The Mechanism Of EphB2 Mediated Intestinal Neuroimmunity In Chronic Colitis

Objects: To explore the expression and immunoregulation of enteric nervous system(ENS)in the intestinal mucosa of mice with chronic DSS colitis.Methods: C57BL/6 mice were given 2% DSS drinking water for 7 days and another normal drinking water for 14 days as a cycle.The model of chronic DSS colitis was established by such three cycles.We randomly divided the mice into two groups: normal control group(NC group)and model group(DSS group).The establishment of model was evaluated by the changes of body weight,colon length,disease index score,histopathological score and other indicators.The density changes of pgp9.5 and gap43 in colonic mucosa of each group of mice were observed by immunofluorescence confocal experiment,and Western blot was used to detect the expression of pgp9.5 and activation level of ENS(c-fos protein).The colon of NC group and DSS group were treated with veratridine(VER)and electrical filed stimulation(EFS)respectively,Western-blot assay was used to detect the expression of inflammatory factors(TNF ? and IL-1 ?)in colon mucosa after the activation of ENS in vitro.After stimulation by EFS,the colon tissues of the two groups were given the intervention of TTX,and the expression of inflammatory factors was detected after the intervention of TTX.By comparing the ? value of the change range of inflammatory factors in NC group and DSS group after the intervention of EFS and(EFS + TTX),we explored the effect of the changed ENS on the inflammatory response in DSS colitis.Results:(1)Compared with NC control group,the colon length and weight of DSS group mice were shortened,but the disease index score and histopathological score were increased.The expression of TNF ? and IL-1 ? inflammatory factors were also significantly increased,and there were significant statistical differences.(2)In the DSS group,the total protein expression of ENS in colon mucosa was increased,along with the increase of nerve fiber density and new nerve fiber density,as well as the expression of c-fos protein was also increased.It was confirmed that the intestinal inflammatory response positively regulated the expression density and activation degree of ENS.(3)After the activation of ENS by VER and EFS,the expression of inflammatory factors in intestinal mucosa of NC group and DSS group were increased.After TTX inhibited the activation of ENS,the absolute value of TNF ? and IL-1 ? expression in DSS group were higher than that in NC group.It was suggested that the severity of intestinal inflammation was related to ENS.Conclusion: the expression of ENS and the activation of ENS are increased in the colonic mucosa of chronic DSS colitis mice,and the expression of ENS is positively correlated with the intestinal inflammation.This suggests that intestinal immunity interacts with ENS in the intestinal mucosa,and ENS is involved in the regulation of inflammatory response.Objective: To explore the relationship between Eph B2 and ENS in intestinal mucosa and the effect of ENS on colitis.Methods: The model of DSS chronic colitis in C57BL/6 mice were established successfully.During the modeling period,some mice were randomly injected with Eph B2-Fc fusion protein,a specific antagonist of Eph B2,or Ephrin B2-Fc protein,a specific agonist of Eph B2,via tail vein.The mice were randomly divided into four groups: normal control group(NC),model group(DSS)and Eph B2 intervention group(DSS + Eph B2-FC)with(DSS+Ephrin B2-Fc).Western-blot was used to detect the protein expression level of Eph B2 and the phosphorylation level of Eph B2(p Eph B2)in the colonic mucosa of three group mice.The effects of Eph B2-Fc or Ephrin B2-Fc on chronic enteritis were observed by recording the changes of colon length and weight,evaluating the disease activity index score,histopathological score,and the expression of inflammatory factors in colon mucosa.The expression of Eph B2 on the nerve fiber membrane of intestinal mucosa and the expression of new nerve fiber of colon mucosa in Eph B2 FC-group were observed by immunofluorescence confocal microscopy.The relationship between Eph B2 and ENS was demonstrated by further comparing the expression levels of total nervous protein and activated nervous protein in colon mucosa of three groups of mice.By comparing the ? value of the change range of inflammatory factors in three groups after the intervention of EFS and(EFS+TTX)respectively,the influence of the change of Eph B2 mediated ENS on colitis was clarified by the calculation method of(TTX intervention value-EFS intervention value)/ EFS intervention value.Results:(1)Eph B2-Fc effectively inhibited the expression and activation of Eph B2.Compared with NC group,the expression of Eph B2 in the colon mucosa of DSS group was higher.Compared with DSS mice,the expression level of Eph B2 in colon mucosa of Eph B2-Fc intervention group was decreased,and the activation of Eph B2 was also inhibited,while p Eph B2 activation was significantly increased in Ephrin B2-Fc intervention group.