线粒体凋亡途径在玉米赤霉烯酮致大鼠睾丸支持细胞凋亡中的作用

Effect of Mitochondria-dependent Pathway on Apoptosis Induced by Zearalenone in Rat Sertoli Cells

为探讨玉米赤霉烯酮(zearalenon,ZEA)雄性生殖毒性作用机理,研究ZEA诱导大鼠睾丸支持细胞(sertoli cell,SC)的凋亡途径,试验选取大鼠原代SC为研究材料,用不同浓度ZEA(0、5、10、20μmol/L)处理SC 24 h后,采用实时荧光定量PCR法检测Bax和Bcl-2 mRNA表达水平;用Western blotting法检测Bax、Bcl-2等线粒体凋亡途径相关蛋白表达;同时检测了20μmol/L ZEA联合Caspase-8抑制剂(Z-IETD-FMK)处理对SC凋亡率和线粒体凋亡相关蛋白的影响。结果显示,与对照组相比,20μmol/L ZEA组细胞Bcl-2转录水平显著下降(P<0.05),Bax转录水平显著上升(P<0.05);10、20μmol/L ZEA组Bax/Bcl-2比值及Cyto-CytC、Cleaved Caspase-9、Cleaved Caspase-3表达量极显著上升(P<0.01),而Mito-CytC表达量极显著下降(P<0.01);与ZEA组相比,ZEA与Z-IETD-FMK联合处理可使ZEA诱导的SC凋亡率极显著下降(P<0.01),tBid/Bid、Bax/Bcl-2、Cyto-CytC、Cleaved-Caspase3、Cleaved-Caspase9蛋白表达显著或极显著下降(P<0.05;P<0.01),而Mito-CytC表达量极显著上升(P<0.01)。综合试验结果,ZEA能够通过线粒体途径诱导大鼠SC发生凋亡。

In order to elucidate the mechanism of zearalenone (ZEA) on male reproductive toxicity and investigate the pathway of sertoli cell (SC) apoptosis induced by ZEA,the primary SC was used as the experiment material,exposed to different concentration of ZEA(0,5,10,20 μmol/L) for 24 h.qRT-PCR was used to detect the level of gene transcription of Bax and Bcl-2;Western blotting method was used to detect the expression of proteins related to mitochondria-dependent apoptotic pathway.The changes of apoptosis rate and apoptosis-related protein were detected by 20 μmol/L ZEA combined with the inhibitor of Caspase-8 (Z-IETD-FMK).The results showed that compared with the control group,the level of gene transcription of Bcl-2 was decreased significantly in the group treated with 20 μmol/L of ZEA ( P <0.05),while the level of gene transcription of Bax increased significantly ( P <0.05).The ratio of Bax/Bcl-2 and the expression of Cyto-CytC,Cleaved caspase-3 and Cleaved caspase-9 were extremely significantly increased while the expression of Mito-CytC was decreased extremely significantly in the group treated with 10,20 μmol/L of ZEA ( P <0.01).Compared with ZEA group,the apoptosis rate induced by ZEA decreased extremely significantly after being treated with ZEA and Z-IETD-FMK ( P <0.01).The ratio of tBid/Bid and Bax/Bcl-2,Cyto-CytC,Cleaved Caspase-3 and Cleaved Caspase-9 were decreased significantly or extremely significantly ( P <0.05;P <0.01),while the Mito-CytC protein expression was increased extremely significantly ( P <0.01).The results showed that ZEA could induce the apoptosis of SC via the mitochondria-dependent apoptotic pathway.