Oxidative stress induced by NOX2 contributes to neuropathic pain via plasma membrane translocation of PKCε in rat dorsal root ganglion neurons

Background Nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown. Methods A spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping. Results SNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKC epsilon but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by psi epsilon RACK (a PKC epsilon activator) and reduced by epsilon V1-2 (a PKC epsilon-specific inhibitor). Importantly, epsilon V1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKC epsilon protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKC epsilon was recapitulated by H2O2 administration. SNI-induced upregulation of PKC epsilon was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, epsilon V1-2 attenuated the mechanical allodynia induced by H2O2 Conclusions NOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKC epsilon.