Multi-level characterization of Wutou decoction highlights its role in gut microbiota and intestinal barrier regulation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder associated with gut microbiota dysbiosis and intestinal barrier impairment. Wutou decoction (WTD), a traditional herbal formulation, shows therapeutic efficacy in RA. This study investigated its mechanisms in cold-damp collagen-induced arthritis (CIA) rats. WTD treatment improved body weight, reduced pro-inflammatory cytokines, increased IL-10, and alleviated ankle injury. H&E staining revealed colon structure restoration and goblet cell increase. Gut microbiota analysis showed enrichment of beneficial genera (Roseburia, Lactobacillus) and reduction of pathogenic taxa (Bacteroides, Helicobacter). Immunofluorescence confirmed enhanced barrier integrity. Flow cytometry indicated improved Treg/Th17 balance in colon, blood, and joints. Correlation analysis linked microbiota changes with cytokines (e.g., IL-22), immune cell proportions, and organ status. FMT further verified microbiota-dependent effects of WTD in repairing barrier damage and joint protection. Western blot demonstrated reduced p38, MLC2, p-ERK, MLCK, p-STAT3, p-JAK2, and increased IL-22 and tight junction proteins. In conclusion, WTD mitigates joint lesions via IL-22-mediated STAT3/ZO-1 and MAPK/MLCK/ZO-1 pathways, regulating Treg/Th17 balance along the gut-blood-joint axis, thereby promoting microbiota-dependent barrier restoration in RA therapy.