Caffeic acid regulates the gut microbiota-tryptophan metabolic axis to improve intestinal barrier function and relieve ulcerative colitis.

Abstract

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease for which current therapies remain inadequate. Caffeic acid (CA), a natural hydroxycinnamic acid polyphenol, possesses anti-inflammatory and antioxidant properties. However, whether CA engages the gut microbiota-tryptophan-aryl hydrocarbon receptor (AHR)-Nrf2 axis in UC has not been established. PURPOSE: To evaluate the anti-UC activity of CA in vivo and in vitro and to elucidate its underlying mechanisms through a multi-platform integrative strategy. STUDY DESIGN: A dextran sulfate sodium (DSS)-induced murine colitis model and lipopolysaccharide (LPS)-stimulated mouse colonic epithelial cells (MCECs) were used in parallel. Mechanism was addressed by combining microbiota, metabolomic, transcriptomic and proteomic profiling with faecal microbiota transplantation, computational and biophysical target validation, and siRNA-mediated AHR knockdown. METHODS: CA was administered at 10 and 20 mg/kg/day, with 5-aminosalicylic acid as positive control. Complementary platforms included 16S rRNA gut microbiota sequencing, untargeted serum metabolomics, colonic transcriptomic profiling, data-independent acquisition mass spectrometry proteomics, human UC single-cell RNA sequencing (GEO: GSE214695), faecal microbiota transplantation into antibiotic-pretreated recipients, molecular docking, 100 ns molecular dynamics simulation, cellular thermal shift assay, dual-luciferase reporter assay, and co-immunoprecipitation. RESULTS: CA dose-dependently alleviated dextran sulfate sodium-induced colitis, suppressed NF-κB activation, and reduced interleukin-6, interleukin-1β, and tumour necrosis factor alpha. Gut microbiota profiling revealed enrichment of Bifidobacterium and Dubosiella with depletion of Escherichia-Shigella. Transplantation of the CA-remodelled microbiota into antibiotic-pretreated recipients reproduced the protective phenotype and restored colonic AHR and cytochrome P450 1A1 expression, demonstrating that the remodelled community is sufficient to confer protection. Metabolomics showed enrichment of serotonin-branch tryptophan metabolites, with 5-hydroxytryptamine among the most significantly enriched differential metabolites. Molecular docking and 100 ns molecular dynamics simulation indicated stable binding of CA within the AHR ligand-binding domain, cellular thermal shift assay confirmed intracellular target engagement, and a dual-luciferase reporter assay demonstrated concentration-dependent activation of dioxin response element-driven transcription, establishing CA as an AHR agonist. CA increased Nrf2, NQO1 and HO-1 and restored Claudin-1, Occludin and zonula occludens-1; AHR knockdown reduced these effects and the suppression of NF-κB, indicating that both actions depend on AHR. CONCLUSIONS: CA ameliorates UC through convergent AHR activation, arising from both microbiota-mediated tryptophan reprogramming and direct receptor binding, with AHR-dependent Nrf2/NQO1/HO-1 induction and NF-κB suppression to restore intestinal barrier integrity. These findings support CA as a multi-target phytochemical candidate for UC treatment.

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