生殖医学杂志

目的 探讨电针刺改善卵巢功能的潜在生物学机制。方法 本研究选择SD雌鼠(8周龄，36只)为研究对象，按照随机数字表法随机分为对照组(12只)、卵巢储备功能减退(DOR)组(12只)、电针刺+DOR组(EAD组，12只)。记录3组大鼠体重变化趋势，ELISA法检测大鼠血清抗苗勒管激素(AMH)、雌二醇(E₂)、卵泡刺激素(FSH)、睾酮(T)、黄体生成素(LH)、孕酮(P)等激素水平。对卵巢组织石蜡包埋切片进行苏木精-伊红(HE)染色观察卵巢的组织学改变并进行卵泡计数，通过Western blot和ELISA等方法测定卵巢组织氧化与抗氧化指标(Nrf2、Keap1、p-p38/p38、ROS、MDA、GSH-Px、SOD)、DNA损伤相关蛋白(ATM、γH2AX、BRCA1)、凋亡水平及相关蛋白(Caspase-3、Bax、Bcl-2)的表达水平。结果 与对照组比较，DOR组大鼠卵巢体积缩小，卵巢重量和卵巢器质比显著降低(P<0.05);与DOR组比较，EAD组电针刺后卵巢体积增大，卵巢重量和器质比显著增加(P<0.05)。与对照组比较，DOR组总卵泡数显著减少、闭锁卵泡数显著增加(P<0.05);与DOR组比较，EAD组电针刺后总卵泡数显著增加，闭锁卵泡数量显著减少(P<0.05)。与对照组比较，DOR组大鼠血清中AMH、E₂水平显著降低(P<0.05),FSH水平显著增加(P<0.01);与DOR组比较，EAD组FSH水平显著降低(P<0.05),AMH、E₂水平显著增加(P<0.05)。与对照组比较，DOR组大鼠血清和卵巢组织中ROS、MDA水平显著增加而抗氧化指标GSH-Px、SOD水平显著降低(P<0.05),Nrf2表达水平显著降低、Keap1表达及p-p38/p38比值显著升高(P均<0.05),导致DNA损伤相关蛋白(ATM、γH2AX)、凋亡相关蛋白(Caspase-3、Bax)表达水平显著增加、抗凋亡蛋白Bcl-2表达显著降低(P均<0.05);与DOR组比较，EAD组电针刺治疗后GSH-Px、SOD水平显著增加(P<0.05),Nrf2表达显著上调、Keap1表达及p-p38/p38水平显著降低(P均<0.05),Bax、Caspase-3表达显著减少(P<0.05),Bcl-2表达显著增加(P<0.05),DNA损伤相关蛋白γH2AX和ATM表达显著降低，修复蛋白BRCA1表达水平显著上调(P<0.05)。结论 电针刺通过调节Nrf2介导的抗氧化通路降低卵巢颗粒细胞凋亡并增加DNA损伤修复水平，减少卵泡闭锁，最终改善卵巢储备功能。

Objective:To explore the potential biological mechanism of improving ovarian function by electroacupuncture.Methods:Thirty-six SD female rats aged 8 weeks were divided into the control group(n=12),diminished ovarian reserve(DOR) group(n=12) and electroacupuncture + DOR(EAD) group by a random number table method. The trend of body weight of rats was recorded, and serum E₂,FSH,LH,anti-Müllerian hormone(AMH),testosterone(T) and progesterone(P) were detected by enzyme-linked immunosorbent assay(ELISA). The hematoxylin and eosin(HE) staining method was used to evaluate the histological changes of the ovaries and count the number of follicles. The oxidative and antioxidant indexes[Nrf2,Keap1,p-p38/p38,reactive oxygen species(ROS),malondialdehyde(MDA),GSH-Px and superoxide dismutase(SOD)],DNA damage and repair proteins(ATM,γ-H2AX and BRCA1) and apoptosis-related proteins(Bax, Bcl-2 and Caspase-3) in ovarian tissue were determined by western blot and ELISA.Results:Compared with the control group, the ovarian volume of the DOR group was reduced, and the ovarian weight as well as the ratio of ovarian organ weight to body weight were significantly decreased(P<0.05). The ovarian volume was increased, and ovarian weight as well as the ratio of ovarian organ weight to body weight were significantly increased after electroacupuncture in the EAD group when compared with the DOR group(P<0.05). The total number of follicles in the DOR group was significantly reduced and the number of atretic follicles was significantly increased when compared with the control group(P<0.05). Compared with the DOR group, the total number of follicles was significantly increased and the number of atretic follicles was significantly decreased after electroacupuncture in the EAD group(P<0.05). Compared with the control group, the serum levels of AMH and E₂ were significantly decreased(P<0.05) and the level of FSH was significantly increased(P<0.01) in the DOR group. The level of FSH was significantly lower(P<0.05),and the levels of AMH and E₂ were significantly higher(P<0.05) in the EAD group than those in the DOR group. Compared with the control group, the levels of ROS and MDA in the serum and ovarian tissue were increased, while the levels of antioxidant indicators in terms of GSH-Px and SOD and the expression level of Nrf2 were significantly decreased(P<0.05),and the expression level of Keap1 and the ratio of p-p38/p38 increased, resulting in a significant increase in the expression levels of DNA damage-related proteins(ATM,γH2AX) and apoptosis-related proteins(Caspase-3,Bax) and a significant decrease in the expression of anti-apoptosis protein Bcl-2 in the DOR group. Compared with the DOR group, after electroacupuncture in the EAD group, the levels of GSH-Px and SOD were significantly increased(P<0.05),the Nrf2-mediated antioxidant pathway was significantly up-regulated(P<0.05),the expression levels of Keapl, p-p38/p38,Bax and Caspase-3 significantly decreased(all P<0.05),and the expression level of Bcl-2 significantly increased(P<0.05),while lower expression levels of DNA damage-related proteins in terms of γ-H2AX and ATM and higher expression level of repair protein BRCA1 were found(P<0.05).Conclusions:Electroacupuncture can regulate the Nrf2-mediated antioxidant pathway to reduce ovarian granulosa cell apoptosis, increase DNA repair level and reduce follicle atresia, which ultimately improves ovarian reserve function.