Effects Of Homocysteine On The Intestinal Permeability By Regulating MLCK Expression In Experimental Colitis

Background: Inflammatory bowel disease?IBD?is a chronic intestinal inflammatory disease which is of unknown etiology.Studies have shown that disorder of tight junction function in intestinal mucosa and increased intestinal permeability contributes to the pathophysiology of IBD.Homocysteine?Hcy?is a thiol containing amino acid which is an important byproduct of methionine metabolism.Studies have indicated that Hcy could damage endothelial barrier and increase endothelial permeability through multiple ways.Hcy levels in serum and tissues of IBD patients were significantly higher than those of normal control.However,whether Hcy influence intestinal permeability and cause IBD by regulating epithelial tight junction remains unclear.The objective of this study was to elucidate the effect of homocysteine on the permeability of instetinal mucosa in colitis rats and Caco-2monolayer and its possible mechanisms.Objective:To elucidate the effect of homocysteine on the permeability of instetinal mucosal in Caco-2 cell and colitis rats.Methods: 1.The Caco-2 cells were treated with different signal inhibitors(ERK inhibitor PD98059,P38 MAPK inhibitor SB203580,Rho inhibitor Y27632,Ca²⁺/Calmodulin inhibitor KN62,PKC inhibitor Staurosporine and MLCK inhibitor ML-7)and Hcy.2.The TEER was recorded and the medium in each lower chamber was collected.The FITC-transmenbrane transport was investigated by Fluorescence spectrophotometer.The expressions of MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin in Caco-2 cells were tested by western blot.2.The experimental colitis model was established in SD rats with enema of2,4,6-trinitrobenzene sulphonic acid?TNBS?/ethanol,the HHcy model was established in rats with subcutaneous injection of Hcy;3.32 healthy male SD rats were divided into 4 groups?n=8?: Group A: control group,Group B:control + Hcy injection group,Group C: colitis model group,Group D: colitis model + Hcy injection group;4.The symptoms and body weights of rats were recorded and DAI was conducted;colon tissues were collected for histopathological index?HI?,HE staning respectively;5.Levels of Hcy and MLCK in colon tissues of rats were examined by ELISA and MPO activity was tested.The expressions of MEK,ERK,p-ERK,MLCK,p-MLCK,ZO-1,Claudin-1,Claudin-2,Occludin in intestine samples were evaluated by immunohistochemistry and western blot.The MLCK m RNA expression was examined by RT-q PCR.Results: Compared with control group,the TEER in the Caco-2 cells pretreated with 50?mol/L Hcy decreased most greatly.Compared with Caco-2 cell pretreated with Hcy only,the TEER decrease in Caco-2 cells which pretreated with PD98059 and ML-7 was slowed down and FITC transmembrane transport was decreased.The expressions of MLCK,p-MLCK,Claudin-2 were decreased while the expressions of ZO-1,Claudin-1,Occludin were increased?P<0.05?,which indicated that Hcy might influence the permeability of Caco-2 cell via MEK-ERK-MLCK pathway.Compared with control group and colitis model group,the DAI,HI,MPO activity,the levels of MLCK in colon tissues were increased in colitis model + Hcy injection group?P<0.05?.The expressions of MEK,ERK,p-ERK,MLCK,p-MLCK and Claudin-2were increased while the expressions of ZO-1,Claudin-1 and Occludin were decreased in intestinal mucosa of colitis rats?P<0.05?,which indicated that Hcy coud regulate the permeability of intestinal mucosa in colitis rats and Caco-2 cell through MEK-ERK-MLCK pathway and eventually increase intestinal permeability.