【作者】 梁尚杰； 尹亚群； 沈梅红；

【Author】 LIANG Shangjie;YIN Yaqun;SHEN Meihong;College of Acupuncture-Moxibustion and Tuina,College of Health and Rehabilitation,Nanjing University of Chinese Medicine;

【机构】 南京中医药大学针灸推拿学院·养生康复学院；

【摘要】 目的:观察电针对早发性卵巢功能不全（POI）大鼠卵巢组织自噬水平及AMPK/ULK1信号通路影响,探讨电针改善POI的潜在机制。方法:动情周期正常SD雌性大鼠随机分为空白组、模型组和电针组,每组9只。采用雷公藤多苷混悬液（TGS）连续灌胃两周制备POI大鼠模型。电针组在灌胃1h后行电针干预,选取双侧"肾俞"或"中脘"、"关元"两组穴位隔日交替电针,2/100Hz疏密波,10min/d,共14d。采用阴道涂片观察大鼠动情周期变化,HE染色观察卵巢组织形态学变化,统计卵泡数量;ELISA法检测血清FSH、LH、E₂、AMH的含量;IHC、q RT-PCR检测卵巢组织中AMPK、ULK1、Beclin1、LC3、ATG5的组织定位、蛋白及mRNA表达,Western blot检测卵巢组织中AMPK、ULK1蛋白表达。结果:与空白组相比,模型组大鼠动情周期紊乱率增加（P<0.01）,各级卵泡数量减少（P<0.01）,闭锁卵泡数量增多（P<0.01）,总卵泡数量减少（P<0.01）,血清FSH、LH含量升高（P<0.001,P<0.05）,E₂、AMH含量降低（P<0.01,P<0.05）,AMPK、ULK1、Beclin1、LC3、ATG5蛋白及m RNA表达降低（P<0.01,P<0.05）;与模型组相比,电针干预后动情周期紊乱率降低（P<0.01）,次级卵泡数量及窦卵泡数量增加（P<0.05,P<0.01）,闭锁卵泡数量减少（P<0.01）,血清FSH水平下降（P<0.05）,E₂、AMH含量升高（P<0.05）,AMPK、ULK1、LC3、ATG5蛋白及mRNA表达升高（P<0.05,P<0.01）结论:电针能够改善POI大鼠卵泡发育,平衡性激素水平,激活卵巢自噬,其作用机制可能与激活AMPK/ULK1信号通路有关。更多还原

【Abstract】 Objective To observe the effect of Electroacupuncture on autophagy and AMPK/ULK1 signaling pathway in ovarian tissue of rats with premature ovarian insufficiency（POI）,and to explore the possible mechanism.Methods Female SD rats with normal estrous cycle were randomly divided into blank,model and EA groups,with 9 rats in each group.TGS was used to prepare POI rats model for 14 days.After 1h of gavage the EA group,bilateral " Shenshu"," Zhongwan"," Guanyuan",two groups of acupoints alternating EA on alternate days,with a sparse-dense wave of 2/100 Hz for 10 min/d for 14 days.Observation of changes in the rat’s estrous cycle by daily vaginal smears,HE staining to observe the morphological changes of rat ovarian tissue,counting the number of follicles.The levels of serum FSH,E₂,AMH and LH were detected by ELISA.The tissue localization,protein and m RNA expressions of AMPK,ULK1,Beclin1,LC3 and ATG5 in ovarian tissues were detected by IHC and q RT-PCR,and the protein expressions of AMPK and ULK1 were detected by Western blot.Results Compared with the control group,rats in the model group showed disturbed estrous cycle（P<0.01）,the number of follicles at all levels decreased（P<0.01）,atretic follicles increased（P<0.01）,total follicle counts much lower than the control group（P<0.01）.The model group had a higher level of FSH and LH（P<0.001,P<0.05）,lower E₂ and AMH levels（P<0.01,P<0.05）.The relative expression levels of AMPK,ULK1,Beclin1,LC3,and ATG5 m RNA and protein expression were reduced in ovarian tissues of the model group（P<0.01,P<0.05）,AMPK,ULK1 protein expression decreased（P<0.05）.Compared with the model group,rats in the EA group showed a lower rate of disorders of the estrous cycle after intervention（P<0.01）,significant improvement in the ovarian index（P<0.05）.In the EA group there was an increase in the number of secondary and antral follicles（P<0.05,P<0.01）,a decrease in the number of atretic follicles（P<0.01）.FSH levels decreased（P<0.05）,E₂ and AMH levels increased（P<0.05）.Relative expression levels of AMPK,ULK1,LC3 and ATG5 m RNA and protein increased（P<0.05,P<0.01）,the average optical density increased（P<0.01,P<0.05）,AMPK,ULK1 protein expression increased（P<0.05,P<0.01）.Conclusion EA can delay the level of ovarian decline,protect the ovarian function in POI rats,and the mechanism of this may be related to the regulation of the AMPK/ULK1 pathway and activation of ovarian autophagy levels.更多还原