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针刺对卵巢低反应小鼠卵巢的保护机制OA北大核心CSTPCDMEDLINE

Study on the protective effect of acupuncture on poor ovarian response in mice

中文摘要英文摘要

目的:观察针刺对卵巢低反应(POR)小鼠卵巢功能的保护作用及对卵巢中Hippo通路关键分子的影响,探讨针刺抑制POR小鼠卵巢细胞凋亡的可能机制.方法:C57BL/6小鼠随机分成正常组、模型组和针刺组.雷公藤多苷混悬液(50 mg·kg-1·d-1)灌胃2周复制POR小鼠模型.针刺组针刺"太冲""三阴交""中极""关元",每次20 min,每日1次,连续治疗10 d.对各组小鼠同时促排卵后取材.阴道脱落细胞涂片观察小鼠的动情周期变化,每周记录小鼠的体质量变化,检测小鼠的获卵数、卵巢湿重与卵巢指数;ELISA法检测血清中抗缪勒管激素(AMH)、促卵泡生成素(FSH)、雌二醇(E2)、促黄体生成激素(LH)的含量;HE染色和TUNEL染色观察卵巢组织形态和卵巢颗粒细胞凋亡情况;荧光定量PCR和Western blot法检测卵巢组织Yes相关蛋白(YAP)、带有PDZ结合基序的转录共激活因子(TAZ)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X的蛋白(Bax)、半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)mRNA和蛋白表达水平.结果:与正常组比较,模型组小鼠动情周期紊乱率升高(P<0.01),体质量降低(P<0.01),卵巢组织各级正常发育卵泡数量减少且形态较差,闭锁卵泡增多,卵巢颗粒细胞凋亡率升高(P<0.01),获卵数下降(P<0.01),卵巢湿重与卵巢指数降低(P<0.01),血清中FSH和LH含量升高(P<0.01),AMH和E2含量下降(P<0.01),卵巢组织Bax和Caspase-3 mRNA和蛋白表达水平升高(P<0.01),YAP、TAZ和Bcl-2 mRNA和蛋白表达水平降低(P<0.01),卵巢组织磷酸化(p)-YAP蛋白表达水平升高(P<0.01);与模型组比较,治疗后的针刺组小鼠动情周期紊乱率降低(P<0.01),动情周期缩短(P<0.05),体质量升高(P<0.05),卵泡数量和形态得到不同程度改善,卵泡闭锁量降低,卵巢颗粒细胞凋亡率降低(P<0.01),获卵数升高(P<0.01),卵巢湿重与卵巢指数提高(P<0.05,P<0.01),小鼠血清FSH和LH含量降低(P<0.05),AMH和E2含量升高(P<0.05),卵巢组织Bax和Caspase-3 mRNA表达水平降低(P<0.01),YAP、TAZ和Bcl-2 mRNA表达水平升高(P<0.01,P<0.05),卵巢组织p-YAP、Bax和Caspase-3蛋白表达水平降低(P<0.01),YAP、TAZ和Bcl-2蛋白表达水平升高(P<0.05,P<0.01).结论:针刺可以抑制POR小鼠卵巢细胞凋亡,改善卵巢功能,其机制可能与调节Hippo通路中的关键分子相关.

Objective To observe the protective effect of acupuncture on ovarian function and its impact on key molecules(Yes associated protein[YAP]and transcriptional coactivator with PDZ binding motif[TAZ])in the Hippo signaling pathway in poor ovarian response(POR)mice,in order to explore its mechanisms underlying improvement of POR by inhibiting ovarian cell apoptosis.Methods The mice with regular motility cycle after screening were randomly divided into normal control,model and acupuncture groups,with 12 mice in each group.The POR model was established by gavage of tripterygium wilfordii polyglycoside suspension(50 mg·kg-1·d-1)for 2 weeks,and the mice of the normal control group was received an equal volume of 0.9%sodium chloride solution by gavage.The mice in the acupuncture group received manual acupuncture stimulation of bilateral"Taichong"(LR3)and"Sanyinjiao"(SP6),and"Zhongji"(CV3)and"Guanyuan"(CV4),with the filiform needles retained for 20 min,once daily for successive 10 days.The estrous cycle was determined by using vaginal exfoliated cell smear,and the body mass was detected weekly.The levels of serum anti Mullerian hormone(AMH),follicle stimulating hormone(FSH),estradiol(E2)and luteinizing hormone(LH)were measured using enzyme-linked immunosorbent assay(ELISA).Histopathological changes of the ovarian tissue were observed after H.E.staining,and the apoptosis of ovarian granulosa cells was measured using terminal deoxynucleotidyl transferase(TdT)dUTP Nick-End Labeling(TUNEL)assay.The expression levels of YAP,TAZ,B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(Bax),cysteine aspartic acid specific protease-3(Caspase-3)mRNAs and proteins were detected using real-time PCR and Western blot,respectively.Results Compared with the normal control group,the model group had an increase in the rate of estrous cycle disorder,estrous cycle,serum FSH and LH content,and apoptosis of ovarian granulosa cells,and expression levels of Bax and Caspase-3 mRNAs and p-YAP,Bax and Caspase-3 proteins(P<0.01),and a decrease in the body mass,number of retrieved oocytes,ovarian wet weight and ovarian index,serum AMH and E2 contents,and the expression levels of YAP,TAZ,Bcl-2 mRNAs and proteins(P<0.01).After acupuncture intervention,modeling induced increase and decrease of indexes mentioned above were completely reversed(P<0.05,P<0.01).H.E.staining showed deformed ovarian structure,reduced number of normal developing follicles and increased number of atretic follicles,disordered arrangement of the granulosa cells with fewer hierarchy in the model group,which was improved in the acupuncture group,such as increase in the number and improvement in the shape of normal ovarian follicles and reduction of the atretic follicles.Conclusion Acupuncture intervention can inhibit the apoptosis of ovarian granulosa cells and improve the ovarian function of POR mice,which may be related to its effects in up-regulating the expressions of YAP,TAZ(key molecules of Hippo signaling pathway).

郝健亨;赵越梦;兰颖;刘璇;王海军;曹玉霞;冀来喜

成都中医药大学针灸推拿学院,成都 610075成都中医药大学附属医院,成都 610075山西中医药大学针灸推拿学院,山西晋中 030619

针刺卵巢低反应Hippo信号通路细胞凋亡性激素

AcupuncturePoor ovarian responseHippo signal pathwayApoptosisSex hormone

《针刺研究》 2024 (010)

1001-1009 / 9

国家自然科学基金项目(No.82105028、82074549)

10.13702/j.1000-0607.20230337

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