Nrf2-dependent Upregulation Of Antioxidative Enzymes: A Novel Pathway For Hypoxic Preconditioning-mediated Delayed Cardioprotection

Objective:To invesgate whether activation of the Nrf2-ARE pathway is responsible for theinduction of antioxidative enzymes by HPC and contributes to the delayedcardioprotection of HPC by constructing Nrf2gene-specific small interfering RNA(siRNA) expression vectors and then transfecting into H9c2cells.Methods:1. The complementary shRNA oligonucleotides targeting the Nrf2gene weredesigned, synthesized, annealed and inserted into the pGPU6/GFP/Neo plasmid togenerate pGPU6/GFP/Neo-Nrf2siRNA recombinant plasmids. Moreover, thenegative control siRNA recombinant plasmid pGPU6/GFP/Neo-NC siRNA was alsoconstructed to examine the specificity of pGPU6/GFP/Neo system. All recombinantplasmid were identified by DNA sequencing analysis.2. H9c2cells were harvested at0,12,24,48, and72h following HPC, afterwhich cytosolic and nuclear fractions were prepared and analyzed for the content ofcytosolic and nuclear Nrf2by western blot. The interaction of Nrf2with AREsequences in the promoter regions of the antioxidant enzymes HO-1and MnSODwere examined at24h after HPC by ChIP assays.3. H9c2cells were transiently transfected with or without negative control (NC)or Nrf2siRNA expression vector for24h and then subjected to HPC. Afterexperimental treatment, the expressions of HO-1, MnSOD, and nuclear Nrf2were,respectively, detected by Western blot.4. H9c2cells were transiently transfected with or without negative control (NC)or Nrf2siRNA expression vector for24h and then subjected to HPC24h before H/R.After experimental treatment, cell viability was measured by Methyl thiazolyltetrazolium (MTT) method. Lactate dehydrogenase (LDH), malondialdehyde (MDA),superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) weredetected by a colorimetric method. And intracellular ROS level was measured by flow cytometry.Results:1The constructions of the recombinant Nrf2siRNA expression vectorpGPU6/GFP/Neo-Nrf2siRNA and the negative control vector pGPU6/GFP/Neo-NCsiRNA were successfully confirmed by the results of DNA sequencing.2. Western blot analysis showed that the Nrf2nuclear translocation appearedapparent as earlier as at12h, reached a peak at24h and maintained high levels till72h after HPC in H9c2cells. The time course of Nrf2nuclear translocation wasconsistent with the established time window of delayed cytoprotection afforded byHPC. Moreover, ChIP assay showed that HPC-induced direct binding of Nrf2to AREon the promoters of HO-1and MnSOD in H9c2cells.3. In untransfected H9c2cells, HPC significantly upregulated HO-1andMnSOD expression in the late phase. However, in Nrf2siRNA expression vectortransfected H9c2cells, Nrf2expression was knocked down, and, as a result, theinduction of HO-1and MnSOD protein expression by HPC was apparently abolished.4. HPC attenuated H/R-induced viability loss and LDH release in untransfectedH9c2cells, In addition, HPC inhibited H/R-induced the elevation of ROS and MDAcontent and alleviated the reduction of cellular antioxidant enzymes (SOD, CAT, andGPx) activities. These results again demonstrate that HPC can inhibite H/R-inducedoxidant stress and induce the delayed cardioprotection. However, in Nrf2siRNAexpression vector transfected H9c2cells, when nuclear Nrf2expression wasspecifically knocked down by siRNA, the inhibitory effect of HPC on H/R-inducedoxidant stress was reversed, and the delayed cardioprotection induced by HPC wasalso abolished.Conclusion:1. We successfully constructed the recombinant Nrf2siRNA expression vectorpGPU6/GFP/Neo-Nrf2siRNA, which lays the foundation for further studying thebiological function of Nrf2in the delayed cardioprotection of HPC.2. We have demonstrated that activation of Nrf2-ARE pathway is a novelmechanism by which HPC upregulates antioxidative enzymes HO-1and MnSOD in H9c2cells, thereby providing a delayed cardioprotection against oxidative stress byH/R.