DJ-1 Mediates The Delayed Cardioprotection Of Hypoxic Preconditioning Via Activation Of Nrf2 And Subsequent Upregulation Of Antioxidative Enzymes

Objective:This study aims to investigate the underlying mechanism by which DJ-1 mediates the delayed cardioprotection of hypoxic preconditioning(HPC) against hypoxia/reoxygenation(H/R)-induced oxidative stress from the perspective of activation of nuclear factor erythroid 2-related factor 2(Nrf2) pathway and subsequent upregulation of antioxidative enzymes(manganese superoxide dismutase, Mn SOD, and heme oxygenase-1, HO-1) in H9c2 cells. Methods:1. To examine the role of DJ-1 in activation of the Nrf2 pathway 24 h after HPC in H9c2 cells. parental H9c2 cells and H9c2/DJ-1 si RNA cells were transfected with p Flag-h DJ-1 or its empty control vector p Flag for 24 h and then subjected to HPC. Twenty-four hours later, the expression of DJ-1 protein was analyzed by western blot. Kelch-like ECH-associated protein-1(Keap1)-Nrf2 interaction was assessed by Co-immunoprecipitation. Nrf2 nuclear translocation was analyzed by western blot. Chromatin immunoprecipitation(Ch IP) assay was performed for the binding of Nrf2 to antioxidant response elements(ARE) on promoters of Mn SOD and HO-1 genes.2. In order to observe the role of DJ-1 in the induction of antioxidative enzymes(Mn SOD and HO-1) 24 h after HPC, parental H9c2 cells and H9c2/DJ-1 si RNA cells were transfected with p Flag-h DJ-1 or its empty control vector p Flag for 24 h and then subjected to HPC. Twenty-four hours later, the expressions of Mn SOD and HO-1 proteins were analyzed by western blot.3. To observe the impact of Nrf2 knockdown on the DJ-1-mediated induction of antioxidative enzymes by HPC, parental H9c2 cells and H9c2/DJ-1 si RNA cells were transfected with p Flag-h DJ-1 or its empty control vector p Flag for 24 h and subsequently with negative control(NC) or Nrf2 si RNA for 24 h and subjected to HPC. Twenty-four hours later, the expressions of Nrf2, Mn SOD, and HO-1 proteins were analyzed by western blot.4. To examine the effects of Nrf2 knockdown on DJ-1-mediated the delayed cardioprotection of HPC against oxidative stress induced by H/R, parental H9c2 cells and H9c2/DJ-1-si RNA cells were transfected with p Flag-h DJ-1 or its empty control vector p Flag for 24 h and subsequently with NC si RNA or Nrf2 si RNA for 24 h and subjected to HPC. Twenty-four hours later, these cells subjected to H/R. Cellular damage was monitored by measuring cell viability and lactate dehydrogenase(LDH) release, and oxidative stress was analyzed by quantifying reactive oxygen species(ROS) and Malondialdehyde(MDA) content. Cell viability was detected by Methyl thiazolyl tetrazolium(MTT) method. LDH and MDA were detected by a colorimetric method. Intracellular ROS level was measured by flow cytometry. Results:1. In normal H9c2 cells, HPC upregulated DJ-1 expression, promoted nuclear factor Nrf2 and its cytoplasmic inhibitor Keap1 dissociation, and resulted in increased nuclear translocation and ARE-binding of Nrf2 24 h after HPC. However, in DJ-1-knockdown H9c2 cells, knockdown of DJ-1 exhibited inhibitory effects on HPC-derived Nrf2 activation. To further confirm that the inhibitory effects is indeed caused by specific downregulation of DJ-1 protein and is not due to off-target effects, we restored DJ-1 expression in the DJ-1-knockdown H9c2 cells by transfection of p Flag-h DJ-1 vector. After transient transfection of p Flag-h DJ-1 for 24 h, DJ-1 expression in DJ-1-knockdown H9c2 cells was restored to a level higher than baseline level. Remarkably, the restoration of DJ-1 expression was also accompanied by the restoration of Nrf2 activation by HPC(Fig. 1-4). In contrast, transfection of p Flag-h DJ-1 markedly increased DJ-1 levels concomitant with an obvious elevation in Nrf2 activation in the parental H9c2 cells.2. In normal H9c2 cells, HPC significantly upregulated HO-1 and Mn SOD expression in the late phase. However, in DJ-1-knockdown H9c2 cells, knockdown of DJ-1 exhibited inhibitory effects on the induction of HO-1 and Mn SOD protein expression by HPC. Likewise, the inhibitory effects were also reversed by restoration of DJ-1 expression.3. Knockdown of Nrf2 by si RNA in H9c2 cells mimicked the effects of DJ-1 knockdown and abolished HPC-derived the induction of antioxidative enzymes(Mn SOD and HO-1). More importantly, knockdown of Nrf2 also reversed the effects of restored DJ-1 expression on induction of antioxidative enzymes by HPC in DJ-1-knockdown H9c2 cells.4. In normal H9c2 cells, HPC attenuated H/R-induced viability loss, LDH release, and elevation of ROS and MDA content. However, in DJ-1-knockdown H9c2 cells, the aforementioned effects of HPC were reversed. More intriguingly, knockdown of Nrf2 by si RNA in H9c2 cells had the same role as DJ-1 knockdown, with an inhibitory effect in the delayed cardioprotection of HPC against H/R-induced oxidative stress. In addition, knockdown of Nrf2 also reversed the restored effects of restored DJ-1 expression on HPC-derived the delayed cardioprotection in DJ-1-knockdown cells. Conclusion:Activation of Nrf2 pathway and subsequent upregulation of antioxidative enzymes(Mn SOD and HO-1) could be a critical mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress in H9c2 cells.