Ameliorative Effect and Mechanism of Huoxiang–Huanglian on TNBS‐Induced Inflammatory Bowel Disease in Zebrafish

The Huoxiang-Huanglian (HH) drug pair has demonstrated efficacy in treating enteritis, yet its underlying mechanisms remain unclear. We integrated UPLC-MS, network pharmacology, and metabolomics to identify HH components and predict targets for inflammatory bowel disease (IBD). Molecular docking assessed interactions between bioactive compounds and core targets. A 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced zebrafish enteritis model was treated with HH (100, 200, and 400 μg/mL) or dexamethasone. Colon histopathology was evaluated via H&E and AB-PAS staining. Inflammatory cytokines, oxidative markers, and mRNA/protein expression of key pathway molecules were analyzed. Integrated analysis revealed 147 potential targets and 45 differential metabolites, identifying ornithine decarboxylase 1 (ODC1) as a key target. Molecular docking confirmed strong binding (below -5.00 kJ/mol) of HH components like retusin and berberine to ODC1. HH treatment reduced interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, nuclear factor kappa B (NF-κB) p65, and ODC1 levels, while increasing interleukin-10 (IL-10), superoxide dismutase (SOD), and aryl hydrocarbon receptor (AhR) expression. Our findings suggest that HH alleviates IBD, which may involve the NF-κB/AhR/ODC1 pathway, highlighting its multitarget therapeutic potential.