Shixiaosan Attenuates Intestinal Epithelial Barrier Injury in TNF-α-stimulated Caco-2 Cells and DSS-induced Colitis Mice

Disruption of the intestinal epithelial barrier is implicated in the development and persistence of inflammatory bowel disease. Although Shixiaosan (SXS) has anti-inflammatory activity, its effects on epithelial barrier injury and the signaling events involved remain insufficiently characterized. We examined SXS in Caco-2 cells challenged with TNF-α and in mice with DSS-induced colitis, focusing on RELA/NF-κB signaling and myosin light-chain (MLC) phosphorylation. Cell experiments used SXS at 25–100 μg/mL and assessed viability, transepithelial electrical resistance (TEER), paracellular tracer flux, inflammatory mediators, and tight-junction proteins. Male C57BL/6 mice received 3% DSS for 7 days and regular water for a further 3 days, together with SXS at 1, 2, or 4 g/kg/day (n = 6 per group). Disease activity, tissue injury, serum barrier-related markers, inflammatory mediators, and colonic tight-junction proteins were evaluated. RELA silencing in the cell model was used to examine pathway involvement. SXS showed no obvious cytotoxicity and counteracted the TNF-α-associated loss of viability and TEER, limited paracellular permeability and inflammatory mediator release, and restored ZO-1, occludin, and claudin-1. In mice, treatment attenuated weight loss, disease activity, histological injury, and colon shortening, while lowering serum permeability markers and restoring colonic tight-junction proteins. Reduced phosphorylation of p65, IκBα, and MLC accompanied these protective effects. RELA knockdown also reduced MLC phosphorylation and partly recovered tight-junction protein expression. Together, these findings indicate that SXS limits experimental intestinal barrier injury and inflammation, with RELA/NF-κB-associated MLC phosphorylation implicated in its effects. Direct validation of MLCK remains necessary.