Macrophage NADPH oxidase inhibition by ultralow-dose dextromethorphan alleviates DSS-induced inflammation and colitis

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic inflammatory condition of the gastrointestinal tract with limited treatment options and no definitive cure. Emerging evidence implicates NADPH oxidase (Nox) in IBD pathogenesis, but the specific subtype involved and its therapeutic potential remain unclear. To assess the efficacy of dextromethorphan (DM), a potent Nox2 inhibitor, we employed a dextran sulfate sodium (DSS)-induced chronic colitis model in mice. Mice exposed to intermittent DSS for 34 days developed IBD-like symptoms, including weight loss, elevated disease activity index (DAI), colon shortening, and epithelial barrier disruption. DM exhibited a bimodal protective effect: both standard-dose DM (10 mg/kg/day) and ultralow-dose DM (ULDM, 10 ng/kg/day) significantly reduced colitis severity, with ULDM proving more effective and selected for further studies. Notably, ULDM remained effective when post-administered after DSS exposure. Histological analyses revealed that ULDM reduced immune cell infiltration, crypt damage, and tissue disruption. It also suppressed pro-inflammatory cytokine expression (MCP-1, IL-6, IL-1β, TNF-α) and oxidative stress markers (myeloperoxidase, malondialdehyde, 8-hydroxydeoxyguanosine), while enhancing anti-inflammatory cytokines (IL-4, IL-10) and antioxidant enzymes (catalase, superoxide dismutase). Importantly, ULDM protected Nox1 KO but not Nox2 KO mice, indicating a Nox2-dependent mechanism. In vitro, ULDM inhibited Nox2 activation in primary macrophages and RAW 264.7 cells by blocking the membrane translocation of p47^phox. Furthermore, our study suggested a feed-forward inflammatory cycle between epithelial cell death and macrophage overactivation that exacerbated colitis. Together, these findings demonstrated that Nox2 played a central role in DSS-induced chronic colitis and identified ultralow-dose dextromethorphan as a promising, mechanism-based therapeutic candidate for IBD.