基于线粒体凋亡途径探讨“秩边透水道”针法对弱精子症小鼠生殖功能的影响  

Effect of"Zhibian"(BL54)-toward-"Shuidao"(ST28)acupuncture on reproductive function in mice with asthenozoospermia based on mitochondrial apoptosis

作　　者：郝健亨 常博雅 任佳 高珍 张艳林 王海军 冀来喜 HAO Jianheng;CHANG Boya;REN Jia;GAO Zhen;ZHANG Yanlin;WANG Haijun;JI Laixi(College of Acupuncture-Moxibustion and Tuina,Chengdu University of TCM,Chengdu 610075,Sichuan Province,China;Second Clinical College,Shanxi University of CM,Jinzhong 030619)

机构地区：[1]成都中医药大学针灸推拿学院,四川成都610075 [2]山西中医药大学第二临床学院,晋中030619

出　　处：《中国针灸》2025年第1期71-81,共11页Chinese Acupuncture & Moxibustion

基　　金：国家自然科学基金资助项目:82074549;山西省中医药管理局基金项目:2023ZYYB048;山西中医药大学博士科研启动基金项目:2023BK17。

摘　　要：目的:观察“秩边透水道”针法对弱精子症小鼠睾丸组织线粒体凋亡途径中关键调控因子的影响,探究其对生殖功能保护作用的可能机制。方法:将30只C57BL/6雄性小鼠随机分成空白组、模型组和针刺组,各10只。模型组和针刺组小鼠给予环磷酰胺(30 mg·kg-1·d-1)腹腔注射7 d制备弱精子症模型。造模成功后针刺组小鼠予“秩边透水道”针法干预,每日1次,每次20 min,共干预2周。观察小鼠一般情况,并记录小鼠造模前、造模后和干预后体质量。干预后,记录小鼠睾丸质量并计算睾丸系数;检测小鼠精子质量;ELISA法检测血清睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)含量;HE染色法观察睾丸组织形态;透射电镜观察睾丸组织线粒体超微结构;JC-1染色检测睾丸组织线粒体膜电位水平;TUNEL染色法观察睾丸组织凋亡细胞情况;实时荧光定量PCR法和Western blot法分别检测睾丸组织B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X的蛋白(Bax)、细胞色素c(Cyt C)、凋亡蛋白酶活化因子1(Apaf-1)、半胱氨酸蛋白酶(Caspase)9、Caspase-3 mRNA和蛋白表达;免疫组化法检测睾丸组织剪切的Caspase-3(Cleaved Caspase-3)阳性表达。结果:与空白组比较,模型组小鼠精神萎靡,毛发粗糙,饮食与活动量减少,体质量减轻(P<0.01);睾丸质量和睾丸系数下降(P<0.01);精子总数、精子活力和精子活率降低(P<0.01);血清T、FSH和LH含量降低(P<0.01);睾丸组织内部分生精小管形态异常,生精细胞数量减少,线粒体数量减少,线粒体内嵴发生断裂缺失,出现空泡现象;线粒体膜电位降低(P<0.01);睾丸组织凋亡细胞阳性率升高(P<0.01);睾丸组织Bax、Cyt C、Apaf-1、Caspase-9、Caspase-3 mRNA和蛋白表达升高(P<0.01,P<0.05),Bcl-2 mRNA和蛋白表达降低(P<0.01),Cleaved Caspase-3平均吸光度值升高(P<0.01)。与模型组比较,针刺组小鼠一般情况好转;睾丸质量和睾丸系数升高(P<0.01);精子总数、精子活力Objective To observe the effects of the"Zhibian"(BL54)-toward-"Shuidao"(ST28)acupuncture on key regulatory factors during mitochondrial apoptosis of testicular tissue in asthenozoospermia mice,and explore the potential mechanism of the protective effect of acupuncture on reproductive function.Methods Thirty C57BL/6 male mice were randomly divided into a blank group,a model group and an acupuncture group,10 mice in each group.In the model and the acupuncture groups,the intraperitoneal injection of cyclophosphamide(30 mg·kg-1·d-1)was delivered for 7 days to prepare the asthenozoospermia model.After the success of modeling,the modeled mice in the acupuncture group were intervened with"Zhibian"(BL54)-toward-"Shuidao"(ST28)acupuncture,once daily and the needles were retained for 20 min.The duration of the intervention was 2 weeks.The general condition of each mouse was observed,and the body mass was recorded before modeling,after modeling and after intervention completion.After intervention,the testicular mass was recorded and the weight coefficient was calculated,and the mouse sperm quality was examined;the serum contents of testosterone(T),follicle stimulating hormone(FSH)and luteinizing hormone(LH)were detected using ELISA,the morphology of testicular tissue was observed using HE,the mitochondrial ultra-microstructure of testicular tissue was observed under transmission electrone microscopy,the mitochondrial membrane potential level of testicular tissue was detected using JC-1 staining,the positive rate of apoptosis cell of testicular tissue was observed using TUNEL;and the mRNA and protein expression of b-cell lymphocytoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cytochrome c(Cyt C),apoptotic protease-activating factor1(Apaf-1),Caspase-9 and Caspase-3 of testicular tissue was detected using real-time quantitative fluorescence PCR and Western blot methods separately;and the positive expression of Cleaved Caspase-3 of the testicular tissue was detected using immunohistochemistry.Results Compared with the blank gro

关 键 词：弱精子症 针刺 秩边透水道 线粒体 凋亡 

分 类 号：R245[医药卫生—针灸推拿学]