马兜铃酸诱导TM3小鼠睾丸间质细胞凋亡机制

The mechanism of apoptosis in mice testicular cells TM3 induced by aristolochic acid

目的:探讨马兜铃酸(AAs)对TM3细胞凋亡的作用及分子机制。方法:用2.5、10、25、100、400、1000μmol/L浓度的马兜铃酸分别对TM3细胞作用24、48、72 h后,CCK-8法测定细胞活力;25、100、400μmol/L的马兜铃酸对TM3细胞作用48 h后,TUNEL检测细胞凋亡率,实时荧光定量法检测Caspase通路相关基因Caspase-3、Caspase-9和Bax mRNA相对表达情况,并通过Western blot法检测凋亡相关蛋白Caspase-3、Caspase-9和Bax蛋白表达水平变化。结果:不同浓度的马兜铃酸对小鼠睾丸间质细胞的活力有一定的抑制作用;用25、100、400μmol/L浓度的马兜铃酸处理TM3细胞48 h后,通过TUNEL染色发现,不同浓度的马兜铃酸能够促进细胞的凋亡,Caspase-3、Caspase-9、Bax mRNA和蛋白表达水平升高。结论:马兜铃酸通过Caspase途径促进TM3细胞的凋亡来影响生殖细胞的发育。

Objective:To study the effects and mechanism of apoptosis in mice testicular cells induced by Aristolochic acids(AAs).Methods:AAs at concentrations of 2.5,10,25,100,400 and 1000μmol/L acted on TM3 cells for 24,48 and 72 h cell viability was determined by CCK-8 assay.TUNEL assay was used to examine apoptosis rate by 25,100,400μmol/L of AAs acted on TM3 cells for 48 h.qRT-PCR detected the relative expressions of Caspase-3,Caspase-9 and Bax mRNA in the caspase pathway.The level of apoptosis-related proteins Caspase-3,Caspase-9 and Bax were detected by Western blot.Results:After TM3 cells were treated with 25,100 and 400μmol/L AAs for 48 h,different concentrations of AAs could promote apoptosis by TUNEL staining.The results of qRT-PCR showed that the expression levels of Caspase-3,Caspase-9 and Bax increased.Similarly,Western blot showed that the expression levels of Caspase-3,Caspase-9 and Bax increased.Conclusion:AAs can promote apoptosis of TM3 cells through caspase pathway to influence the development of leydig cells.