Total polysaccharides of the Sijunzi decoction attenuate tumor necrosis factor-α-induced damage to the barrier function of a Caco-2 cell monolayer via the nuclear factor-κB-myosin light chain kinase-myosin light chain pathway

Aim:To explore the protective effects and underlying mechanisms of total polysaccharides of the Sijunzi decoction (TPSJ) on the epithelial barriers in vitro.

Methods:Caco-2 cell monolayers were treated with or without TPSJ in the presence or absence of TNF-α, and paracellular permeability and transepithelial electrical resistance (TEER) were measured to evaluate the epithelial barrier function. Immunofluorescence and western blotting were respectively used to evaluate the distribution and expression of the tight junction proteins claudin 1, claudin 2, zo3, and occludin in Caco-2 cells. Western blotting was also used to evaluate the cellular expression of myosin light chain (MLC), phosphorylated MLC (pMLC), MLC kinase (MLCK), and nuclear factor (NF)-κB p65.

Results:TPSJ promoted the proliferation of Caco-2 cells and inhibited TNF-α-induced secretion of pro-inflammatory cytokines. Furthermore, TPSJ significantly ameliorated both the reduction of TEER and the increased paracellular permeability observed in tumor necrosis factor (TNF)-α-damaged Caco-2 monolayers. Furthermore, TPSJ remarkably attenuated TNF-α-induced morphological changes, downregulated the expression of claudin 1, claudin 2, zo3, and occludin, and markedly suppressed TNF-α-mediated upregulation of p-MLC and MLCK expression. Finally, TPSJ inhibited the activation and expression of NF-κB p65.

Conclusion:Our results demonstrate that TPSJ alleviates the TNF-α-induced impairment of the intestinal epithelial cell barrier function by suppressing NF-κB p65-mediated phosphorylation of MLCK and MLC.

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