基于AMPK介导的线粒体自噬研究黄精赞育胶囊治疗少弱精子症的分子机制  

Molecular Mechanisms of Huangjing Zanyu Capsule in Treating Oligoasthenospermia:A Study Based on AMPKMediated Mitophagy

作　　者：高源 郭怡阳 武默涵 郑燕飞[2] GAO Yuan;GUO Yiyang;WU Mohan;ZHENG Yanfei(School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;National Institute of Traditional Chinese Medicine Constitution and Preventive Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学中医学院,北京100029 [2]北京中医药大学国家中医体质与治未病研究院,北京100029

出　　处：《四川大学学报(医学版)》2025年第1期74-82,共9页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金面上项目(No.82174389)资助。

摘　　要：目的基于AMPK介导的线粒体自噬方面探究国医大师王琦院士研发的治疗男性不育的国家中药三类新药黄精赞育胶囊治疗少弱精子症细胞层面的分子机制。方法选择丙烯醛(acrolein,ACR)作用于GC-2spd(ts)小鼠精母细胞制作少弱精子症细胞模型,通过CCK8细胞活性检测确定后续造模用ACR浓度和造模时间。造模成功后加入不同浓度含黄精赞育胶囊的完全培养基进行细胞培养,于24 h后CCK8细胞活性检测,根据细胞活力确定后续给药浓度。GC-2spd细胞贴壁后分为NC组、造模组和ACR+HJZY给药组,通过共聚焦荧光显微镜观测黄精赞育胶囊对线粒体自噬的影响;将上述3组细胞分别转染siRNA-NC和siRNA-AMPK,分为siRNA-NC+control、siRNA-NC+ACR、siRNANC+ACR+HJZY、siRNA-AMPK+control、siRNA-AMPK+ACR、siRNA-AMPK+ACR+HJZY 6组,通过Western blot验证黄精赞育胶囊对AMPK介导的p-AMPK、LC3B、P62、PINK1、Parkin、TBK1、ULK1等线粒体自噬相关蛋白的调控作用。结果通过细胞活性检测实验确定ACR的造模浓度为34μmol/L,造模时间为20 min,HJZY给药浓度为160μmol/L。共聚焦荧光显微镜显示黄精赞育胶囊对受损生精细胞线粒体膜电位具有一定正向调节作用,造模组线粒体膜电位较NC组大幅降低,ACR+HJZY给药组给药后细胞膜电位较造模组有所升高,差异有统计学意义(P<0.05);Western blot检测结果显示siRNA-NC+ACR组p-AMPK/AMPK和PINK1蛋白表达水平较siRNA-NC+control组下降(P<0.001),siRNA-NC+ACR组Parkin蛋白水平较siRNA-NC+control组有所下降,但差异无统计学意义,HJZY给药后这3种蛋白表达水平均较siRNANC+ACR组上升(P<0.001);siRNA-NC+ACR组LC3B、P62、TBK1和ULK1蛋白表达水平均较siRNA-NC+control组上升(P<0.01),siRNA-NC+ACR+HJZY组上述蛋白表达水平均较siRNA-NC+ACR组有所下降(P<0.05)。转染敲减基因siRNAAMPK后,siRNA-AMPK+ACR组p-AMPK/AMPK、PINK1和Parkin蛋白表达水平较siRNA-AMPK+control组有所下降(P<0.01);H J Z Y给�Objective To investigate the molecular mechanism of Huangjing Zanyu Capsule(HJZY),a new class-Ⅲtraditional Chinese medicine for the treatment of male infertility developed by Wang Qi,an academician of the Chinese Academy of Engineering,based on AMPK-mediated mitophagy in the treatment of oligoasthenospermia.Methods Acrolein(ACR)was used to treat GC-2spd(ts)mouse spermatocytes to establish a cell model of oligoasthenospermia.The optimal ACR concentration and exposure time for subsequent modeling were determined by CCK8 cell viability assay.After successful modeling,the cells were cultured in complete medium containing different concentrations of HJZY.Then,cell viability was assessed by CCK8 assay after 24 hours,and the subsequent treatment concentration was determined based on the cell viability.After the GC-2spd cells adhered to the wall,they were divided into a normal control(NC)group,a modeling group,and an ACR+HJZY treatment group.The effect of HJZY on mitophagy was observed by confocal fluorescence microscopy.The three groups of cells were transfected with siRNA-NC and siRNA-AMPK,respectively,and divided into six groups,including siRNA-NC+control,siRNA-NC+ACR,siRNANC+ACR+HJZY,siRNA-AMPK+control,siRNA-AMPK+ACR,and siRNA-AMPK+ACR+HJZY groups.Western blot was performed to validate the regulatory effect of HJZY on mitophagy-related proteins,such as p-AMPK,LC3B,P62,PINK1,Parkin,TBK1,and ULK1,which were all proteins mediated by AMPK.Results Through the cell viability assay,34μmol/L was selected as the the modeling concentration of ACR,and 20 minutes was selected as the modeling time The treatment concentration of HJZY was 160μmol/L.Confocal fluorescence microscopy showed that HJZY had,to a certain degree,a positive regulatory effect on the mitochondrial membrane potential of damaged spermatogenic cells.The mitochondrial membrane potential of the model group decreased significantly compared with that of the NC group.After exposure to treatment,the cell membrane potential of the ACR+HJZY treatment group increased

关 键 词：男性不育 少弱精子症 AMPK 线粒体自噬 蛋白免疫印迹 

分 类 号：R285[医药卫生—中药学]