Betulinic Acid Enema Alleviates Colitis by Targeting Vitamin D Receptor to Protect the Intestinal Mucosal Barrier and Modulate Mucosa‐Associated Microbiota

ABSTRACT Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with limited effectiveness and safety of current drug therapies. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid present in traditional Chinese medicines used clinically as retention enemas for UC, but its direct effects and mechanisms remain unclear. This study evaluated the therapeutic impact of BA enema in dextran sulfate sodium (DSS)‐induced colitis by monitoring disease activity index (DAI), colon length, and histopathology, and by assessing inflammatory cytokines, mucosal tight junction, and mucin‐2 (MUC2) expression. Colonic mucosa‐associated microbiota were profiled by 16S rRNA gene sequencing. Potential BA targets were predicted using Swiss Target Prediction and validated by molecular docking, molecular dynamics simulations, cellular thermal shift assay, and biolayer interferometry. The functional relevance of the vitamin D receptor (VDR) was examined using a VDR antagonist in vivo and in vitro, and transcriptomic datasets from UC patients were analyzed to define VDR expression patterns. BA enema markedly attenuated DSS‐induced colitis, such as reducing the DAI, inhibiting colonic damage, and improving colon length shortening. The BA also significantly restored the tight junction proteins, associated mucin proteins, and mucin‐2 (MUC2). The BA reduced potentially pathogenic bacteria in the colonic mucosa. In addition, BA significantly restored the VDR proteins, and the VDR antagonist weakened the effects of BA in vivo and in vitro. The binding site between VDR and BA was Trp286. Transcriptomic analyses confirmed VDR downregulation in UC. These findings indicate that BA enema alleviates experimental colitis in part by targeting VDR to protect the intestinal mucosal barrier and modulate mucosa‐associated microbiota, supporting BA as a promising candidate for UC treatment.