关元命门序贯针刺激活FSHR/cAMP/PKA通路促进早发性卵巢功能不全模型大鼠颗粒细胞增殖的机制研究  

Study on the Mechanism of Guanyuan Mingmen Sequential Acupuncture Activating FSHR/cAMP/PKA Pathway to Promote Granulosa Cell Proliferation in POI Model Rats

作　　者：许江虹[1,2] 陈跃来 尹平 赵雪丹 郑慧敏 胡俊威[1] 刘璐慜 XU Jiang-Hong;CHEN Yue-Lai;YIN Ping;ZHAO Xue-Dan;ZHENG Hui-Min;HU Jun-Wei;LIU Lu-Min(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Dept.of Gynecology,Jing’an District Hospital of Traditional Chinese Medicine,Shanghai 200072,China;Sleep Medicine Center of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学,上海201203 [2]上海市静安区中医医院妇科,上海200072 [3]上海中医药大学附属龙华医院睡眠医学中心,上海200032

出　　处：《广州中医药大学学报》2024年第4期965-972,共8页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：上海市静安区中医药临床重点专科建设项目(编号:JA2020-Z006);上海市静安区不孕不育专病诊疗中心项目(编号:JA2021-ZLZX001)。

摘　　要：【目的】观察关元命门序贯针刺方案对早发性卵巢功能不全(POI)模型大鼠的治疗作用及机制。【方法】将雌性SD大鼠分为空白组、模型组、蛋白激酶A(PKA)抑制剂(H89)+针刺组、针刺组各12只。除空白组,其他3组大鼠采用雷公藤多苷片灌胃制备POI模型。模型成功建立后,空白组和模型组每日捆绑一次;针刺组大鼠在动情间期取关元穴针刺,在动情前期取命门穴针刺;H89+针刺组按照针刺组针刺方案干预,在每次针刺前30 min内腹腔注射H89。连续干预20 d。各组大鼠分别在干预后第1个动情间期和动情前期取材。酶联免疫吸附分析(ELISA)检测动情间期促卵泡激素(FSH)、雌二醇(E2)水平,Western Blot法检测动情间期促卵泡激素受体(FSHR)、芳香化酶P450(P450arom)蛋白表达,细胞计数试剂盒8(CCK-8)法检测动情间期和动情前期颗粒细胞活性,免疫组织化学法检测动情前期增殖细胞核抗原(PCNA)蛋白表达水平。【结果】(1)与空白组比较,模型组和H89+针刺组血清FSH水平显著升高(P<0.01),E2水平显著降低(P<0.001);H89+针刺组FSH水平与模型组无差异(P>0.05),E2水平低于模型组(P<0.05);针刺组FSH水平低于模型组和H89+针刺组(P<0.05),与空白组无差异(P>0.05),E2水平显著高于模型组和H89+针刺组(P<0.01),仍低于空白组(P<0.05)。(2)模型组和H89+针刺组FSHR、P450arom蛋白表达均低于空白组(P<0.01);H89+针刺组FSHR蛋白表达水平与模型组无差异(P>0.05),P450arom蛋白表达水平低于模型组(P<0.05);针刺组FSHR、P450arom蛋白表达水平均高于模型组和H89+针刺组(P<0.05),但仍低于空白组(P<0.05)。(3)模型组和H89+针刺组GCs活性和PCNA平均光密度值均低于空白组(P<0.05);H89+针刺组GCs活性和PCNA平均光密度值均低于模型组(P<0.05);针刺组的GCs活性和PCNA平均光密度值显著高于模型组和H89+针刺组(P<0.05或P<0.01)。【结论】关元命门序贯针刺方案可通过上调促卵泡激�Objective To observe the therapeutic effects and mechanisms of Guanyuan Mingmen Sequential Acupuncture on rats with premature ovarian insufficiency(POI)model.Methods Female SD rats were divided into the blank group,the model group,the protein kinase A(PKA)inhibitor(H89)+acupuncture group,and the acupuncture group,with 12 rats in each group.Except for the blank group,the POI model was prepared by gavage with Tripterygium Glycosides Tablets in the other three groups of rats.After the model was successfully established,the blank group and the model group were bundled once a day;in the acupuncture group,Guanyuan(RN4)point was taken during the intermotility period,and in the pre-motility period,Mingmen(DU4)point was taken;in the H89+acupuncture group,the intervention was performed in accordance with the acupuncture protocol of the acupuncture group,and H89 was injected intraperitoneally for 30 minutes prior to each acupuncture session.Continuous intervention was performed for 20 days.Samples were taken from each group of rats in the first estrus period and in proestrus period after intervention.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of follicle stimulating hormone(FSH)and estradiol(E2)during the estrous phase,Western Blot was used to measure the protein expressions of follicle stimulating hormone receptor(FSHR)and aromatase P450(P450arom)during the estrous phase,and the activity of granulocytes during the estrous phase and the proestrus phase were measured using the cell-counting kit 8(CCK-8)method.The immunohistochemistry method was used to detect the protein expression of pre-motility proliferating cell nuclear antigen(PCNA).Results(1)Compared with the blank group,the serum FSH level of the model group and H89+acupuncture group was significantly increased(P<0.01),and the E2 level was significantly decreased(P<0.001);there was no difference between the FSH level of the H89+acupuncture group and that of the model group(P>0.05),and the E2 level of the H89+acupuncture group was lower than

关 键 词：序贯针刺 早发性卵巢功能不全 关元穴 命门穴 促卵泡激素受体(FSHR) 芳香化酶P450(P450arom) 大鼠 

分 类 号：R245.31[医药卫生—针灸推拿学]