Norcantharidin Ameliorates Experimental Ulcerative Colitis Through Epigenetic and Metabolic Reprogramming Involving IL-6/DNMT1/SOCS3 and AMPK/SIRT1/FOXO3a-Nrf2 Signaling

Ulcerative colitis (UC) is a chronic inflammatory disorder associated with cytokine imbalance, epigenetic alterations, and metabolic–redox dysfunction. Despite the widespread use of mesalazine (5-ASA), therapeutic limitations remain. Norcantharidin (NCTD), a synthetic cantharidin analogue, may provide multi-target protection against UC. Experimental colitis was induced in male Sprague-Dawley rats by intrarectal administration of 4% acetic acid (AA). Rats received oral NCTD (10 mg/kg), 5-ASA (100 mg/kg), or their combination for 8 days. Disease severity was assessed by disease activity index, body weight, colon length, colon weight/length ratio, and histopathology. Colonic biomarkers were evaluated using ELISA, qRT-PCR, Western blotting, and immunohistochemistry. Fe2+ and malondialdehyde (MDA) were measured as indicators of iron accumulation and lipid peroxidation. Molecular docking suggested that NCTD may adopt plausible binding poses within the binding pockets of AMPK, SIRT1, and DNMT1, providing structural support for potential protein–ligand interactions. NCTD significantly ameliorated AA-induced colitis, improving clinical and histological outcomes. These effects were associated with reduced IL-6, TNF-α, DNMT1, Fe2+, and MDA levels, restoration of SOCS3, activation of p-AMPK/SIRT1/FOXO3a signaling, and enhancement of Nrf2/HO-1 defenses. Combined NCTD/5-ASA treatment produced greater clinical and histological protection, with differential effects on molecular markers. Docking studies suggested favorable interactions of NCTD with AMPK, SIRT1, and DNMT1. NCTD treatment was associated with protection against experimental colitis, linked to modulation of inflammatory, epigenetic, metabolic, and antioxidant pathways.