氰戊菊酯过度激活Ca^(2+)/CaM/CaMKⅡ信号通路诱发线粒体损伤干扰TM3细胞睾酮合成  

Overactivation the Ca^(2+)/CaM/CaMKⅡsignaling pathway by fenvalerate induces mitochondrial damage and interferes with testosterone synthesis in TM3 cells

作　　者：姚金玲 李廷兵 胡静 万小榆 孔德营 Yao Jinling;Li Tingbing;Hu Jing;Wan Xiaoyu;Kong Deying(Department of Physiology,School of Basic Medicine,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学基础医学院生理学教研室,贵州遵义563099

出　　处：《遵义医科大学学报》2023年第11期1032-1040,共9页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31760339);贵州省科技计划项目[NO:黔科合基础-ZK(2022)一般594];遵义医科大学大学生创新创业训练计划项目(NO:ZYDC202202170)。

摘　　要：目的利用胞内Ca^(2+)螯合剂BAPTA-AM和CaM阻断剂TFP探讨Ca^(2+)/CaM/CaMKⅡ信号通路在氰戊菊酯(Fen)诱发小鼠睾丸间质细胞(TM3细胞)线粒体损伤和睾酮合成障碍中的作用。方法TM3细胞实验分为对照组、Fen暴露组(25μmol/L Fen)、Fen+BAPTA-AM组(25μmol/L Fen+5 mmol/L BAPTA-AM)、Fen+TFP组(25μmol/L Fen+10μmol/L TFP),Fen暴露时长为24 h。采用ELISA法检测TM3细胞的睾酮和环磷酸腺苷(cAMP)水平,化学比色法检测TM3细胞的ATP水平,Flou-4/AM探针测定细胞内Ca^(2+)水平,JC-1染色法测定TM3细胞的线粒体膜电位(MMP)改变,蛋白免疫印迹法检测CYP11A1、3β-HSD、StAR、CaM、p-CaMKⅡ、CaMKⅡ、Bax和Bcl-2的蛋白表达水平。结果Fen暴露组TM3细胞的T、ATP和cAMP含量下降,MMP水平降低,睾酮合成的相关蛋白和酶CYP11A1、3β-HSD、StAR表达减少(P<0.01);同时,TM3细胞胞内的Ca^(2+)水平显著上升(P<0.01),CaM、p-CaMKⅡ/CaMKⅡ、Bax蛋白表达升高(P<0.01),Bcl-2蛋白表达下降(P<0.01)。Fen+BAPTA-AM组和Fen+TFP组TM3细胞的睾酮、ATP和cAMP含量上升,MMP水平升高(P<0.01),睾酮合成相关的蛋白和酶CYP11A1、3β-HSD、StAR表达增加(P<0.01),CaM、p-CaMKⅡ/CaMKⅡ及Bax蛋白表达下降,Bcl-2蛋白表达增加(P<0.01)。结论Fen诱发的TM3细胞mPTP开放以及睾酮合成障碍可能与Fen暴露后TM3细胞胞内Ca^(2+)超载、CaM和p-CaMKⅡ表达上调引起的Ca^(2+)/CaM/CaMKⅡ信号通路过度激活有关。Objective To investigate the role of the Ca^(2+)/CaM/CaMKⅡsignaling pathway in fenvalerate(Fen)-induced mitochondrial damage and testosterone synthesis disorders in mouse testicular interstitial cells(TM3 cells).Methods TM3 cells were exposed to Control(0μmol/L Fen),Fen(25μmol/L Fen),Fen+Ca^(2+)chelator BAPTA-AM(25μmol/L+5 mmol/L BAPTA-AM),and Fen+CaM blocker TFP(25μmol/L Fen+10μmol/LTFP)for 24 h.The levels of testosterone and cyclic adenosine monophosphate(cAMP)in TM3 cells were analyzed by ELISA,the contents of ATP by biochemical colorimetry,the intracellular Ca^(2+)level by Flou-4/AM probe,the changes of mitochondrial membrane potential(MMP)by JC-1 staining,and the protein expression levels of CYP11A1,3β-HSD,StAR,CaM,p-CaMKⅡ,CaMKⅡ,Bax and Bcl-2 by Western blot.Results The content of testosterone,ATP,cAMP,the MMP levels,and the expression of proteins and enzymes involved in testosterone synthesis(CYP11A1,3β-HSD and StAR)in TM3 cells were decreased in the Fen exposure group(P<0.01);Meanwhile,the intracellular Ca^(2+)level,the expression of CaM,p-CaMKⅡ/CaMKⅡ,and Bax proteins in TM3 cells were significantly increased(P<0.01),while the expression of Bcl-2 protein decreased(P<0.01)in the Fen exposure group.The content of testosterone,ATP,cAMP,the MMP level,and the expression of proteins and enzymes involved in testosterone synthesis(CYP11A1,3β-HSD and StAR)in TM3 cells were increased in the Fen+BAPTA-AM group and Fen+TFP group(P<0.01);the intracellular Ca^(2+)level,the expression of CaM,p-CaMKⅡ/CaMKⅡ,and Bax proteins in TM3 cells were significantly decreased(P<0.01),while the expression of Bcl-2 protein increased in the Fen+BAPTA-AM group and Fen+TFP group(P<0.01).Conclusion The mitochondrial permeability transition pore(mPTP)opening and testosterone synthesis impairments induced by Fen in TM3 cells may be related to the Ca^(2+)/CaM/CaMKⅡsignaling pathway overactivation caused by intracellular Ca^(2+)overload,and upregulation of CaM and p-CaMKⅡexpression after Fen exposure in TM3 cells.

关 键 词：氰戊菊酯 TM3细胞 睾酮 Ca^(2+)/CaM/CaMKⅡ信号通路 mPTP开放 

分 类 号：R339.2[医药卫生—人体生理学]