针刺对雷公藤多苷片所致卵巢功能减退大鼠氧化应激及凋亡的影响

Effect of acupuncture on oxidative stress and apoptosis in ovarian hypofunction induced by Tripterygium Glycosides

目的:探索针刺是否通过调节大鼠抗氧化应激状态及颗粒细胞凋亡来改善雷公藤多苷片所致的卵巢功能减退(DOR)。方法:将有正常动情周期的SD雌性大鼠随机分为空白组、模型组、激素组、针刺组,每组10只。采用雷公藤多苷片混悬液灌胃(50 mg·kg^-1·d^-1,连续14 d)制备DOR大鼠模型。激素组予雌孕激素序贯疗法干预,针刺组针刺双侧"肾俞"或"关元""中脘"(隔日轮流进行),连续14 d。第9天开始观察各组大鼠的动情周期;实验结束后观察各组大鼠卵巢指数;采用HE染色法观察各组大鼠卵巢形态;ELISA法检测各组大鼠血清雌二醇(E2)、卵泡刺激素(FSH)、黄体生成素(LH)、丙二醛(MDA)及超氧化物歧化酶(SOD)的含量;免疫组织化学法检测各组大鼠卵巢颗粒细胞凋亡蛋白Bax及Bcl-2表达水平;荧光定量PCR法检测各组大鼠卵巢内Bax及Bcl-2 mRNA相对表达量。结果:与空白组比较,模型组卵巢内成熟卵泡减少,闭锁卵泡增多,颗粒细胞形态多出现异常,动情周期紊乱比例升高(P<0. 01),卵巢湿重及卵巢指数明显降低(P<0. 01),血清E2含量显著降低(P<0. 01),FSH、LH、MDA含量明显升高(P<0. 05,P<0. 01),血清SOD含量明显降低(P<0. 01),卵巢组织Bax蛋白及mRNA表达水平显著升高(P<0. 01),卵巢组织Bcl-2蛋白及mRNA表达水平显著降低(P<0. 05,P<0. 01)。与模型组比较,激素组及针刺组卵巢内成熟卵泡增多,闭锁卵泡减少,颗粒细胞形态异常情况明显减少,动情周期紊乱比例显著下降(P<0. 01),卵巢湿重及卵巢指数显著升高(P<0. 01),血清E2含量升高(P<0. 05),FSH、LH、MDA含量降低(P<0. 01,P<0. 05),血清SOD含量明显升高(P<0. 05,P<0. 01),卵巢组织Bax蛋白及mRNA表达水平显著降低(P<0. 01),卵巢组织Bcl-2蛋白及mRNA表达水平显著升高(P<0. 01)。与激素组比较,针刺组SOD含量显著升高(P<0. 01),Bax及Bcl-2蛋白表达水平上升(P<0. 05,P<0. 01),Bcl-2 mRNA表达水平下降(P<0. 01)。结论:针刺可以提高大鼠对抗卵巢氧化应激的能力,降低颗粒细胞的凋亡情况,从而减轻雷公藤多苷片所致的DOR损伤。

Objective To explore whether manual acupuncture stimulation of"Shenshu "(BL23),"Guanyuan"(CV4),"Zhongwan"(CV12) can improve the ovarian function by resisting oxidant stress and reducing apoptosis of granulosa cells in rats with ovarian hypofunction.MethodsForty female SD rats with normal estrous cycles were randomly divided into blank control,model,hormone therapy and acupuncture groups(n=10 rats in each group). The ovarian hypofunction model was established by gavage of Tripterygium Glycosides suspension(50 mg·kg^-1·d^-1)for 14 successive days. Rats of the hormone therapy group were treated by gavage of estrogen and progesterone,and those of the acupuncture group treated by manual acupuncture stimulation of bilateral BL23 or CV4 and CV12 alternatively by using uniform reinforcing-reducing method for 10 s every 5 min(3 times in 10 min). The treatment was performed once daily for 14 days. The blank group was given equal volume of normal saline daily. On the 9 thday,the estrous cycle of each rat was observed by means of vaginal smear test. The ovarian index,serum estradiol(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH),malondialdehyde(MDA)and superoxide dismutase(SOD)contents were detected by ELISA. Histopathological changes of the ovary tissue were observed by H. E. staining. The expressions of apoptosisrelated proteins Bax and Bcl-2 and their mRNAs of the ovaries were determined by immunohistochemical staining and quantitative real time-PCR separately.ResultsThe menstrual disorder rate was 100% in the model group,and was significantly higher than those in the hormone therapy(30%) and acupuncture(40%) groups(P<0. 01). Following modeling,the ovarian wet weight and index,E2 and SOD contents,Bcl-2 protein and mRNA expressions were significantly decreased(P<0. 01,P<0. 05),and serum FSH,LH and MDA levels,Bax protein and mRNA expressions were significantly increased(P<0. 05,P<0. 01)in the model group in comparison with the blank control group. H. E. staining showed a large amount of connective tissue in the ovary,with fewer mature follicles,increase of atresia follicles,significant reduction of luteal tissue,and appearance of scarring tissue in the model group. Compared with the model group,there were more mature follicles,fewer atresia follicles,lower abnormal granulosa cell morphology and lower estrous cycle disorder ratio in both hormone therapy and acupuncture groups. After the interventions,the decreased ovarian wet weight and index,serum E2 and SOD contents,expressions of Bcl-2 protein and mRNA were significantly increased(P<0. 05,P<0. 01),and the increased levels of FSH and LH,MDA,expressions of Bax protein and mRNA were significantly decreased in both hormone therapy and acupuncture groups(P<0. 05,P<0. 01). The effect of acupuncture was significantly superior to that of hormone in up-regulating SOD,Bax and Bcl-2 protein expressions(P<0. 01).Conclusion Acupuncture can improve the menstrual disorder in rats with ovarian hypofunction,which is closely related to its effects in improving antioxidant stress ability and regulating the expressions of apoptosis-related protein and mRNA of ovarian granulosa cells.