DJ-1 Participates In Hypoxic Preconditioning-induced Delayed Cardioprotection Through Maintaining The Activity Of Mitochondrial Complex ?

Objective:This study aims to investigatethe underlying mechanism bywhich DJ-1 mediates the delayed cardioprotection of hypoxic preconditioning(HPC)against hypoxia/reoxygenation(H/R)injury from the perspective that mitochondrial respiratory chain complex I activity variation exerts influence on mitochondrial function and occurrence of oxidative stress in H9c2 cells.Methods:1.To analyze the effects of HPC treatment on the expression level and mitochondrial distribution of DJ-1,H9c2 cells were subjected to HPC.Twenty-four hours later,proteins in cytoplasm and mitochondria in H9c2 cells with or without H/R treatment were separated from each other.The content of DJ-1 protein in each part was analyzed by Western blot.2.To observe the role of DJ-1 in HPC against H/R-induced impairment of mitochondrial function,H9c2 cells were transfected with pFLAG-rDJ-1 or Si-DJ-1 RNAs for 24 h and then subjected to HPC.Twenty-four hours after HPC treatment,the expression levels of DJ-1 protein,the mitochondrial membrane potential and the mitochondria-derived superoxide in H9c2 cells with or without H/R treatment were analyzed by Western blot,JC-1 staining and MitoSOX RED incubation respectively.3.In order to observe the impact of mitochondrial complex I inhibition on the DJ-1-mediated antioxidant effect of HPC,H9c2 cells were transfected with pFLAG-rDJ-1 or Si-DJ-1 RNAs,followed by HPC,complex I inhibition and H/R treatments at the time point of 24 h,36 h and 48 h after the transfection.At the end of H/R procedure,the expression of DJ-1 protein was analyzed by Western blot.The activities of mitochondrial complex I and the antioxidant enzymes(SOD,CAT and GPx)were assessed by spectrophotometry.The total intracellular reactive oxygen species(ROS)production was detected by DCFH-DA probe.4.To detect the effects of mitochondrial complex I inhibition on the DJ-1-mediated delayed cytoprotection of HPC against H/R-induced cell injury,H9c2 cells were transfected with pFLAG-rDJ-1 or Si-DJ-1 RNAs,followed by HPC,complex I inhibition and H/R treatments at the time point of 24 h,36 h and 48 h after the transfection.After H/R procedure,cell injury was measured by cell counting kit-8(CCK8)and lactate dehydrogenase(LDH)release was detected by a colorimetric method.Results:1.Whether the cells were received H/R treatment or not,twenty-four hours after HPC treatment,the expression level of DJ-1 in H9c2 cells was up-regulated.Additionally,the content of mitochondrial DJ-1 was also elevated.2.In parental H9c2 cells,HPC significantly alleviated the H/R-induced collapse of mitochondrial membrane potential and accumulation of mitochondrial-derived superoxide.The HPC-afforded protection of mitochondrial function was mimicked by pFLAG-rDJ-1 transefection.In contrast,Si-DJ-1 RNAs transfection totally abolished the beneficial effects of HPC treatment.3.The H/R-induced decrease in mitochondrial complex I activity,impairment of antioxidant enzymes(SOD,CAT and GPx)and accumulation of intracellular ROS in parental H9c2 cells were significantly improved by pretreated HPC.These HPC-afforded beneficial effects were mimicked by DJ-1 overexpression and blocked by DJ-1 knockdown.More intriguingly,the delayed protection of HPC against H/R-induced antioxidant enzymes impairment and ROS burst was totally abolished by mitochondrial complex I inhibition.The HPC-like antioxidant effect which was observed in DJ-1 overexpressed H9c2 cells was also abolished by mitochondrial complex I inhibition.4.The delayed protection of HPC against H/R-induced cell damage was mimicked by DJ-1 overexpression and abolished by DJ-1 knockdown.Moreover,the cytoprotective effect which was afforded by HPC and DJ-1 overexpression was blocked by mitochondrial complex I inhibition.Conclusion:Maintaining the activity of mitochondrial complex I and subsequent protecting the mitochondrial function and inhibiting the oxidative stress could be an essential mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced cell injury.