| BackgroundThe incidence of Inflammatory bowel disease (IBD) increases each year, however, its exact pathophysiological mechanism remains largely unknown. It may be associated with intestinal flora, immune mediated tissue damage, genetic susceptibility, or other related factors. Though many studies have shown that bacterium is a precipitating factor for IBD. This is because the main component of bacterial flagellum is flagellin(CBirl), which is also the toxic component of bacteria. However, the relationship between IBD and bacterial flagellin is less frequently studied.ObjectiveThrough establishing a IBD animal model, observing the disease activity situation of mice, analyzing the expression level of CBirl,TLR5,mast cell tryptase β2(MCT), and histamine in the serum and tissues of this animal model. And that by making an intervention on the model group we aim to research the role of CBirl in the pathogenesis of IBD, which is of great significance in understanding the diagnosis of IBD and evaluating the grade of inflammatory activity, therefore enabling us to better explore the pathogenesis of IBD. MethodsSPF male BALb/c mice were randomly divided into6groups; each group consisting of12mice.①normal control group:wild-type BALb/c mice were given an unrestricted diet.②saline group:mice were put in a fasted state for24h then given unrestricted water after having received a100μl saline enema and given an unrestricted diet. In8days and15days this was repeated.③50%ethanol group: mice were given50%100μl ethanol solution enema.④TNBS+50%ethanol group: mice were given2.0mg TNBS/50%ethanol100μl enema on the1st day,2.5mg TNBS/50%ethanol100μl enema on the8st day, and3.0mgtnbs/50%ethanol100μl enema on the15th day.⑤ketone Ketotifen+TNBS+50%ethanol group:0.5mg ketotifen was dissolved in100μl saline. Then each mouse was administered an intraperitoneal injection of100μl Ketotifen solution before a30minute enema.⑥LPS+OVA+TNBS+50%ethanol group:LPS10μg+OVA20μg dissolved in100μl saline. Each mouse was treated with an intraperitoneal injection of100μl of this solution during days7,9,12, and15. OVA50μg was dissolved in100μl saline, and each mouse was given intraperitoneal injection of100μl of this solution during days18,19,20, and21. DAI was score for each group, and the mice were terminated by having their heads cut off on day22. Anti-CBirl, MCT, and histamine concentrations were measured in serum by enzyme-linked immuno sorbent assay (ELISA). The colon tissues were examined by routine HE and scored for HI. The expression of CBirl in the colon, TLR5and MCT was detected by immunohistochemistry.The numerical data was recorded by statistical software SPSS13.0.All the measurement data were expressed in mean±and standard diviation (x±s) Levene test of homogeneity of varianc was used in the data of statistics analysis. The measurement data of multiple group were compared with single factor analysis of variance (the ANOVA), and the comparison between two groups was performed with LSD-t test. P<0.05was considered statistically significant (a=0.05). Then Pearson correlation was used to describe the relationship between the variables. Results(1)We found that the colonic mucosal epithelial were intact, the glands were regular in arrangement, and there was almost no inflammatory cell infiltration in the mucosa of the normal control group and saline group. Only a tiny amount of lymphocytes were found in the mucous membrane of the50%ethanol group. We also observed that the epithelium cells were broken, goblet cells were reduced, and large amounts of inflammatory cells were infiltrated in the mucosal layer and muscularis,at the same time,there was a piece of necrosis in the mucous membrane and a dermal proliferation of small blood vessels stemming from the muscularis mucosa into the submucosa. All of which were appeared in the TNBS+50%ethanol group. Furthermore, a few inflammatory cells were found in the mucosal layer and muscularis of the Ketotifen+TNBS+50%ethanol group. The elongated normal structures were discovered, and the inflammation was covered with all intestinal wall and the lamina propria was thickened, all of which were discovered in LPS+OVA+TNBS+50%ethanol group. We also found that the deeper the extent of the injury of intestinal mucosa and histology, the higher expression in intestinal mucosa tissue and/or serum of CBirl (or anti-CBirl), MCT, and/or the TLR5and/or histamine was found.(2)The DAI score、HI score, and the expression level of CBirl (or anti-CBirl), MCT, and/or the TLR5and/or histamine in TNBS+50%ethanol group,Ketotifen+TNBS+50%ethanol group and LPS+OVA+TNBS+50%ethanol group were higher than those in the normal group, with significant difference (P<0.05). All of which in the Ketotifen+TNBS+50%ethanol group were lower than in the TNBS+50%ethanol group,of which the differences had statistical significance (P<0.05). The expression level in the TNBS+50%ethanol group was lower than in the LPS+OVA+TNBS+50%ethanol group, of which the differences also had statistical significance (P<0.05). When compared with the normal control group, little statistical significance was found (P>0.05) between the saline group and the50%ethanol group.(3)The concentration of anti-CBirl in serum of IBD model groups was positively correlated with MCT (r1=0.648, F1<0.01). And the concentration of anti-CBirl in serum was also positively correlated with histamine (r2=0.751,P2<0.01).Conclusion(1) CBirl was involved in the pathogenic process of experimental IBD;(2) CBirl can activate mast cells to release chemical mediators and induce intestinal inflammation;(3) The expression level of CBir1,TLR5,MCT and histamine was correspondent to the severity of colitis in IBD models;... |
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