TFEB介导的溶酶体功能障碍在ZEA致TM3细胞凋亡中的作用  

Role of TFEB-mediated lysosomal dysfunction in ZEA-induced apoptosis of TM3 cells

作　　者：杨黛祺 侯凡蓉 郑豪 邹辉[1] 顾建红[1] 袁燕[1] 刘学忠[1] 刘宗平[1,2] 卞建春 YANG Dai-qi;HOU Fan-rong;ZHENG Hao;ZOU Hui;GU Jian-hong;YUAN Yan;LIU Xue-zhong;LIU Zong-ping;BIAN Jian-chun(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2023年第7期932-938,共7页Chinese Veterinary Science

基　　金：国家重点研发计划支持项目(2016YFD0501208);高等学校学科创新引智计划资助(D18007);扬州大学大学生学术科技创新基金项目(X20210686);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：为揭示玉米赤霉烯酮(ZEA)的雄性生殖毒性机理,对转录因子EB(TFEB)介导的溶酶体功能障碍在ZEA致TM3细胞凋亡中的作用进行了研究,试验以小鼠睾丸间质细胞株(TM3细胞)为材料,用0(对照组)、5、10μmol/L ZEA分别处理细胞24 h,流式细胞术检测TM3细胞内Lysotracker水平;Western-blot检测凋亡相关蛋白CC3、CC9、Bax、Bcl2,溶酶体相关膜蛋白LAMP1、LAMP2,组织蛋白酶B(CTSB)、D(CTSD)以及细胞质内TFEB蛋白等的表达水平。过表达TFEB,Western-blot检测CC3、CC9、Bax、Bcl2表达水平。结果显示,与对照组相比,ZEA处理组Bax、CC3、CC9表达水平显著上升而Bcl2表达水平显著下降(P<0.05);流式细胞术检测结果显示溶酶体膜稳定性下降;CTSD和CTSB表达水平显著升高(P<0.05),LAMP2和TFEB表达水平显著降低(P<0.05),LAMP1表达水平在ZEA为5μmol/L时显著降低(P<0.05)。与ZEA组相比,TFEB过表达后ZEA组的Bax、CC3和CC9蛋白表达水平显著下降(P<0.05),而Bcl2表达水平显著上升(P<0.05),过表达TFEB可缓解ZEA引起的凋亡。结果表明,ZEA可引起TM3细胞凋亡增加,其作用机制与破坏TFEB信号通路,导致溶酶体出现功能障碍有关。To uncover the mechanism of male toxicity of zearalenone(ZEA),the role of TFEB-mediated lysosomal dysfunction in ZEA-induced apoptosis of TM3 cells was investigated.TM3 cells(mouse Leydig cell line)were used as materials.Cells were treated with 0(control group),5 and 10μmol/L ZEA for 24 h.The level of Lysotracker in TM3 cells was detected by flow cytometry.Western-blot was used to detect the expression levels of apoptosis-related proteins CC3,CC9,Bax,Bcl 2,lysosome-associated membrane proteins LAMP1 and LAMP2,cathepsin B(CTSB),D(CTSD)and TFEB protein in the cytoplasm.The TFEB in TM3 cells was overexpressed.Western-blot was used to detect the expression levels of CC3,CC9,Bax,Bcl_(2),etc.The results showed that compared with the control group,the expression levels of Bax,CC3 and CC9 in the ZEA treatment group increased significantly while the expression level of apoptotic protein Bcl 2 decreased significantly(P<00.05).Flow cytometry showed that lysosome membrane stability decreased.The expression levels of CTSD and CTSB were significantly increased(P<0.05).The expression levels of LAMP2andTFEBweresignificantlyreduced(P<00.05),andtheexpressionlevelofLAMP1wassignificantly reduced when the ZEA was 5μmol/L(P<0.05).Compared with the ZEA group,the expression levels of Bax,CC3 and CC9 proteins in the ZEB group decreased significantly(P<0.05).The expression levels of apopto-tic Bcl2 protein increased significantly(P<0.05),indicating that the overexpression of TFEB could alleviate apoptosis caused by ZEA.The results showed that ZEA could cause increased apoptosis in TM3 cells,and its mechanism of action was related to the destruction of TFEB signaling pathway,resulting in lysosomal dysfunction.

关 键 词：玉米赤霉烯酮(ZEA) 小鼠睾丸间质细胞(TM3) 转录因子EB 凋亡 溶酶体 线粒体 

分 类 号：S859.8[农业科学—临床兽医学]