| BackgroundInflammatory bowel disease(IBD)is a chronic relapsing intestinal inflammatory disease,whose etiology and pathogenesis have not yet been fully elucidated.According to different clinical syndroms and pathological manifestations,this disease has two forms,ulcerative colitis(UC)and Crohn’s disease(CD).Increasing evidence shows that P2X7 purinoceptor is closely related to inflammation and is widely expressed in intestinal cells.Mast cells(MCs)are known effector cells in allergic and inflammatory diseases.It was reported that mast cells expressing P2X7 purinoceptors increased significantly in patients with CD.Furthermore,studies have shown that ceramide-CD300 f binding inhibits immunoglobulin E-dependent and mast celldependent allergic responses.However,the role of CD300 f in mast cell mediated colitis is not well understood.Our understanding of the relationship between P2X7 purinoceptor and IBD has continued to evolve.Therefore,further exploration of the regulation of P2X7 purinoceptor,and the relationship between P2X7 purinoceptor and CD300 f will provide new insights into clinical treatment of IBD.ObjectiveTo build the model of IBD,mice were given DSS supplemented in sterile,distilled water for 7 days.Mice were intraperitoneally injected with ceramide and myriocin to activate and inhibit CD300 f separately.The disease activity index(DAI)score,colon morphology and histology changes were detected to evaluate the severity of colitis.The expression of P2X7 purinoceptor,mast cell(MC),CD300 f,tryptase,histamine,and the co-localization of P2X7 purinoceptor and activating transcription factor-2(ATF2)were measured to explore the role of P2X7 purinoceptor in MC-mediated colitis,and to provide new treatment for IBD.Methods1.To build the model of IBD,male mice on the BALB/c background aged 6 to 8 weeks were given DSS supplemented in sterile,distilled water for 7 days.2.All mice were randomly divided into 4 groups,and 12 in each group.The mice in group DSS,DSS+Ceramide(CD300f agonist),DSS+Myriocin(CD300f antagonist)were given DSS supplemented in sterile,distilled water for 7 days.Control group mice were given sterile,distilled water only.The group DSS+Ceramide were given 100μg/day of ceramide on day 2 and day 6.The group DSS+ Myriocin were given 5μg/day of myriocin on day 2.3.The weight loss,stool consistency,bleeding,water and food intake were observed every day.All the mice were sacrificed on day 8 to collect serum and colon specimen.4.A disease activity index(DAI)score was determined by daily assessment of weight loss,stool consistency and bleeding.The colon histology changes were detected to evaluate the severity of colitis.The expression of P2X7 purinoceptor,MC,CD300 f in the intestinal mucosa were detected by immunohistochemistry and western blot,the levels of tryptase and histamine in serum were measured by ELISA,and the co-localization of P2X7 purinoceptor and ATF2 were tested by immunofluorescence.5.The data were analysed by SPSS 21.0 software,and were presented as mean ± standard deviation(SD).The difference between groups was determined by the Student t test or one-way analysis of variance(ANOVA)if more than two groups.P<0.05 was regarded statistically significant.Results1.Disease activity index(DAI)As compared with the control group,the group DSS saw weight loss,bloody stools,and diarrhea.Symptoms in group DSS+ Ceramide were milder than those in group DSS,while symptoms in group DSS+ Myriocin were more severe than those in group DSS.DAI in group DSS was higher than control group(P<0.05),while group DSS+Ceramide was lower than DSS group(P<0.05).There was no significant difference between DSS+ Myriocin group and DSS group.2.Colon lengthCompared with the control group,the colon length in group DSS was shorter(P<0.05),while group DSS+Ceramide was longer than DSS group(P<0.05).There was no significant difference between DSS+ Myriocin group and DSS group.3.Histological indexHistological index in group DSS was higher than control group(P<0.05),while group DSS+Ceramide was lower than DSS group(P<0.05).There was no significant difference between DSS+ Myriocin group and DSS group.4.The expression of P2X7 purinoceptor,MC,CD300 f in the intestinal mucosa were detected by immunohistochemistryThe expression of P2X7 purinoceptor,MC,CD300 f in group DSS were higher than control group(P<0.05),while the expression of P2X7 purinoceptor and MC in group DSS+Ceramide were lower than DSS group(P<0.05),and the expression of CD300 f in group DSS+Ceramide were higher than DSS group(P<0.05).There was no significant difference between DSS+ Myriocin group and DSS group.5.The expression of P2X7 purinoceptor,MC,CD300 f in the intestinal mucosa were detected by western blotThe expression of P2X7 purinoceptor in group DSS+Ceramide was lower than DSS group(P<0.05).There was no significant difference between control group,DSS group,and DSS+ Myriocin group.The expression of MC in group DSS+Ceramide was lower than DSS group(P<0.05).There was no significant difference between control group,DSS group,and DSS+ Myriocin group.The expression of CD300 f in group DSS was higher than control group(P<0.05),while group DSS+Ceramide was higher than DSS group(P<0.05).There was no significant difference between DSS+ Myriocin group and DSS group.6.The levels of tryptase and histamine in serum were measured by ELISAThe levels of tryptase in group DSS were higher than control group(P<0.05),while group DSS+Ceramide were lower than DSS group(P<0.05),and the DSS+ Myriocin group were higher than DSS group(P<0.05).There was no significant difference about the level of histamine between the four groups.7.The co-localization of P2X7 purinoceptor and ATF2 were tested by immunofluorescenceCompared with the control group,the group DSS showed co-localization of P2X7 purinoceptor and ATF2.Conclusions1.P2X7 purinoceptor is involved in mast cell-mediated colitis.2.Ceramide-CD300 f binding inhibits experimental colitis by suppressing ATP-P2X7-mediated mast cell activation.3.ATF2 probably regulate the expression of P2X7 purinoceptor. |
PDF Full Text Request