A Preliminary Study On Effects Of EPA On Tight Junction In Intestinal Epithelial Cells Induced By Ecoli LF82

?Objectives? The tight junction protein is an important structural basis for the formation of the intestinal epithelium barrier and plays an important role in the regulation of molecular trans-cellular bypass transport.Normal physiological condition,tight junction protein is complete,when the tight junction protein structure damage,intestinal mucosal permeability will increase,toxic substances through the intestinal mucosal barrier and microbial translocation,these changes can activate the intestinal mucosal immune system,will lead to Inflammatory bowel disease(IBD)and systemic inflammatory response,and most of the drugs for the treatment of intestinal diseases are mainly used to restore intestinal mucosal permeability.Therefore,maintaining the intestinal barrier function is essential for maintaining human health.At present,a number of studies have confirmed that patients with inflammatory bowel disease in the intestinal mucosa have more adhesion of invasive E.coli(adherent-invasive Escherichia coli,AIEC)compared with the control group.Eicosapentaenoic acid(EPA)can effectively prevent the destruction of tight junction protein structure,and has anti-inflammatory effect,but the role of EPA on the intestinal barrier function caused by bacterial infection remains to be further studied.Human colon cancer cell line Caco-2 cells,conventional culture conditions,are able to differentiate and fuse into continuous monolayer cells,which function and structure similar to intestinal epithelial cells.In this study,we treated Caco-2 cells with EPA and E.coli LF82 strains.RT-q PCR and western blot were used to detect Effect of eicosapentaenoic acid on the expression of tight junction protein ZO-1 in intestinal epithelial cells induced by adherent-invasive Escherichia coli LF82.The level of TNF-? and INF-? in culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA).To explore the protective effect and mechanism of EPA on cell tight junction protein after E.coli LF82 infection,So as to clarify the protective effect of EPA on intestinal epithelial barrier injury-related diseases.?Methods? 1.Caco-2 cell line was used to establish the model of intestinal epithelial barrier in vitro,the Caco-2 cell model was evaluated by observing the morphological characteristics of cells,determining the growth curve of the cells and comparing the alkaline phosphatase(ALP)activity on both sides of the cell membrane,the effect of E.coli LF82 on the tight junction of ZO-1 was studied by RT-q PCR,the effect of E.coli LF82 on inflammatory cytokines in supernatant of cell culture was studied by ELISA.2.The effect of EPA on the survival rate and apoptosis rate of Caco-2 cells was detected by MTT and flow cytometry to determine the safe concentration of EPA on Caco-2 cells,the effect of EPA on the expression of tight junction protein ZO-1 m RNA was detected by RT-q PCR,and the lowest effective concentration of EPA was determined.3.To investigate the effect of EPA on the infection of E.coli LF82 and its possible mechanism.The effect of EPA on the expression of tight junction protein ZO-1 was studied by RT-q PCR and western blot,the effect of EPA on the secretion of TNF-? and INF-? in culture supernatant of E.coli LF82 was detected by ELISA,the protective effect and possible mechanism of EPA on intestinal epithelial barrier junction proteinsafter E.coli LF82 infection were confirmed.?Results? 1.Caco-2 cells from colon cancer were able to simulate the physiological status of the intestinal epithelium in vitro after 21 days of culture,E.coli LF82 can down-regulate the expression of ZO-1m RNA in intestinal epithelial cells and increase the secretion of inflammatory cytokines TNF-? and INF-? in intestinal epithelial cell barrier.2.MTT and flow cytometry showed that 25,50 ?mol/L EPA significantly increased the survival rate of Caco-2 cells in a concentration-dependent manner,100,200?mol/L EPA had a significant inhibitory effect on Caco-2 cell proliferation and was dose-dependent,when the concentration of EPA was 0,25,50,100,200?mol/L,the apoptotic rate increased with the increase of EPA concentration,however,when the concentration of EPA was 25 and 50?mol/L,the apoptotic rate was not statistically significant compared with the control group(0?mol/L),when the concentration of EPA was 100,200?mol/L,the apoptosis rate was significantly higher than that of the control group(0?mol/L),and 50?mol/L EPA could increase the expression of tight junction protein ZO-1m RNA.3.EPA can increase the expression of close junction protein ZO-1 after E.coli LF82 infection,effectively prevent the damage of intestinal epithelial barrier,EPA can reduce the secretion of inflammatory cytokines TNF-? and INF-? after intestinal epithelial barrier of E.coli LF82 infection,inhibit the secretion of inflammatory factors induced by infection and alleviate the damage of inflammatory factors to tight junction proteins.?Conclusion? In this study,we found that low dose of EPA could induce the proliferation of Caco-2 cells,but the high dose of EPA could induce apoptosis.E.coli LF82 could decrease the expression of Caco-2 cell tight junction protein ZO-1 and stimulate the secretion of inflammatory factors.EPA can effectively prevent the effect of LF82 infection on tight junction proteins and inhibit the secretion of proinflammatory cytokines induced by LF82 infection in a concentration-dependent manner.n summary,the EPA may protect theintestinal mucosal barrier by inhibiting the secretion of proinflammatory cytokines after LF82 infection.It is speculated that supplementing the appropriate concentration of EPA may have the effect of preventing infection and intestinal inflammatory response and intestinal mucosal barrier injury,these can be further studied by animal experiments.