Effects And Mechanisim Of Tnf-α On Intestinal Epithelial Cellular Permeability

Background and objectives:In addition to being the organ responsible for digestion and absorption of nutrients, the intestine also serves as a critical component of the innate immune system, it's called intestinal mucosal barrier, and intestinal epithelial barrier is the most important component of it. Breakdown of the intestinal epithelial barrier is implicated in several illnesses such as inflammatory bowel disease, sepsis, and burn. Among these cinditions, the serum TNF-a increases significantly, which is related with the intestinal epithelial barrier impairment, while anti-TNF-a can restore the impaired barrier. In early days, people thought that TNF-a might affect the intestinal epithelial cell(IEC) through cellular apoptosis, but recently people focus more on a apoptosis-independent manner, which may involve protein kinase C(PKC), nuclear factor-Kappa B(NF-κB), myosin light chain kinase(MLCK), and mitogen-activated protein kinase(MAPK). Therefore, we detect the effect of TNF-a on intestinal permeability and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells, and study the mechanisms involved in tight junction discharged assembly induced by TNF-a. MethodsThe Caco-2 cells were cultured in vitro to establish intestinal epithelial barrier and transfected with Mu-IκB plasmid or not. Then the cells were treated with TNF-a(100μg/L) for 0,3,6,12,24 h respectively. The permeability of intestinal epithelial cells was determined by transepithelial electrical resistance (TEER) of Caco-2 cell monolayer. Then we detected NF-κB activity through Luciferase-reporter gene, phosphorylation of myosin light chain (MLC) by western blot, and F-actin by Rhodamine-phalloidin direct staining according to different treatments.ResultsTNF-αreduces the TEER of Caco-2 cells in a time-dependent manner. TNF-αcan also activates NF-κB. Transfection of the Mu-IκB plasmid into Caco-2 cells leads to down-regulation of NF-κB activity, accompanied by less decrease in Caco-2 cell TEER. Meanwhile, TNF-αleads to phosphorylation of MLC in cells, but inhibition of NF-κB activity by Mu-IκB delays such phosphorylation. TNF-αinterrupts the integrity of F-actin in Caco-2 cells. While inhibition of NF-κB activity delays such interruption.ConclusionHyperpermeability of intestinal epithelial cell induced by TNF-αis partially due to the phosphorylation of MLC and F-actin rearrangment in IEC, in which NF-κB may participate.