旋毛虫HSP70蛋白诱导大鼠心肌细胞线粒体凋亡机制的研究

Effects of Trichinella spiralis HSP70 protein on apoptosis related protein expression in rat cardiomyocytes

为了解旋毛虫HSP70致心肌损伤的机制,本研究将不同浓度旋毛虫重组HSP70蛋白(Ts-Hsp70)与大鼠心肌细胞H9c2共孵育后,采用CCK-8试剂盒筛选Ts-Hsp70作用H9c2细胞的最适浓度。以最适浓度Ts-Hsp70与H9c2细胞共孵育不同时间后,采用微孔板法检测细胞培养上清液中乳酸脱氢酶(LDH)的含量,采用比色法检测细胞培养上清液中心肌细胞损伤标志物肌酸激酶(CK)的含量,采用免疫荧光检测Ts-Hsp70作用后H9c2细胞活性氧(ROS)的释放,采用qPCR和western blot方法检测Ts-Hsp70作用后的H9c2细胞线粒体凋亡与抗凋亡途径中的Bax、Caspase-3、Caspase-9、Cytochrome C与Bcl-2的基因转录水平与蛋白表达水平。结果显示,经最适浓度为60μg/mL的Ts-Hsp70分别刺激H9c2细胞6 h、12 h和18 h后,随着Ts-Hsp70对H9c2细胞作用时间的延长,LDH、CK、ROS含量与Bax、Cytochrome C及激活状态下的Caspase-3、Caspase-9基因转录水平与蛋白表达水均升高,且均于作用12 h时达峰值,显著高于对照组(P<0.01),而线粒体抗凋亡基因Bcl-2基因转录水平与蛋白表达水平随着Ts-Hsp70对H9c2细胞作用时间的延长均逐渐降低,且均于作用12 h时达谷值,显著低于对照组(P<0.01)。本研究首次证实Ts-Hsp70可增加细胞中LDH、CK、ROS的含量,激活并调节H9c2细胞中的线粒体凋亡通路,该研究结果为进一步研究旋毛虫致心肌损伤机制提供了基础数据。

To investigate the mechanism of cardiac injury induced by Trichinella spiralis,the recombinant protein of Trichinella spiralis HSP70(Ts-HSP70)was applied to rat H9c2 myocardial cells.The optimum concentration of Ts-Hsp70 stimulated H9c2 cells was dertermined by CCK-8 kit.The amount of lactate dehydrogenase(LDH)was determined by microplate method,and methodcreatine kinase(CK)as marker of myocardial injury was measured by colorimetry.ROS release was detected by immunofluorescence as well as western blot.qPCR were used to detect the expression of Bax,Caspase-3,Caspase-9,Cytochrome C,and Bcl-2 in mitochondrial apoptosis and anti-apoptosis pathways.The results showed that when the optimal concentration of Ts-HSP70 at 60μg/mL stimulated H9c2 cells for 6 hours,12 hours and 18 hours,with the extension of the culturing time of Ts-HSP70 on H9c2 cells,the amount of LDH,CK,ROS and the gene transcription and protein expression level of Bax,Cytochrome C and activated Caspase-3,Caspase-9 gene were significantly higher than that of control group(P<0.01),and reached the peak at 12 hours.Meanwhile,the expression of mitochondria antiapoptotic gene Bcl-2 was significantly lower than control group(P<0.01),and reached the lowest at 12 hours.The results for the first time showed that Ts-Hsp70 could increase the amount of LDH,CK and ROS in H9c2 cells,and activate and regulate the mitochondrial apoptosis pathway in H9c2 cells.The results provided basic infromation for further research on the mechanism of cardiac injury induced by Trichinella spiralis.