The Molecular Mechanism Of DJ-1 Mediating The Delayed Cardioprotection Of Hypoxic Preconditioning Through Preserving Mitochondrial Complex Ⅰ In Hypoxic-Reoxygenated Cardiomyocytes

Objective:This study aims to investigate the underlying mechanism by which DJ-1mediates the delayed cardioprotection of hypoxic preconditioning(HPC)against hypoxia/reoxygenation(H/R)-induced oxidative stress through preserving mitochondrial Complex Ⅰ activity in H9c2 cells from the perspective of the interactions between DJ-1 and ND1/NDUFA4 subunits of mitochondrial Complex Ⅰ.Methods:1.To observe whether HPC promotes the interactions between DJ-1 and ND1/NDUFA4 by upregulating DJ-1 expression in H9c2 cells subjected to H/R,H9c2 cells were transfected with NC-siRNA or DJ-1-siRNA for 24 h and then subjected to HPC 24 h prior to H/R,or transfected with pFlag or pFlag-DJ-1 for 24 h followed by H/R.Subsequently,the association of DJ-1 with ND1/NDUFA4 was evaluated by co-immunoprecipitation and immunofluorescence assays.2.To observe whether DJ-1-mediated the protection of HPC on mitochondrial Complex Ⅰ is associated with the interactions between DJ-1 and ND1/NDUFA4 in H9c2 cells subjected to H/R.H9c2 cells were transfected with NC-siRNA or DJ-1-siRNA for 24 h and then subjected to HPC 24 h prior to H/R,or transfected with pFlag or pFlag-DJ-1 for 24 h followed by H/R.Subsequently,the activity of mitochondrial Complex Ⅰ was detected by spectrophotometry.3.To observe whether DJ-1-mediated the inhibition of HPC on mitochondrial ROS production is associated with the interactions between DJ-1 and ND1/NDUFA4 in H9c2 cells subjected to H/R.H9c2 cells were transfected with NC-siRNA or DJ-1-siRNA for 24 h and then subjected to HPC 24 h prior to H/R,or transfected with pFlag or pFlag-DJ-1 for 24 h followed by H/R.Subsequently,the mitochondrial ROS generation was measured by a Mito SOX Red fluorescent staining.4.To observe whether DJ-1-mediated the delayed cardioprotection of HPC against H/R-induced oxidative stress is associated with the interactions between DJ-1and ND1/NDUFA4 in H9c2 cells.H9c2 cells were transfected with NC-siRNA or DJ-1-siRNA for 24 h and then subjected to HPC 24 h prior to H/R,or transfected with pFlag or pFlag-DJ-1 for 24 h followed by H/R.Subsequently,oxidative stress was monitored by quantifying intracellular ROS and Malondialdehyde(MDA)content,and the cell damage was analyzed by cell viability and lactate dehydrogenase(LDH)release.The intracellular ROS level was assessed by flow cytometry using DCFH-DA fluorescent staining.MDA content was determined by using the corresponding assay kit.Cell viability was detected by CCK8 method.LDH release was evaluated by a colorimetric method.Results:1.HPC remarkably up-regulated DJ-1 expression and enhanced the interaction between DJ-1 and ND1/NDUFA4 in H9c2 cells subjected to H/R.These effects were reproduced in H9c2 cells transfected with pFLAG-DJ-1 recombinant plasmid and inhibited in H9c2 cells transfected by DJ-1-siRNA.2.HPC significantly increased mitochondrial Complex Ⅰ activity in H9c2 cells subjected to H/R.The protective effect of HPC was inhibited in DJ-1-siRNAtransfected H9c2 cells but mimicked in pFLAG-DJ-1-transfected H9c2 cells.3.HPC significantly reduced H/R-induced mitochondria ROS generation in H9c2 cells.The above effect was inhibited in DJ-1-siRNA-transfected H9c2 cells but reproduced in pFLAG-DJ-1-transfected H9c2 cells.4.HPC obvionsly decreased ROS and MDA levels,increased cell viability,and decreased LDH release in H9c2 cells subjected to H/R.The above effects of HPC were also inhibited in DJ-1-siRNA-transfected H9c2 cells but mimicked in pFLAG-DJ-1-transfected H9c2 cells.Conclusion:This work revealed that up-regulating DJ-1 expression and subsequently promoting the interaction between DJ-1 and ND1/NDUFA4 could be a key mechanism by which HPC preserves mitochondrial Complex Ⅰ and induced the delayed cardioprotection of HPC against H/R-induced oxidative stress damage in H9c2 cells.