过调节Nrf2信号通路的传导有关；针刺、艾灸在改善卵巢储备功能作用中有相同的上调或下调miRNA谱，也有独有的miRNA。 .结论：针刺、艾灸均可通过调控Nrf2信号途径，阻抑雷公藤多苷致卵巢损伤，发挥整体保护效应，但两者略有不同，可能与不同miRNA机制有关。.科学意义：本研究通过探讨针灸保护卵巢储备功能的Nrf2信号通路介导的抗氧化、抗炎、抗凋亡作用，寻找针灸保护卵巢储备功能效应中卵巢组织的特征性miRNA，为针灸防治绝经综合征、卵巢储备功能减退、卵巢早衰的机制研究提供新思路，为推广针灸改善卵巢功能，延缓卵巢衰老的临床应用和提高临床疗效提供科学的实验依据。