| BackgroundInflammatory bowel disease(IBD)-ulcerative colitis and Crohn’s disease-is emerging as a worldwide epidemic.its exact pathophysiological mechanism remains largely unknown. It was shown recently that an impairment of intestinal barrier is associated with Crohn’s disease and ulcerative colitis. Simultaneously,the environmental factor implicated in the pathophysiology of gut inflammation, which is undergoing increased scrutiny, is the intestinal flora. flagellin is the main component of bacterial flagellum,which is also the toxic component of bacteria.However,the relationship between intestinal barrier and bacterial flagellin is less frequently studied.ObjectiveT84cell monolayers were cultured for simulating intestinal epithelial barrier. Transepithelial electric resistance(TER) of the T84monolayers and flow of HRP was determined after treatment, so that permeability of the T84monolayers was got, therefore enabling us to better explore the relationship between intestinal barrier and bacterial flagellin. Methods1. T84monolayers were employed in this study. To evaluate the transport of flagellin by T84monolayers and the influence of paracellular permeability of T84monolayer by bacterial flagellin, flagellin and HRP was added to the apical chambers of transwell systems.flagellin and HRP in samples were determined by enzyme-linked immune assay(ELISA). HRP flux and TER were performed to evaluate the permeability of T84monolayers. Mast mast cell line (HMC-1) derived from human mast cell leukemia cells,which have mast cell characteristics after activation, were co-cultured with T84monolayers in transwell systems.2. Flagellin with different concentrations was added to the transwell systems. HRP flux and TER were performed2h after the addition of flagellin to evaluate the permeability of T84monolayers.3. Transwell systems were randomly divided into five groups:the naive control group, the flagellin groups, HMC-1group, flagellin+HMC-1group, ketotifen intervention group. HRP flux and TER were performed2h after the addition of flagellin and collectioning the basal chambers liquid, histamine and mast cell tryptase (MCT) levels in basolateral liquid were determined by ELIS A.4. To observe the uptake of flagellin by T84monolayers, flagellin with different concentrations was added to the plates which were cultured T84. Cells were collected from the plates2h after the addition of flagellin.,then intracytoplasmic flagellin were determined by immunocytochemistry. In order to observe the transport of flagellin by T84monolayers, flagellin in the basal chambers were determined by ELISA.5. The data was recorded by statestecal software SPSS17.0.Measurement data were expressed as means±standard diviation. Shapiro-Wilk of normality test was used in the data of statistics analysis. Levene test of homogeneity of varianc was used in the data of statistics analysis too.Three or more groups were analyzed with the analysis of variance(ANOVA) and the comparison between two groups was performed with LSD-t test. Differences between two groups were analyzed with the Student’s t-test.Then Spearman correlation was used to describe the relationship between the variables.Differences between means at a level of P<0.05were considered significant. Result1. Flagellin with different concentrations added to the apical chambers of transwell. When flagellin concentration less than8mg/L, to increase the flagellin concentration with luminal side, does not cause a decline with TER and a increases with HRP flow. When flagellin concentration greater than8mg/L, with the concentration of flagellin increases, The transmembrane resistance gradually decline and HRP flow gradually increase, compare with naive control group and the small concentration flagellin group, the difference was statistically significant.2. The results of each group on the barrier function of the intestinal epithelial cell line T84monolayers:①The results showed that the HMC-1group did not alter TER and HRP in T84monolayers as compared with naive controls, differences were not considered significant (P>0.05).②The comparative TER value of flagellin group was(59.73±7.00)%compared with the naive controls decreased significantly (P<0.05). At the same time, the HRP flow of flagellin group results (0.08±0.01)%compared with the naive controls, differences were considered significant(P<0.05).③The comparative TER value of flagellin+HMC-1group was (39.35±0.81)%compared with the flagellin group decreased significantly (P<0.05). At the same time, the HRP flow of flagellin+HMC-1group results(0.27±0.03)%compared with the flagellin group, differences were considered significant P<0.05).④The comparative TER value of ketotifen intervention group was (45.25±3.64)%compared with the flagellin+HMC-1group decreased significantly (P<0.05). At the same time, the HRP flow of ketotifen intervention group results(0.19±0.04)%compared with the flagellin+HMC-1group, differences were considered significant(P<0.05).3.Immunocytochemistry results were observed under an optical microscope:T84cells were incubated by flagellin (flagellin group) their cytoplasm was stained brown while the cytoplasm of the normal growth of the T84cells (negative control group) as a pale blue. It is indicate that flagellin were uptaked by T84cells in cytoplasm. It is indicate that OD value of flagellin group’s flagellin in basal chambers was significantly higher than the naive controls, differences were considered significant(P <0.05). And the mean of OD value was more than1.5times as greater as the OD value of naive controls.That is flagellin can be detected in basal chambers. 4. Histamine and tryptase detection is used in the naive control group, HMC-1group, flagellin+HMC-1group,ketotifen intervention group, differences among each group were considered significant(P<0.05). The comparison between two groups is indicate that HMC-1don’t secretion or a small amount of secretion of histamine and tryptase in no pyrogen stimulation. But HMC-1secretion of histamine and tryptase in flagellin stimulation that can be inhibition by ketotifen.5. The results of correlation analysis with histamine concentration and HRP flow was r=0.86, P<0.05, It is indicate that there is a positive correlation between histamine concentration and HRP flow.The results of correlation analysis with MCT levels and HRP flow was r=0.94, P<0.05, It is indicate that there is a positive correlation between MCT levels and HRP flow.Conclusion1. Flagellin increase human intestinal epithelial cell line T84monolayers paracellular permeability.2. Flagellin can be intake and transport to the basolateral by T84. then, flagellin activates mast cells and dysfunction the T84monolayers. |
PDF Full Text Request