Abstract
BackgroundImpaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway.MethodsIPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA.ResultsThe results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88.ConclusionTryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.
Highlights
Enterotoxigenic Escherichia coli (ETEC) invasion causes intestinal damage and diarrhea in children and young animals
We found that ETEC K88 enhanced the contents of tumor necrosis factor-alpha (TNF-a) and IL-8, which is in agreement with a previous article that ETEC K88 induced intestinal proinflammatory response in pigs [8]
We found that NPS2143 reversed the enhancement effect of tryptophan on the protein concentrations of zonula occludens-1 (ZO-1), occludin, claudin-1, and calcium-sensing receptor (CaSR) and the TEER and decrease of permeability and IL-8 and TNF-a contents
Summary
Enterotoxigenic Escherichia coli (ETEC) invasion causes intestinal damage and diarrhea in children and young animals. Piglets are inclined to ETEC K88-induced diarrhea because the bacteria can produce enterotoxins. These substances destroy the intestinal mucosal layer and tight junction (TJ) structure, which increases the permeability of the intestine, causing intestinal inflammation [1,2,3,4]. ETEC K88 can modulate epithelium barrier function by inducing cellular signals such as the toll-like receptors (TLR) and p38/mitogen-activated protein kinase (MAPK) signaling pathway in intestinal epithelial cells [5, 6]. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/ phospholipase Cg1 (PLC-g1) signaling pathway
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