Inhibition of NADPH oxidase during ischemia-reperfusion protects rat cardiomyoblasts from cellular death.

Oxidative stress is increased in cellular models of ischemiareperfusion (VR) injury, with reperfusion thought to be the most potent stimulus for Reactive Oxygen Species (ROS) production. In order to ascertain the biochemical bases of these effects, aim of the study was to evaluate NADPH oxidase-dependent superoxide production during I/R and its ability to trigger oxidative stress-induced kinases (ERK, JNK and p-3 8) pathway. H9c2 cells were subjected to simulated ischemia (SI) for 24 h and to t h reperfusion. Increases in NADPH oxidase activity, intracellular ROS content, and MDA level, a marker of lipid peroxidation, were observed after IiR (4.I6-fold vs. control , p10.01; 4.I3-fold vs. control , p10.01; 1.817-fold vs.control , p 10.001 , respectively). In the presence of 10 pM DPI, a NADPH oxidase inhibitor, we found a significant reduction of these parameters as we obtained with 10 mM Tiron, a superoxide scavenger. Only JNK was markedly activated by IiR (2.25-fold vs. control , P < 0.01) and this activation well correlated with both the percentage of cellular necrosis, assessed by Trypan Blue dye exclusion assay (p:0.039, r:0.897) and the LDH release (p:0.0028, r:0.982). DPI treatment reduced JNK activation to the same level obtained with 20 pM SP600125, a JNK phosphorylation inhibitor, and showed a significantly protective effect from cellular death. Taken together, our data suggest a pivotal role of NADPH oxidase, during VR injury, in triggering JNK pathway and the possibiliry to limit myocardial damage through combined effect of inhibitors of NADPH oxidase and JNK pathway.