The Protective Effect Of Butyrate On TNBS Induced Colitis In Mice

BackgroundThe incidence of inflammatory bowel disease(IBD)is increasing year by year,and its exact pathogenesis is still not very clear.Besides being closely related to the genetic susceptibility of individual organisms,it may also be influenced by many factors such as imbalance of intestinal flora and imbalance of immune regulation,or inflammatory injury of intestinal tissue.Especially the T lymphocyte and the intestinal tissue macrophages in the spleen play a key role in the pathogenesis of IBD.As an important part of the short chain fatty acids produced by the intestinal mucosal probiotics,butyrate can not only provide energy and nutrients in the intestinal cavity,but also plays a good role in regulating and protecting the whole intestinal tract.However,the study of butyrate on the specific pathogenesis of IBD and the protection and treatment of IBD has little research so far,so it is more worthy to have further exploration of this.ObjectiveIn this experiment,BALB/c mice induced by TNBS were used to establish a colitis model that could simulate the pathogenesis of human IBD.During the modeling process,butyrate solution and the same amount of Clostridium butyricum were given intragastric administration.Therefore,whether butyrate regulates the immune balance of Th1/Th2 in spleen tissue of mice with colitis,and whether it inhibits the proliferation of neutrophils in the intestinal tract and the imbalance of M1/M2 macrophages is preliminarily explored.Then it further explores whether butyrate regulates the PD-1/PD-L1 signaling pathway in the spleen.So it is more clear that butyrate has a certain degree of regulation and protection in colitis mice,and it also provides a new method and basis for the clinical treatment of IBD.Methods60 male BALB/c mice were randomly divided into four groups:(1)the normal control group,(2)the TNBS model group,(3)the TNBS+butyrate group and(4)the TNBS+ Clostridium butyricum group.During the feeding process,A general examination of the weight and mortality etc in each group of mice were examined and scored by DAI.After three weeks,all mice were killed and the colonic tissues of each group were compared with HE staining and HI scores.Then the expression of MPO in intestinal tissue and the concentration of IFN-?,IL-4,IL-17,TGF-? and IL-10 in the serum of mice in each group were detected by ELISA kit.The expression of Foxp3 protein was detected by Western Blot and the expression of mRNA in PD-1 and PD-L1 was detected by qPCR in the mice spleen tissues of each group.Finally,the expression of CD86 and CD206 was detected by immunohistochemical in colon tissues of each group,and the expression of chemokine receptor and ligand CCL2,CCR2,CCL17 and CX3CR1 were detected by ELISA.All the data in this experiment were processed with SPSS 21.0 statistical software.The measurement data is expressed by means of mean±standard deviation(x ±s).A single factor analysis of variance was used among the multiple groups of data.When the analysis of variance was meaningful,the LSD-t test was further compared between two groups.Chi square test was used to compare the immunohistochemistry results of each group.The level of test was ?=0.05,and p<0.05 was statistically significant.Results(1)The normal control group was generally in good condition,the body weight was slightly increased,no death was found and the DAI and HI score were in the normal range.Compared with(1)the normal control group,(2)the TNBS model group had poorer general condition,higher mortality and weight loss,and the DAI and HI scores also increased.However,the general condition,body weight and mortality rate of(3)TNBS+butyrate group and(4)the TNBS+ Clostridium butyricum group were all alleviated compared with those of(2)the TNBS model group,and the scores of DAI and HI were also decreased.The differences are statistically significant(p<0.05).Compared with(1)The normal control group,the ELISA value of MPO in intestinal tissue of(2)the TNBS model group increased,the ELISA value of IFN-? and IL-17 in the serum of(2)the TNBS model group increased,while the ELISA value of TGF-? and IL-10 decreased.The ELISA value of intestinal tissue MPO was recovered in(3)TNBS+butyrate group and(4)the TNBS+ Clostridium butyricum group compared with those in(2)the TNBS model group.The ELISA value of IFN-? and IL-17 in serum decreased,and the ELISA value of TGF-? and IL-10 also increased.The differences are statistically significant(p<0.05).However,there was no significant difference in the ELISA value of IL-4 in the serum of the four groups(p>0.05).Compared with(1)The normal control group,the expression of Foxp3 in spleen tissue of(2)the TNBS model group decreased,and the mRNA expression level of PD-1 and PD-L1 decreased correspondingly.However,the expression of Foxp3 in spleen tissues in(3)TNBS+butyrate group and(4)the TNBS+ Clostridium butyricum group was higher than in(2)the TNBS model group,and the mRNA expression level of PD-1 and PD-L1 also increased.The differences are statistically significant(p<0.05).Compared with(1)The normal control group,the immunohistochemical expression of CD86 in colonic tissue of(2)the TNBS model group increased,and the immunohistochemical expression of CD206 in the colon tissue decreased.The ELISA value of CCL2 and CCR2 in the intestinal tissue increased and the ELISA value of CCL17 and CX3CR1 decreased.In contrast of(2)the TNBS model group,the expression of CD86 in(3)TNBS+butyrate group and(4)the TNBS+ Clostridium butyricum group decreased with the increase of CD206 expression.In addition,the ELISA value of CCL2 and CCR2 in the intestinal tissue decreased and the ELISA value of CCL17 and CX3CR1 increased relatively.The differences are statistically significant(p<0.05).Conclusion1.Butyrate can not only improve the clinical manifestation and basic pathology of colitis mice,but also improve and regulate the number of Treg cells in spleen tissue and the corresponding immune balance.At the same time,the proliferation of neutrophils in the colon tissue and the imbalance of the macrophage typing were inhibited.2.Butyrate can promote the proliferation of Treg cells by inducing the PD-1/PD-L1 signaling pathway in the spleen tissue.It can also regulate the balance of M1/M2 macrophages in colonic tissue by inhibiting the expression of chemokine receptor and ligand of macrophages in the colon tissue.3.Clostridium butyricum as an important probiotics in the intestine,can also play an anti-inflammatory effect and protective regulation in the TNBS induced colitis mouse model.