(2)Eph B2-Fc significantly inhibited intestinal inflammation.Compared with DSS group,the colon length,body weight,disease activity index and histopathological score of Eph B2-FC group mice increased significantly,and the expression level of TNF ? and IL-1 ? decreased significantly.While the inflammatory responses in Ephrin B2-Fc group were significantly increased.(3)Eph B2 was expressed on the nerve fiber membrane of colon mucosa and regulated the density and activation of ENS in the intestinal mucosa.Compared with DSS group,the density of nerve fibers in intestinal mucosa of Eph B2-Fc intervention group decreased,and the activation degree of mucosal ENS decreased significantly.However,the activation degree of mucosal ENS in Ephrin B2-Fc group was significantly increased.(4)ENS activation mediated by Eph B2-Fc alleviated the colitis response.After the activation of ENS by EFS,the expression of inflammatory factors in the Eph B2-FC intervention group was significantly lower than that DSS group,and also lower than that in NC group.On the contrary,the expression of inflammatory factors in Eph B2-FC group was higher than that in EFS alone after intervention of(EFS + TTX),and the value of ? was positive,which was significantly higher than the ? negative absolute value of DSS.Conclusion:(1)The inhibition of Eph B2 expression and activation is negatively correlated with colonic inflammation.(2)Inhibition of Eph B2 expression can reduce the expression and activation of ENS in colonic mucosa of mice with chronic colitis,indicating the positive regulatory effect of Eph B2 on ENS.(3)It demonstrated that the inhibition of EphB2 expression in colitis could reduce the intestinal inflammation by mediating the change of ENS.Objective: To explore the mechanism of Eph B2-medidiated ENS in regulating intestinal inflammatory response?Methods: C57 BL / 6 mice were randomly divided into three groups: NC normal control group,chronic DSS colitis model group and(DSS + Eph B2-FC)intervention group.Western-blot experiment was used to detect the expression of n NOS labeled nitrogen nerves and CHAT labeled cholinergic nerves in the colonic mucosa of the three groups.Immunofluorescence confocal assay was used to observe the location and expression of CHAT labeled nerve fibers and GAP43 labeled new nerve fibers in the colon mucosa of Eph B2-FC mice.In vitro,three groups of mice stimulated by EFS were treated with n NOS specific antagonist 7NI and chat cholinergic receptor pathway inhibitor MLA,and Western-blot was used to detect the expression of inflammatory factors in the colon mucosa of the three groups.By comparing the ? value of the change range of inflammatory factors in three groups of mice after the intervention of(EFS + 7NI)and(EFS + MLA),the calculation method of(7NI/ MLA intervention value-EFS intervention value)/ EFS intervention value was used to clarify the mechanism of the effect of Eph B2 mediated ENS change on intestinal inflammation.Results:(1)Compared with NC group,the expression of n NOS and CHAT in the colon mucosa of DSS group were significantly increased.Compared with DSS group,the expression of n NOS in colon mucosa of Eph B2-FC group was decreased,but the expression of CHAT nervous was increased,and the co-staining of CHAT and gap43 nerve fibers were observed by immunofluorescence confocal method in Eph B2-FC group.(2)After the intervention of(EFS + 7NI)in vitro,the expression of inflammatory factors in colon mucosa of three group mice were lower than that of single EFS stimulation.After the intervention means of(EFS + MLA)in vitro,the expression of inflammatory factors in colon mucosa of three groups of mice was higher than that of single EFS stimulation.It is suggested that inhibition of intestinal mucosal n NOS expression can reduce intestinal inflammation,and inhibition of the CHAT cholinergic nerve-receptor pathway can aggravate inflammation.(3)Compared with DSS group,the absolute value of ? negative value of inflammatory factor expression in Eph B2-FC group after EFS and(EFS + 7NI)intervention was significantly lower than that in DSS group,but there was no significant difference between Eph B2-FC group and NC group.After the intervention of EFS and(EFS + MLA),the ? value of the change of inflammatory factor expression was positive in Eph B2-Fc group,which was significantly greater than the absolute value of ? in DSS group and also greater than the absolute value of ? in NC group,and there were statistical differences.This suggested that the decrease of Eph B2-Fc-mediated n NOS nerve and the increase of CHAT nerve were the main mechanism for ENS to regulate colitis response.Conclusion:(1)By observing the inhibition of Eph B2 expression,the expression of n NOS was reduced and the expression of CHAT was promoted in colitis,which clarified the differentiation and regulation of Eph B2 on ENS in the intestinal mucosa.(2)It was demonstrated that the regulatory role of EphB2-mediated nNOS and CHAT nerves on the colitis response.(3)The role of Eph B2 in regulating the differentiation of n NOS and CHAT nerves was further explained,and the mechanism of the role of CHAT cholinergic neuralreceptor pathway in the intestinal nerve immunity mediated by Eph B2 was elucidated.