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

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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Affiliations

Authors

Contributions

Tang XD, Wang FY and Chen T designed the study, Chen T carried out the experiments and draft of the manuscript, Yin XL co-participated in draft and publication of the paper. Kang N participated in the experimental design and Wang XG coordinated the experiments. Ji HJ, Bian LQ and Zhang BH involved in discussion of experiment. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xu-dong Tang.

Ethics declarations

The authors declare no financial or commercial conflict of interest.

Additional information

Supported by the National Nature Science Foundation of China (No. 81373580, 81173209, 81704070), Visiting Scientist Program of China Academy of Chinese Medical Sciences (No. ZZ070801) and “Ten Diseases and Ten Drugs” Program of Beijing Municipal Science and Technology Commission (No. Z161100000116046)

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Chen, T., Yin, Xl., Kang, N. et al. 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. Chin. J. Integr. Med. 26, 745–753 (2020). https://doi.org/10.1007/s11655-019-3034-6

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Keywords

  • 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