Indirubin attenuates DSS-induced colitis in mice and is associated with partial gut microbiota shifts and altered intestinal metabolic profiles.

چکیده اصلی

BACKGROUND: Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease for which safe and durable treatment options remain limited. Indirubin (IDB) is a natural bisindole constituent of Indigo naturalis, Folium Isatidis, and Radix Isatidis with reported anti-inflammatory activity, but the contribution of gut microbiota-host metabolic interactions to its effects in colitis remains unclear. METHODS: Male C57BL/6 mice with dextran sulfate sodium (DSS)-induced colitis received IDB at 10, 20, or 40 mg/kg, with 5-aminosalicylic acid as a positive control. Disease activity, colon histopathology, intestinal barrier proteins, inflammatory mediators, and oxidative stress indices were evaluated. 16S rRNA sequencing and untargeted metabolomics of cecal contents and colon tissue were performed in the control, DSS, and medium-dose IDB groups. Absolute bacterial-load qPCR, antibiotic treatment and fecal microbiota transplantation (FMT) were used to assess microbiota dependence. Network pharmacology, molecular docking and dynamics, and Western blotting were used to explore candidate host pathways. RESULTS: IDB attenuated body-weight loss, disease activity index and colonic mucosal damage index scores, colon shortening, histological injury, inflammatory cytokine production, and oxidative stress, while preserving ZO-1, Occludin, and Claudin-3. IDB treatment was associated with partial shifts in DSS-disrupted microbial community structure and enrichment of taxa including Bacteroidia, Muribaculaceae, Bifidobacterium and Actinomycetales. Absolute qPCR confirmed a marked reduction in total bacterial load after antibiotic treatment. The protective phenotype was markedly attenuated in antibiotic-treated mice and was partially transferred by fecal material from IDB-treated donors. Untargeted metabolomics identified candidate changes in bile acid, fatty acid, amino acid, purine, and tricarboxylic acid cycle-related pathways in cecal contents and colon tissue. These metabolic features were correlated with selected microbial taxa and disease indices. IDB also increased AMPKα and ACC1 phosphorylation in colon tissue. CONCLUSIONS: IDB alleviated DSS-induced colitis through anti-inflammatory, antioxidant, and barrier-preserving effects. The integrated functional and multi-omics findings support a microbiota-associated working model linking IDB treatment with partial gut-community shifts, altered intestinal metabolic profiles, and AMPK pathway activation.

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