艾灸预处理对早发性卵巢功能不全大鼠的卵巢保护作用

Protective effect of moxibustion preconditioning in rats with premature ovarian insufficiency

目的:观察艾灸预处理对早发性卵巢功能不全(POI)大鼠卵巢功能、生育力及卵巢颗粒细胞凋亡的影响,探讨艾灸预处理对POI大鼠卵巢功能的保护作用及其抗凋亡效应机制。方法:SD大鼠随机分为空白组、模型组和预灸组,每组14只。预灸组造模前予14 d温和灸干预,“肾俞”和“关元”“中脘”两组穴位隔日交替进行,每日1次,每次10 min。采用雷公藤多苷混悬液75 mg·kg^(-1)·d^(-1)灌胃14 d制备POI大鼠模型。观察大鼠动情周期、妊娠率和胚胎数量,称量卵巢湿重并计算卵巢指数;HE染色观察卵巢组织形态学变化及卵泡计数;ELISA法检测大鼠血清雌二醇(E2)、卵泡刺激素(FSH)、黄体生成素(LH)、抗苗勒管激素(AMH)含量;TUNEL染色观察卵巢颗粒细胞凋亡率;免疫组织化学法和实时荧光定量PCR法分别检测卵巢组织中半胱氨酸天冬氨酸蛋白酶(Caspase)-3和Caspase-9蛋白和mRNA表达水平。结果:与空白组相比,模型组动情周期紊乱,妊娠率和胚胎数量降低(P<0.01);卵巢湿重及卵巢指数降低(P<0.01),总卵泡数及各级卵泡数量均减少(P<0.01,P<0.05),闭锁卵泡数量增多(P<0.01);血清E2、AMH含量降低(P<0.01),FSH、LH含量升高(P<0.01);卵巢细胞凋亡率升高(P<0.01);卵巢Caspase-3和Caspase-9蛋白及mRNA表达水平升高(P<0.01)。与模型组相比,预灸组动情周期紊乱大部分恢复正常(P<0.01),妊娠率和胚胎数量升高(P<0.01);卵巢湿重升高(P<0.01),总卵泡数及初级卵泡数量增多(P<0.05),闭锁卵泡数减少(P<0.01);血清AMH含量升高(P<0.05),FSH含量降低(P<0.05);卵巢细胞凋亡率降低(P<0.05);卵巢Caspase-3和Caspase-9蛋白及mRNA表达显著降低(P<0.01,P<0.05)。结论:艾灸预处理可以减少卵巢颗粒细胞凋亡,有效保护POI大鼠卵巢功能,提高生育力。

Objective To observe the effects of moxibustion preconditioning on ovarian function,fertility and ovarian granulosa cell apoptosis in rats with premature ovarian insufficiency(POI),so as to investigate its underlying mechanism in improving POI.Methods Forty-two female SD rats with two complete estrous cycles were randomly divided into control group,model group and pre-moxibustion group,with 14 rats in each group.The pre-moxibustion group was pretreated with mild moxibustion for 14 days before POI model establishment at 1)“Guanyuan”(CV4)and“Zhongwan”(CV12)and 2)bilateral“Shenshu”(BL23)as two sets of acupoints on alternate days,once each day,for 10 min each acupoint.After 14-day mild moxibustion intervention,75 mg·kg^(-1)·d^(-1)tripterygium glycoside tablet suspension was administered to rats in the pre-moxibustion group and the model group by gavage,for 14 consecutive days,while equivalent saline was given to rats in the control group in the same way.After modeling,the effect of moxibustion preconditioning on ovarian reserve function was evaluated by the estrous cycles,pregnancy rate and embryo number,morphological changes of ovaries,and serum sex hormone levels.TUNEL staining was used to detect the rate of granulosa cell apoptosis in ovaries.Immunohistochemistry and real time quantitative PCR were used to detect the relative expression of Caspase-3 and Caspase-9 proteins and mRNA levels in ovaries.Results Compared with the control group,the estrous cycles were disturbed;the pregnancy rate and number of embryos,the wet weight of ovary and ovarian index,the number of total follicles and different level of follicles,serum Estradiol(E2)and anti-mullerian hormone(AMH)levels were all significantly decreased(P<0.01,P<0.05),while the number of atretic follicles,serum follicule-stimulating hormone(FSH)and luteinizing hormone(LH)levels,the number of TUNEL-positive granulosa cells,the expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were significantly increased(P<0.01)in the model group.Compared with the model group,the disordered estrous cycles were improved;the pregnancy rate,the embryo numbers,the wet weight of ovary,and the total follicle number and primary follicle number,serum AMH level were significantly increased(P<0.01,P<0.05),while the number of atretic follicles,serum FSH level,the number of TUNEL-positive granulosa cells,expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were all significantly decreased(P<0.01,P<0.05)in the moxibustion group.Conclusion Moxibustion preconditioning could improve ovarian function and improve fertility of POI rats,which may be associated with reducing the apoptosis of ovarian granulosa cells.