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| Chang'an Ⅱ Decoction (肠安Ⅱ 号方)-Containing Serum Ameliorates Tumor Necrosis Factor-α -Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats |
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| KeyWord:myosin light chain kinase-myosin light chain, signaling pathway, intestinal epithelial cells, tight junction, tumor necrosis factor-α , Chang'an Ⅱ Decoction, drug-containing serum |
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| Abstract: |
| 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)-labeled 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 Western 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. |
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