ObjectiveThe integrity of the intestinal mucosal barrier is critical in the pathogenesis of Inflammatory Bowel Disease (IBD). While Astragaloside IV (ASIV) possesses potent anti-inflammatory properties, its specific effects on intestinal epithelial barrier function and the underlying mechanisms involving the Myosin Light Chain Kinase (MLCK) and Myeloid Differentiation Primary Response 88/TNF Receptor-Associated Factor 6 (MyD88/TRAF6) pathways in colitis remain elusive. This study aimed to investigate the protective effects of ASIV on tight junction (TJ) proteins and inflammatory signaling in dextran sulfate sodium (DSS)-induced colitis.MethodsA chronic colitis model was established in wild-type C57BL/6 mice using four cycles of DSS administration. ASIV (30 mg/kg/day) was administered orally starting from day 14. Disease activity index (DAI), colon length, and histopathological changes were assessed. Intestinal barrier function was evaluated by measuring bacterial translocation, colonic permeability to fluorescein isothiocyanate (FITC)-dextran, and serum lipopolysaccharide (LPS) levels. The expression of TJ proteins (ZO-1, Occludin, JAM-A, Claudin-2) and signaling molecules (MLCK, phosphorylated myosin light chain (p-MLC), MyD88, TRAF6) was determined by Western blot and quantitative real-time PCR (qRT-PCR). Representative immunofluorescence localization of Claudin-2 and ZO-1 in colonic tissues was included as supplementary qualitative evidence of tight junction remodeling.ResultsASIV treatment significantly attenuated DSS-induced weight loss, colon shortening, and histopathological damage (p < 0.05). Furthermore, ASIV markedly reduced colonic permeability, serum LPS levels, and bacterial translocation to mesenteric lymph nodes (p < 0.01). At the molecular level, ASIV reversed the DSS-induced downregulation of sealing TJs (ZO-1, Occludin, JAM-A) and suppressed the upregulation of the pore-forming protein Claudin-2 (p < 0.05). These protective effects were accompanied by significant inhibition of TNF-α mRNA expression, reduced MLCK protein expression, decreased MLC phosphorylation, and suppression of MyD88/TRAF6 signaling.ConclusionASIV effectively restores intestinal epithelial barrier integrity in experimental colitis. These protective effects are associated with rebalancing of TJ protein expression and localization and may be partially linked to attenuation of the TNF-α/MLCK/p-MLC and LPS/MyD88/TRAF6 signaling axes. These findings support ASIV as a promising barrier-protective candidate for mucosal homeostasis in IBD, while future cellular and pathway-intervention studies are needed to establish direct causality.