| OBJECTIVE The purpose of this study was to investigate the effect of troglitazone-induced toxicity in human cardiomyocytes,and explore its mechanism related to mitochondrial oxidative stress and autophagy.METHODS AC 16 cells were treated with different concentrations of troglitazone for 24 hours.The morphological changes was observed under a microscope,cell viability was examined by CCK-8assay,whereas LDH leakage was estimated by assay kit.Mitochondrial membrane potential and intracellular reactive oxygen species were detected by high content analysis using TMRM and CM-H2DCFDA fluorescent probes.Mitochondrial mass and Mitochondrial ROS were detected by Flow cytometry using Mito tracker green and Mito SOX Red fluorescent probes.The content of ATP was detected by luciferase chemiluminescence and mitochondrial mtDNA copy number were detected by RT-qPCR to evaluate the drug mitochondrial toxicity.The protein expression of PGC-1?,Nrf2 and autophagy markers LC3-II and LC3-I ratio and p62 was detected by Western blotting.RESULTS As compared to the cells in the control group,troglitazone induced morphological changes,reduction of cells viability?r=-0.928,P<0.05?,and increase of LDH leakage?r=0.746,P<0.05?by a concentration-dependent manner.MMP,mtDNA copy number and ATP content were decreased while ROS were increased in the cells treated with 10 and 20?M of troglitazone?P<0.05?.After 24 h exposure,when treated with 10 and 20?M of troglitazone was found significantly decrease PGC-1?and its downstream proteins and activate Nrf2 antioxidant pathway?P<0.05?.Meanwhile,troglitazone was found to significantly increase the ratio of LC3-II/LC3-I?P<0.05?and up-regulate the protein expression of p62?P<0.05?.CONCLUSION troglitazone can induce cytotoxicity and mitochondrial dysfunction in a concentration-dependent manner,which is possible related to mitochondrial oxidative stress and inhibiting autophagosome degradation in human cardiomyocytes. |
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