针刺治疗对卵巢早衰大鼠PI3K/Akt/mTOR信号通路的影响

Effects of acupuncture on PI3K/Akt/mTOR signaling pathway in rats with premature ovarian failure

目的:探讨针刺与药物治疗对卵巢早衰大鼠PI3K/Akt/mTOR信号通路的影响。方法:50只SPF级雌性SD大鼠随机取10只为正常组,其余40只连续5d腹腔注射环磷酰胺30mg/kg,建立卵巢早衰动物模型。将造模成功的35只大鼠随机分为模型组(9只)、药物组(9只)、针刺Ⅰ组(9只)、针刺Ⅱ组(8只)。正常组、模型组不干预;药物组采用乙烯雌酚灌胃治疗,每天1次;针刺Ⅰ组和针刺Ⅱ组分别取不同腧穴进行针刺治疗,每天2次,均治疗4周。治疗结束后,采用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)试剂盒检测各组血清雌二醇(estradiol,E2)、孕酮(progesterone,P)、促卵泡激素(follicle stimulating hormone,FSH)和促黄体生成素(luteotropic hormone,LH)水平,卵巢组织进行病理苏木精-伊红(HE)染色以及RNA和蛋白提取,分别检测雌激素受体α(estrogen receptor alpha,ERα)、雌激素受体β(estrogen receptor beta,ERβ)、磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase/serine/threonine kinase,PI3K)、丝氨酸苏氨酸蛋白激酶(protein kinase B,PKB,Akt)和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)mRNA的表达量。结果:大剂量短期内给予环磷酰胺可以建立卵巢早衰动物模型,成功率为87.5%。与正常组比较,模型组大鼠阴道细胞涂片呈雌激素缺乏征象,早期卵泡减少,卵泡结构破坏,成熟卵泡数目减少,血清E2水平明显降低(P<0.05),P、FSH和LH水平升高(均P<0.05),与雌激素相关的ERβ、PI3K、Akt和mTOR mRNA表达量均降低(均P<0.05);与模型组相比,药物组和针刺组成熟卵泡有所增加,血清E2水平明显升高(均P<0.05),FSH含量下降明显(均P<0.05),相关信号通路的PI3K、Akt和mTOR mRNA表达量呈升高趋势(均P<0.05);针刺组与药物组各指标组间比较差异无统计学意义(均P>0.05)。结论:针刺治疗卵巢早衰具有一定的优势,并且取得与雌激素相当的治疗效果,作用机制可能与上调PI3K/Akt/mTOR信号通路相关基因和蛋白表达水平有关。

Objective To explore the effects of acupuncture and medication on PI3K/Akt/mTOR signaling pathway in rats with premature ovarian failure. Methods Ten of fifty SPF-grade female SI） rats were randomly selected into a normal group, and the remaining 40 rats were treated with intraperitoneal injection of eyelophospha- mide （30 mg/kg） for consecutive 5 days to establish rat model of premature ovarian failure. Thirty-five successful rat models were randomly divided into a model group （9 cases）, a medication group （9 cases）, an acupuncturegroup A （9 cases） and an acupuncture group B （8 cases）. The rals in the model group and normal group did not receive any treatment. The rats in the medication group were treatment with inlragastric administration of diethylstil bestrol, once a day. The rats in tbe acupuncture group A and acupuncture group Bwere respectively treated with acupuncture at different acupoints, twice a day. All the treatment was given for 4 weeks. After the treatment, en zyme-linked immunosorbent assay （ELISA） was applied to test the levels of estradiol （E2）, progesterone （P）, follicle stinulating hormone （FSH） and luteotropic hormone （LH）. Tbe ovarian tissue sample was processed with bematoxylin eosin （HE） staining as well as RNA and protein extraction to lest the mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERβ）, phosphatidylinositol 3-kinase/serine/threonine kinase （PI3K）, protein kinase B （Akt） and mammalian larget of rapamycin （mTOR）. Results High-dose short-term in- tervention of cyclophosphamide could establish rat model of premature ovarian failure with a successful rate of 87.5%. Compared with the normal group, the vaginal smear in the model group was featured with signs of estro gen deficiency, early follicle reduction, structural damage to the follicle, and reducing number of mature follicles; the level of E2 was significantly reduced （P〈0.05）, levels of P, FSH and LH were increased （all P〈0.05）, and mRNA expression of estrogen-related ERβ, PI3K, Akt and mTOR were all reduced （all P〈0.05）. Compared with the model group, the number of mature follicle was increased in the medicalion group and acupuncture groups, the levels of E2 was obviously increased （all P〈50.05）, level of FSH was reduced （all P〈0.05）, and mRNA expression of PI3K, Akt and mTOR all showed an increasing trend （all P〈0.05）. The differences of each index result between acupuncture groups and medication group were not significant （all P〉 0.05）. Conclusion Acupuncture has certain advantage for the treatment of premature ovarian failure, which achieves similar therapeu tic effect as estrogen; the possible mechanism may be related to up-regulation of gene and protein expression in PI3K/Akt/mTOR signaling pathway.