Protective Effect Of Carboxytmethylpachymaran On TNF-a-Induced Damage Of Intestinal Epithelial Barrier In Caco-2 Cell Monolayers

BackgroundPoria cocos is a medicinal mushroom which was written in "Sheng Nong’ s herbal classic".Poria cocos has been used to treat gastroenteric catarrh,insomnia,oedema.edema,and so on.Modern pharmacological studies show that Poria cocos has the effects of anti-tumor,and immune regulation.In recent years,Poria cocos for the treatment of inflammatory bowel disease(IBD)are being paid more and more attention,but the active ingredient and its mechanism of action remains unclear.Carboxytmethylpachymaran(CMP)is the main active constituents of Poria cocos,but there is no report about the treatment of inflammatory bowel disease for recent.In this study An intestine epithelial barrier damage model was established in Caco-2 monolayers by exposing the monolayers to tumor necrosis factor alpha(TNF-α).After that,the protective effect of CMP on this model was investigated.ObjectivesTo explore the mechanism of CMP in protecting TNF-α-induced damage in human colonic epithelial Caco-2 cells,and to provide new drugs and experimental support for the prevention and treatment of IBD.Methods1.TNF-a on Caco-2 cell growth proliferation effects:the effects on growth and proliferation in Caco-2 cells by MTT method to detect the effect of different concentrations of TNF-α.2.Effect of TNF-a on the structure of Caco-2 cells tight junction:the method of detecting the transmembrane resistance of cells to observe the effects of different concentrations of TNF-α on the tight junction structure of human colonic epithelial Caco-2 cells.3.The effect of TNF-a on the expression of mRNA in Caco-2 cells tight junction:the effect of TNF-α on the expression of mRNA in Caco-2 cells was detected by qPCR technique,4.Effect of TNF-α on the expression of tight junction protein in Caco-2 cells:the effect of TNF-α on the expression of tight junction proteins in Caco-2 cells was detected by immunofluorescence assay.5.Effect of CMP on growth and reproduction of Caco-2 cells:to detect the influence of different concentrations of CMP on the growth and reproduction of Caco-2 cells by MTT.6.Effect of CMP on the tight junction structure of damaged intestinal epithelial barrier:the effect of CMP on the tight junction structure of human colonic epithelial Caco-2 cells was observed by the method of detecting the transmembrane resistance value of TNF-α.7.Effect of CMP on the damaged intestinal epithelial barrier tight junction function:detecting cell phenol red through rates to observe the effect of CMP on TNF-α-induced Caco-2 cells tight junction function.8.The effect of CMP on the damaged intestinal epithelial cell tight junction Occludin and ZO-3 mRNA:the effect of CMP on TNF-a-induced human colonic epithelial Caco-2 cell Occludin and ZO-3 mRNA expression was observed by qPCR.9.The effect of CMP on the damaged intestinal epithelial tight junction proteins Occludin and ZO-3 expression:the effect of CMP on TNF-α-induced human colonic epithelial Caco-2 cell Occludin and ZO-3 protein expression was observed by western blot.10.The effect of CMP on the damaged intestinal epithelial tight junction proteins Occludin and ZO-3 distribution:the effect of CMP on TNF-α-induced human colonic epithelial Caco-2 cell Occludin and ZO-3 protein distribution was observed by immunofluorescence.11.The effect of CMP on NF-KB,MLCK,p-MLC signaling pathway:the effect of CMP on TNF-α-induced human colonic epithelial Caco-2 cell NF-KB,MLCK,p-MLC signaling pathway was observed by Western blot.Results1.0ng/ml-150ng/ml TNF-α had no effect on the growth of Caco-2 cells.2.50ng/ml-150ng/ml TNF-α reduced Caco-2 cell transmembrane resistance.3.50ng/ml-150ng/ml TNF-α down-regulated the expression of ZO-3 and Occludin mRNA.4.50ng/ml-150ng/ml TNF-α down-regulated the expression of Occludin protein.5.50 μ g/ml-150 μg/ml CMP did not promote or inhibit the growth of Caco-2 cells.6.CMP ameliorates TNF-a-induced injury of intestinal epithelial barrier structure by increasing transmembrane resistance value.7.CMP ameliorates TNF-a-induced injury of intestinal epithelial barrier function by decreasing phenol red through rate.8.CMP ameliorates TNF-a-induced injury of intestinal epithelial barrier by increasing the expression of Occludin and ZO-3 mRNA.9.CMP ameliorates TNF-a-induced injury of intestinal epithelial barrier by increasing the expression of Occludin and ZO-3 proteins.10.Caco-2 cell monolayers without any drugs TJ Occludin and ZO-3 protein distributions were continuous,complete along the cell membrane surface like a hive.Damaged by TNF-a,the Caco-2 cell monolayers TJ Occludin and ZO-3 protein distributions did not become to connected,and the intensity of fluorescence got to weak.However,the damaged monolayers were obviously recovered,if they were pretreated by CMP.11.CMP ameliorates TNF-a-induced injury of intestinal epithelial barrier by blocking NF-κB,MLCK,p-MLC signaling pathway.ConclusionCMP protective TNF-α-induced human colon epithelial cells barrier by maintaining the tight junction structure,functional integrity,up-regulating the expression of Occludin,ZO-3 mRNA and protein,maintaining Occludin,ZO-3 protein distribution,blocking NF-κB,MLCK,p-MLC signaling pathway.Our study suggest that CMP protect intestinal epithelial barrier function,and this study lays the fundation for the clinical treatment of IBD.