Rosmarinic acid alleviates colitis and repairs mucus barrier damage in a gut microbiota-dependent manner

Rosmarinic acid (RA) significantly alleviates DSS-induced colitis by repairing the damaged colonic mucus barrier and reversing gut microbiota dysbiosis. However, it remains unclear whether the gut microbiota is required for this protective effect. To address this, pseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to evaluate whether the gut microbiota is required for RA to restore mucus barrier integrity and alleviate colitis. The study showed that transplanting fecal microbiota from RA-pretreated donor mice into pseudo-germ-free recipients with colitis markedly alleviated colitic pathology. This was reflected by an 52% reduction in the disease activity index (DAI) score, improved histological scores, markedly lower serum pro-inflammatory cytokines (IL-6 and TNF-α), and upregulated mRNA expression of anti-inflammatory cytokines (IL-10 and IL-25). Moreover, the mucus barrier was substantially restored in recipient mice: goblet cell numbers increased approximately 5-fold (RH-FMT (recipient mice were transplanted with microbiota derived from the high-dose RA treatment group) vs. DSS group), mucus coverage rose from 5.24% (DSS) to 35.4% (RHF), and mRNA levels of tight junction proteins and mucins (e.g., ZO-1, MUC1) were elevated. Short-chain fatty acid (SCFA) levels recovered, with total SCFAs increasing by about 130.77%. Meanwhile, the gut microbiota of recipient mice was remodeled: beneficial bacteria such as Akkermansia and Limosilactobacillus increased, while harmful bacteria like Proteobacteria and Klebsiella decreased. However, direct oral administration of RA (at either high or low doses) to pseudo-germ-free mice did not produce these protective effects. Further correlation analysis revealed that the abundance of SCFA-producing beneficial genera-including Akkermansia, Limosilactobacillus, and Blautia_A-in recipient mice was significantly positively correlated with the expression of genes involved in the mucus barrier and tight junctions, as well as with SCFA levels. Thus, RA alleviates colitis through mucus barrier repair and modulation of SCFAs metabolism, a process that critically depends on the gut microbiota.