Inhibition of NOX2 signaling limits pain-related behavior and improves motor function in male mice after spinal cord injury: Participation of IL-10/miR-155 pathways

Abstract

NADPH oxidase (NOX2) is an enzyme that induces reactive oxygen species (ROS) and serves as a switch between the pro-inflammatory and neurorestorative microglial/macrophage phenotypes; such changes play an important role in neuropathic pain and motor dysfunction. Increased NOX2 expression after spinal cord injury (SCI) has been reported, and inhibition of NOX2 improves motor function. However, the underlying mechanisms of NOX2 in post-traumatic pain and motor deficit remain unexplored. In the present study, we report that depletion of NOX2 (NOX2−/−) or inhibition of NOX2 using NOX2ds-tat significantly reduced mechanical/thermal cutaneous hypersensitivity and motor dysfunction after moderate contusion SCI at T10 in male mice. Western blot (WB, 3 mm lesion area) and immunohistochemistry (IHC) showed that SCI elevates NOX2 expression predominantly in microglia/macrophages up to 8 weeks post-injury. Deletion of NOX2 significantly reduced CD11b+/CD45hiF4/80+ macrophage infiltration at 24 h post-injury detected by flow cytometry and 8-OHG+ ROS production at 8 weeks post-injury by IHC in both lesion area and lumbar enlargement. NOX2 deficiency also altered microglial/macrophage pro-inflammatory and anti-inflammatory balance towards the neurorestorative response. WB analysis showed robust increase of Arginase-1 and YM1 proteins in NOX2−/− mice. Furthermore, qPCR analysis showed significant up-regulation of anti-inflammatory cytokine IL-10 levels in NOX2−/− mice, associated with reduced microRNA-155 expression. These findings were confirmed in CD11b+ microglia/macrophages isolated from spinal cord at 3 days post-injury. Taken together, our data suggest an important role for IL-10/miR-155 pathway in regulating NOX2-mediated SCI-dysfunction. Thus, specific targeting of NOX2 may provide an effective strategy for treating neurological dysfunction in SCI patients.