INTRODUCTION: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by mucosal inflammation in the colon. Maintaining intestinal mucosal barrier integrity is a fundamental therapeutic objective in managing UC. Swertiamarin (STM) exhibits diverse biological activities and holds promise for the treatment of UC. Autophagy is a critical mechanism that safeguards the integrity of the intestinal mucosal barrier, yet it remains uncertain whether STM ameliorates UC and modulates autophagy to preserve this barrier.
METHODS: An acute colitis mouse model was induced by administering dextran sulfate sodium (DSS) in drinking water. The protective effects of different concentrations of STM against colitis were evaluated by monitoring body weight changes, disease activity index (DAI), colon length, spleen index, histological score, and AB-PAS staining. Colonic inflammation severity was assessed using ELISA and qPCR, while intestinal permeability was evaluated through FITC-dextran permeability assays, Western blotting, and immunohistochemistry. Caco-2/HIEC-6 cell monolayers were treated with 2% DSS, and the effects of STM on cellular inflammation and barrier function were assessed using Western blotting, qPCR, and a FITC-dextran permeability assay. Potential mechanisms and targets of STM for alleviating UC through intestinal epithelial cells were predicted using online databases and network pharmacology analysis. These predictions were subsequently validated through Western blotting. To elucidate the roles of autophagy and the PI3K/AKT/mTOR pathway in STM's protective effects, functional rescue experiments were conducted. These experiments utilized the autophagy inhibitor 3-methyladenine (3-MA) and the PI3K-specific agonist 740YP. Autophagic structures were directly visualized by transmission electron microscopy (TEM).
RESULTS: STM dose-dependently alleviated DSS-induced colitis in mice. It reversed body weight loss, improved the DAI score, and inhibited colon shortening. Treatment also reduced the spleen index, attenuated colonic pathological damage, increased goblet cell counts, suppressed cytokine production, and protected epithelial barrier function. In vitro experiments demonstrated that STM effectively protected the intestinal epithelial barrier and mitigated inflammatory responses. Network pharmacology analysis revealed that STM safeguards the intestinal barrier by promoting intestinal epithelial cell autophagy and mitigating UC through the PI3K/AKT/mTOR-signaling pathway. DSS treatment elevated p62 expression and suppressed LC3-II levels in colonic tissues, suggesting impaired autophagy. Conversely, STM induced autophagy and inhibited the PI3K/AKT/mTOR-signaling pathway in both in vivo and in vitro models. TEM revealed a significantly higher number of autophagosomes in the STM-treated group. Treatment with the autophagy inhibitor 3-MA significantly abrogated the protective effects of STM. Treatment with the PI3K agonist 740YP significantly increased phosphorylation levels of PI3K, AKT, and mTOR. This was accompanied by p62 accumulation, decreased LC3-II expression, and reduced tight junction protein (ZO-1 and Occludin) levels, indicating that PI3K activation counteracts the protective effects of STM on autophagy and barrier function.
CONCLUSION: STM mitigates colonic inflammation in DSS-induced colitis mice by reducing pro-inflammatory cytokine levels. This effect is likely mediated through enhanced autophagy, achieved by inhibiting the PI3K/AKT/mTOR pathway, ultimately ameliorating intestinal barrier dysfunction.
