GYY4137 Attenuates Sodium Deoxycholate-Induced Intestinal Barrier Injury

Objective:The integrity of intestinal epithelial barrier is an important precondition for maintaining the homeostasis of body.Once the barrier function of intestinal epithelium is damaged,it will lead to a series of serious diseases,such as inflammatory bowel disease,endotoxemia and necrotizing enteritis,which have been proved to be closely related to the damage of barrier function.People on a long-term high-fat diet have a significantly increased risk of IBD and also have a significantly increased concentration of secondary bile acids in the enteric cavity.Deoxycholate acid,the major component of secondary bile acids,has been shown to have a disruptive effect on intestinal epithelial barrier function.Therefore,substances that can antagonize intestinal barrier function damage caused by deoxycholate acid have great potential value for the prevention and treatment of these diseases.Hydrogen sulfide,as the third gas signaling molecule in human body,has been widely studied in recent years for its physiological functions,such as anti-apoptosis,anti-inflammation and vasodilation.However,there are still few studies on the protective effect of hydrogen sulfide on intestinal barrier function.Therefore,this study aims to explore the protective effect of an hydrogen sulfide donor,GYY4137,on intestinal barrier function damage caused by SDC and its related mechanisms.Methods:In order to select the optimal concentration and time to establish cell model damaged by SDC,Caco-2 cells cultured in Transwell were treated with SDC at a concentration gradient(0.4mmol/L,0.8mmol/L,1.2mmol/L,1.6mmol/L,2.0mmol/L,2.4mmol/L),and the TEER value was measured every 30min to evaluate the intestinal epithelial barrier function.The protective effect of GYY4137 with concentration gradient(50umol/L,100umol/L,200umol/L)on intestinal barrier function impairment caused by SDC was verified by evaluating TEER value and FD-40 permeability.Total protein of Caco-2 cells in each treatment group was extracted,and WB experiment was used to semi-quantitatively detect the expression levels of ZO-1 and Occludin.ZO-1 and Occludin were labeled by immunofluorescence staining to observe the arrangement and distribution of TJP in the intercellular space of each treatment group.The semi-quantitative analysis of P-MLCK,MLCK,P-MLC2 and MLC2 levels was performed with WB to verify the state of P-MLCKP-MLC2 pathway in the process of processing.The mouse model of intestinal barrier function injury by SDC was established,and the mice were divided into control group,SDC treatment group,GYY4137 treatment group and SDC+GYY4137 treatment group.Body weight,plasma FD-4 concentration,intestinal mucosal histological morphology and score in each treatment group were recorded.Intestinal mucosal proteins of mice in each treatment group were collected,and the expression levels of ZO-1 and Occludin were semi-quantitatively detected by WB.Bile regurgitation is of great significance in the occurrence of reflux esophagitis.In the Gene Expression Omnibus(GEO)database,GSE13378 treated the squamous esophageal cell line of human with 300uM deoxycholate acid and normal media.Then they extracted mRNA in two groups respectively.We conducted mRNA microarray analysis using bioinformatics methods and screened of differentially expressed genes.The enrichment analysis of gene ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)was also conducted.Results:As the minimum concentration and shortest treatment time of SDC that could significantly reduce TEER value,2mmol/L SDC was treated for 30min to establish a cell model of intestinal barrier function injury.The pres function ence of GYY4137 significantly alleviated the decrease of monolayer TEER value and the increase of FD-40 permeability of Caco-2 cells induced by SDC.Treatment with SDC or GYY4137 for 30min did not change the expression levels of ZO-1 and Occludin.However,the presence of SDC would disarrange the distribution of ZO-1 and Occludin in the intercellular space,while GYY4137 could reverse this effect to a certain extent.P-MLCK-P-MLC2 pathway is activated during the action of SDC,and the presence of GYY4137 can inhibit the activation of this pathway,which is also believed to be the specific mechanism of GYY4137 regulating the disorder of TJP arrangement caused by SDC.In animal experiments,SDC can cause weight loss,intestinal mucosal histological score and plasma FD-4 concentration increase in experimental mice.GYY4137 could reverse the weight loss,histological score and plasma FD-4 concentration increase induced by SDC.WB results suggested that in animal experiments,SDC could decrease the expression of ZO-1 and Occludin,while GYY4137 could up-regulate the expression of TJP.The results of bioinformatics suggested that deoxycholate acid could lead to the differential expression of 132 genes,which was involved in multiple physiological processes of cell line.Conclusions:In cell experiments,the relatively short time of SDC treatment can make the distribution of TJP disorder,and then damage the intestinal epithelial barrier function.GYY4137 can regulate the distribution of TJP by inhibiting the activation of P-MLCK-PMLC2 pathway and play a protective role for the intestinal barrier function.In animal experiments,SDC treatment for a relatively long time can destroy the intestinal mucosal barrier and down-regulate the expression level of TJP in mice.GYY4137 can protect the intestinal epithelial barrier function injury caused by SDC and up-regulate the expression of TJP.In bioinformatics analysis,the deoxycholate acid can lead to differential expression of multiple genes of gastrointestinal mucosa.These genes are involved in various physiological processes.