DJ-1 Upregulates Antioxidant Enzymes And Attenuates Hypoxia/Reoxygenation-Induced Oxidative Stress By Activation Of Nrf2-ARE Signaling Pathway

Objective:To investigate the role of Nrf2 signaling pathway in the DJ-1-induced upregulation of anti-oxidative enzymes and cardioprotection against H/R-induced oxidative stress in H9c2 cells by molecular biological methods such as gene transfection and RNA interference. Methods:1. To determine the role of DJ-1 in regulation of cellular antioxidant enzymes(MnSOD, GPx, and CAT) expression. The negative control(NC) or DJ-1 siRNA, or pFlag-DJ-1 or its empty control vector pFlag were transiently transfected for 24 h into H9c2 cells. DJ-1 expression and antioxidant enzymes(MnSOD, GPx, and CAT) levels were examined by western blot.2. To investigate the effects of DJ-1 on H/R-induced oxidative stress injury in H9c2 cells. The negative control(NC) or DJ-1 siRNA, or pFlag-DJ-1 or its empty control vector pFlag were transiently transfected for 24 h into H9c2 cells and followed by H/R. Subsequently, cell damage was estimated by LDH release and cell viability, and oxidative stress was evaluated by quantifying MDA and ROS content.3. To investigate the role of DJ-1 in regulation Keap1-Nrf2 interaction, Nrf2 nuclear translocation, ARE-binding, and its transcriptional activity. The negative control(NC) or DJ-1 siRNA, or pFlag-DJ-1 or its empty control vector pFlag were transiently transfected for 24 h into H9c2 cells.(A) Keap1-Nrf2 interaction was assessed by Co-immunoprecipitation.(B) Nrf2 nuclear translocation was analyzed by western blot.(C) ChIP assay was implemented for the exploration of ARE-binding activity of Nrf2.(D) The transcriptional activity of Nrf2 was tested by Luciferase reporter assay.4. To investigate the effects of Nrf2 knockdown on the DJ-1-mediated induction of antioxidative enzymes(MnSOD, GPx, and CAT) in H9c2 cells. pFlag-DJ-1 or its empty vector pFlag were transiently transfected into H9c2 cells for 24 h and subsequently with or without the negative control(NC) or Nrf2 siRNA for 24 h. The overexpression of DJ-1 was tested by western blot using DJ-1 and Flag antibodies, Nrf2 and antioxidant enzymes(MnSOD, GPx, and CAT) levels were examined with Nrf2, MnSOD, CAT, and GPx antibodies, respectively.5. To investigate the effects Nrf2 knockdown on DJ-1-mediated cardioprotection against H/R-induced oxidative stress in H9c2 cells. pFlag-DJ-1 or its empty vector pFlag were transiently transfected into H9c2 cells for 24 h and subsequently with or without the negative control(NC) or Nrf2 siRNA for 24 h and followed by H/R. The quantifying of MDA and ROS content were used to assess Oxidative stress, and cell viability and LDH release were used to evaluate cellular damage. Results:1. The transfection of pFlag-DJ-1 markedly increased DJ-1 levels concomitant with an obvious elevation in MnSOD, CAT and GPx expression in H9c2 cells. In addition, the 24-h transfection of pFlag-DJ-1 markedly attenuated H/R-induced viability loss, LDH release as well as elevation of ROS and MDA content. Conversely, in DJ-1 siRNA-transfected H9c2 cells, knockdown of DJ-1 significantly reduced endogenous MnSOD, CAT and GPx protein levels. Concomitantly, these DJ-1-deficient cells were more susceptible to H/R-induced oxidative stress. These findings demonstrated that DJ-1 can induce the expression of anti-oxidant enzymes and attenuate H/R-induced oxidative stress in H9c2 cells.2. In transfected pFlag-DJ-1 H9c2 cells, DJ-1 overexpression promoted Nrf2-Keap1 dissociation and resulted in increased nuclear translocation, ARE-binding and transcriptional activity of Nrf2. However, in DJ-1-siRNA-transfected H9c2 cells, knockdown of DJ-1 exhibited inhibitory effects on the Keap1-Nrf2 dissociation, and subsequent nuclear translocation, ARE-binding and transcriptional activity of Nrf2. These results indicated that DJ-1 has an obligatory role in the activation of the Nrf2 pathway in H9c2 cells.3. DJ-1 overexpression upregulated anti-oxidative enzymes(MnSOD, CAT and GPx), whereas simultaneously knocking down Nrf2 significantly attenuated DJ-1-mediated induction of antioxidative enzymes. These results indicated that activation of the Nrf2 pathway is required for DJ-1-mediated induction of anti-oxidative enzymes.4. DJ-1 overexpression protected H9c2 cells against H/R-mediated oxidative stress as shown by attenuation of H/R-induced viability loss, LDH release, as well as ROS and MDA content, whereas simultaneously knocking down Nrf2 significantly attenuated DJ-1-mediated cytoprotection. These results clearly indicated an essential role of Nrf2 activation for DJ-1-mediated cytoprotection against H/R-induced oxidative stress. Conclusion:These findings shown that activation of the Nrf2 signaling pathway is a crucial mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2 cells.