艾灸对卵巢储备功能减退大鼠Nrf2/HO-1信号通路的影响

目的:观察艾灸对卵巢储备功能减退(DOR)大鼠核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路的影响,探讨艾灸对卵巢储备功能的保护效应机制。方法:将40只SD大鼠随机分为空白组、模型组、艾灸组、激素组,每组10只。模型组、艾灸组、激素组均采用雷公藤多苷片混悬液灌胃制备DOR大鼠模型,空白组灌胃等体积的0.9%氯化钠溶液,每日1次,连续灌胃14 d。激素组在造模即日起采用激素序贯疗法连续干预14 d;艾灸组在造模即日起每日给予双侧"肾俞"或"关元""中脘"艾灸治疗,两组穴隔日交替,每次10 min,连续14 d。每日采用阴道脱落细胞涂片观察大鼠的动情周期,统计各组大鼠动情周期紊乱率。干预结束后,采用HE染色观察各组大鼠卵巢组织学形态;ELISA法检测各组大鼠血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、抗苗勒管激素(AMH)和超氧化物歧化酶(SOD)、丙二醛(MDA)含量;免疫组化法检测各组大鼠卵巢组织Nrf2和HO-1蛋白水平;实时PCR(TaqMan探针法)检测各组大鼠卵巢组织Nrf2和HO-1 mRNA的表达。结果:与空白组比较,模型组大鼠动情周期紊乱率升高(P<0.01);与模型组比较,艾灸组和激素组大鼠动情周期紊乱率降低(P<0.01)。与空白组比较,模型组大鼠血清FSH、LH、MDA含量增多(P<0.01),血清E2、AMH、SOD含量减少(P<0.01);与模型组比较,艾灸组和激素组大鼠血清FSH、LH、MDA含量减少(P<0.01,P<0.05),血清E2、AMH、SOD含量增多(P<0.01)。与空白组比较,模型组大鼠卵巢组织Nrf2和HO-1蛋白及mRNA表达减少(P<0.01);与模型组比较,艾灸组和激素组大鼠卵巢组织Nrf2和HO-1蛋白及mRNA表达增多(P<0.01)。结论:艾灸可以降低DOR大鼠的动情周期紊乱率,改善血清性激素水平和抗氧化应激能力,其机制可能与调控Nrf2/HO-1信号通路有关。

Effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve

Objective To observe the effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve(DOR), and to explore the protective mechanism of moxibustion on ovarian reserve function. Methods Forty SD rats were randomly divided into a blank group, a model group, a moxibustion group and a hormone group, 10 rats in each group. The rats in the model group, moxibustion group and hormone group were treated with intragastric administration of tripterysium glycosides turbid liquid to prepare DOR model. The rats in the blank group were treated with intragastric administration of sodium chloride solution with the same volume, once a day for 14 days. The rats in the hormone group were treated with hormone sequential therapy for 14 days from the day of modeling;the rats in the moxibustion group were treated with moxibustion at bilateral "Shenshu"(BL 23) or "Guanyuan"(CV 4) and "Zhongwan"(CV 12) from the day of modeling, and the two groups acupoints were alternated every other day, 10 min each time, for 14 consecutive days. The estrus cycle was observed every day by vaginal exfoliated cell smear, and the estrus cycle disorder rate in each group was calculated. After the intervention, the HE staining was used to observe the histological morphology of ovaries;ELISA was used to detect the contents of follicle stimulating hormone(FSH), luteinizing hormone(LH), estradiol(E2), anti Mullerian hormone(AMH), superoxide dismutase(SOD) and malondialdehyde(MDA);the protein levels of Nrf2 and HO-1 in ovarian tissue were detected by immunohistochemistry;real-time PCR(TaqMan probe method) was used to detect the expression of Nrf2 and HO-1 mRNA. Results Compared with the blank group, the rate of estrus cycle disorder in the model group was increased(P<0.01);compared with the model group, the rate of estrus cycle disorder in the moxibustion group and hormone group was decreased(P<0.01). Compared with the blank group, the serum contents of FSH, LH and MDA in the model group were increased(P<0.01), and the serum contents of E2, AMH and SOD were decreased(P<0.01). Compared with the model group, the serum contents of FSH, LH and MDA in the moxibustion group and hormone group were decreased(P<0.01, P<0.05), and the serum contents of E2, AMH and SOD were increased(P<0.01). Compared with the blank group, the protein and mRNA expression of Nrf2 and HO-1 in the model group were decreased(P<0.01);compared with the model group, the protein and mRNA expressions of Nrf2 and HO-1 in the moxibustion group and hormone group were increased(P<0.01). Conclusion Moxibustion could reduce the rate of estrus cycle disorder, improve the level of serum sex hormones and antioxidant stress in DOR rats, and the mechanism may be related to the regulation of Nrf2/HO-1 signaling pathway.