Methylated tirilazad alleviates DSS-induced colitis in mice through reciprocal microbiome-metabolome

Ulcerative colitis (UC) pathogenesis involves complex interactions between inflammatory reactions, gut dysbiosis, metabolic disruption, and barrier dysfunction. Current therapies primarily target inflammation but fail to correct the underlying dysbiotic ecosystem. We hypothesized that methylated tirilazad (MT), a synthetic 21-aminosteroid with the antioxidant properties of tirilazad, would alleviate colitis by orchestrating a beneficial restructuring of the gut microbiome and its metabolic output. Using a DSS-induced murine colitis model in C57BL/6 mice, we evaluated MT via integrated multi-omics approaches, including 16S rRNA gene sequencing and untargeted metabolomics, coupled with correlation network analysis. In vitro experiments using human intestinal Caco-2 cells were further performed to verify the direct anti-inflammatory effects of MT. MT treatment ameliorated clinical severity, suppressed systemic and colonic inflammation (reducing IL-6, TNF-α; elevating IL-10), restored gut barrier integrity (increasing Occludin, ZO-1), and mitigated oxidative stress. 16S rRNA sequencing revealed that MT reversed DSS-induced dysbiosis, uniquely enriching for the probiotic species Lactobacillus johnsonii (8.4-fold) while suppressing pathobionts like Desulfovibrio fairfieldensis (13.3-fold reduction). Metabolomic analysis showed that MT normalized colitis-associated metabolic disturbances, specifically downregulating the pro-inflammatory eicosanoid 12R-HETE and upregulating barrier-supportive dipeptides (e.g., Gly-Tyr). Integrated correlation analysis established 12R-HETE as a key node, positively linked to pathogenic bacteria and inflammation, and negatively to barrier proteins. In vitro cell experiments confirmed that MT directly inhibited LPS-induced pro-inflammatory cytokine expression in Caco-2 cells. Our findings demonstrate that MT alleviates colitis not merely through direct anti-inflammatory action, but via a reciprocal microbiome-metabolome reprogramming loop, wherein microbial restructuring drives metabolome correction, which in turn reinforces barrier integrity and immune homeostasis. This positions MT as a novel microbiota-metabolome-directed therapeutic candidate that addresses both the symptomatic and root causes of UC.