Objective:To investigate the effect of Chang’an Ⅱ Decoction(肠安 Ⅱ 号方)-containing serum on intestinal epithelial barrier dysfunction in rats.Methods:Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium.Caco-2 monolayers were treated with blank serum and Chang’an Ⅱ Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang’an Ⅱ Decoction intragastrically at doses of 0.49,0.98,1.96 g/(kg·d) for 1 week,respectively.After preparation of containing serum,cells were divided into the normal group,the model group,the Chang’an Ⅱ-H,M,and L groups (treated with 30 ng/mL TNF-α and medium plus 10％ high,middle-,and low-doses Chang’an Ⅱ serum,respectively).Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-Iabeled dextran.Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs).Immunofluorescence of zonula occludens-1 (ZO-1),claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution.The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction.The expression levels of MLCK,myosin light chain (MLC) and p-MLC were determined by Westem blot.Results:Chang’an Ⅱ Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α.It alleviated TNF-α-induced morphological alterations in TJ proteins.The increases in MLCK mRNA and MLCK,MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang’an Ⅱ-H group.Additionally,Chang’an Ⅱ Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus.Conclusion:High-dose Chang’an Ⅱ-containing serum attenuates TNF-α-induced intestinal barrier dysfunction.The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.