针刺对卵巢低反应小鼠卵巢的保护机制  

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

作　　者：郝健亨 赵越梦 兰颖[2] 刘璇 王海军 曹玉霞 冀来喜 HAO Jian-heng;ZHAO Yue-meng;LAN Ying;LIU Xuan;WANG Hai-jun;CAO Yu-xia;JI Lai-xi(College of Acupuncture-Moxibustion and Massage,Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China;The Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610075;College of Acupuncture-Moxibustion and Massage,Shanxi University of Traditional Chinese Medicine,Jinzhong 030619,Shanxi Province)

机构地区：[1]成都中医药大学针灸推拿学院,成都610075 [2]成都中医药大学附属医院,成都610075 [3]山西中医药大学针灸推拿学院,山西晋中030619

出　　处：《针刺研究》2024年第10期1001-1009,共9页Acupuncture Research

基　　金：国家自然科学基金项目(No.82105028、82074549)。

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�ObjectiveTo observe the protective effect of acupuncture on ovarian function and its im pact on key m olecules(Yes associated protein[YAP]and transcriptional coactivator with PDZ binding m otif[TAZ])in the Hippo signaling pathway in poor ovarian response(POR)m ice,in order to explore its m echanism s underlying im provem ent of POR by inhibiting ovarian cell apoptosis.MethodsThe m ice with regular m otility cycle after screening were random ly divided into norm al control,m odel and acupuncture groups,with 12 m ice in each group.The POR m odel was established by gavage of tripterygium wilfordii polyglycoside suspension(50 m g·kg^(-1)·d^(-1))for 2 weeks,and the m ice of the norm al control group was received an equal volum e of 0.9%sodium chloride solution by gavage.The m ice in the acupuncture group received m anual acupuncture stim ulation of bilateral“Taichong”(LR3)and“Sanyinjiao”(SP6),and“Zhongji”(CV3)and“Guanyuan”(CV4),with the filiform needles retained for 20 m in,once daily for successive 10 days.The estrous cycle was determ ined by using vaginal exfoliated cell sm ear,and the body m ass was detected weekly.The levels of serum anti M ullerian horm one(AM H),follicle stim ulating horm one(FSH),estradiol(E2)and luteinizing horm one(LH)were m easured using enzym e-linked im m unosorbent assay(ELISA).Histopathological changes of the ovarian tissue were observed after H.E.staining,and the apoptosis of ovarian granulosa cells was m easured using term inal deoxynucleotidyl transferase(TdT)dUTP Nick-End Labeling(TUNEL)assay.The expression levels of YAP,TAZ,B-cell lym phom a-2(Bcl-2),Bcl-2 associated X(Bax),cysteine aspartic acid specific protease-3(Caspase-3)m RNAs and proteins were detected using real-tim e PCR and W estern blot,respectively.ResultsCom pared with the norm al control group,the m odel 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 m RNAs and p-YAP,

关 键 词：针刺 卵巢低反应 Hippo信号通路 细胞凋亡 性激素 

分 类 号：R245[医药卫生—针灸推拿学]