| Objective: The pathogenesis of IBD is still unclear,and current studies suggest that it may be related to infection,environment,genetics,immunity and other factors.At present,the main treatment of IBD include: amino salicylic acid preparation,glucocorticoid,immunosuppressant,biological preparation.Intestinal immune function plays an important role in the occurrence,development and treatment of IBD,which including the innate immune system and the acquired immune system.Intestinal innate immune system mainly includes intestinal mucosal barrier,innate immune cells and their secreted cytokines.Innate immune system abnormality is related to the incidence of IBD.TLRs can be expressed in a variety of intestinal cells,mainly mediating the interaction between microorganisms and cells,and playing an important role in both innate and acquired intestinal immunity.TLR2 mainly recognizes peptidoglycan of Gram-positive bacteria,lipoproteins of Mycobacterium and Treponema,some components of yeast and mycoplasma,etc.Studies have shown that TLR2 and its signaling pathway were highly expressed in IBD,and inhibition of TLR2 signaling pathway can alleviate DSS-induced colitis in mice.My D88 is an important adaptor protein of TLRs signaling pathway,which mediates TLRs family signaling pathway except TLR3.The binding of TLR and its ligands on the surface of cell membrane leads to the junction of TLR and My D88,the phosphorylation of IRAK-1,the phosphorylation of IκB,finally the activation of NF-κB,and then regulate the transcription of target genes,and cause the release of inflammatory factors.NF-κB is the convergence point of various signaling pathways,which is closely related to inflammation,tumor,infection,autoimmune diseases and so on.T cells play an important role in the intestinal acquired immune system,including Th,Treg,Tc.Previous studies have suggested that CD and UC were related to Th1 and Th2 cell-mediated immune responses respectively.Th17 cells mainly secrete IL17,IL21,IL22 and other cytokines.IL17 is its main effector,which can induce the pro-inflammatory factors IL6,TNF-α,chemokines KC,MCP-1,MIP-2 and MMPs,and promote the inflammation by combining acquired immunity and innate immunity.RORγt is the transcription factor of Th17 cells,which plays a key role in the differentiation of primary T cells into Th17 cells.Researches have found that after interference with the TLRs signaling pathway,related factors that affecting the differentiation of primary T cells were affected,which further affected the differentiation of primary T cells.Probiotics are more and more used in the treatment of IBD as active microbial agents beneficial to human body.The possible mechanisms are regulating intestinal flora,improving intestinal mucosal barrier function,regulating intestinal immune function and so on.Some studies have shown that probiotics such as Bifidobacterium can promote the differentiation of Treg cells and inhibit the differentiation of Th1 cells and Th2 cells.Clostridium butyricum(C.butyricum)as a Gram-positive bacterium,has been shown that its metabolites can affect the expression of TLRs,which indicate that there is a close relationship between C.butyricum and TLRs.The purpose of this study is to investigate the protective effect of C.butyricum on colitis mice induced by DSS in vivo,to observe its effect on TLR2,TLR2/My D88/NF-κB signaling pathway,and Th17 cells differentiation and function,in order to further explore the possible immune regulatory mechanism of C.butyricum.To explore the mechanism of C.butyricum on TLR2/My D88/NF-κB signaling pathway and the possible immune mechanism of Th17 cells interference in cell experiment.Through the above studies,the immune regulation mechanism of C.butyricum on IBD was deeply discussed,which provids more theoretical basis for the treatment of IBD and potential therapeutic targets for the treatment of IBD.Methods: 1.Forty 8-week-old BALB/c mice were randomly divided into five groups of eight mice for 7 days.Control group: double distilled water 0.4ml by gavage once a day;DSS group: free drank 5% DSS solution,double distilled water 0.4ml by gavage once a day;DSS+C.butyricum(1×109CFU)group: free drank 5% DSS solution,C.butyricum(1×109CFU)0.4ml by gavage once a day;DSS+C.butyricum(1×108CFU)group: free drank 5% DSS solution,C.butyricum(1×108CFU)0.4ml by gavage once a day;DSS+C.butyricum(1×107CFU)group: free drank 5% DSS solution,C.butyricum(1×107CFU)0.4ml by gavage once a day.We assessed the DAI scores and histological damage scores.The expression levels of TLR2,IL17,IL23 and RORγt were determined through immunohistochemical staing,western blot and q RT-PCR.The expression levels of CD3+CD4+IL17+(Th17)cells in peripheral blood were measured by flow cytometry.2.Forty 8-week-old BALB/c mice were randomly divided into five groups of eight mice for 7 days.Control group: double distilled water 0.4ml by gavage once a day;DSS group: free drank 5% DSS solution,double distilled water 0.4ml by gavage once a day;DSS+C.butyricum group: free drank 5% DSS solution,C.butyricum(1×109CFU)0.4ml by gavage once a day;DSS+C.butyricum+anti-TLR2 group: free drank 5% DSS solution,C.butyricum(1×109CFU)0.4ml by gavage once a day,CD282 10 ug intraperitoneal injection at 1,3,5,7 day;DSS+anti-TLR2 group: free drank 5% DSS solution,double distilled water 0.4ml by gavage once a day,CD282 10 ug intraperitoneal injection at 1,3,5,7 day.We assessed the DAI socres and histological damage scores.The expression levels of TLR2,My D88,NF-κBp65,IL17,IL23 and RORγt were detected through western blot and q RT-PCR.The expression levels of Th17 cells in peripheral blood were measured by flow cytometry.3.Colon cells HCT-116 were treated with C.butyricum supernatant.TLR2 blocker(CD282)and TLR2 agonist(Pam3CSk4)were used to modify the TLR2 signaling pathway.The expression levels of TLR2,My D88,NF-κBp65,and RORγt were determined through western blot and q RT-PCR.The phospho-NF-κBp65 protein level was determined by western blot.Results: 1.Body weight: DSS group was the lowest in all,C.butyricum groups were higher than DSS group(P< 0.05).DAI scores: the DAI scores began to increase from the 4th day,and peaked at the 7th day;DSS group had the highest DAI scores in all,with the DSS+C.butyricum(1×109CFU)group has lowest among the C.butyricum groups,(P<0.05).Colon length: except control group,the other four groups were all shortened,DSS group was the shortest in all,length of the C.butyricum treated groups were all improved(P<0.05),but there was no significant difference between the different concentrations of C.butyricum.HE staining showed that the lesions of DSS group mainly involved in the mucosa and submucosa,even in the serosa layer;ulcer,hemorrhage,granulation tissue,crypt inflammation and crypt abscess were all observed.Histological damage scores: DSS group was the highest in all,DSS+C.butyricum(1×109 CFU)group was the lowest within the C.butyricum treated groups(P<0.05).According to flow cytometry,Th17 cells level was lowest in the control group,highest in the DSS group,in addition,C.butyricum dose-dependently decreased the levels of Th17 cells in peripheral blood(P<0.05).RORγt was expressed in the lymphocytes of intestinal lamina propria and nuclear staining;IL17 and IL23 were stained with cell membrane and cytoplasm of the colon cells;TLR2 was expressed on the intestinal epithelial cells,crypt epithelial cells and lamina propria lymphocytes cells,and was stained with cell membrane and cytoplasm.C.butyricum dose-dependently down-regulated the m RNA and protein levels of TLR2,IL17,IL23 and RORγt in mouse colon tissue(all P< 0.05).Moreover,the effect of C.butyricum on TLR2 was positively correlated with IL17,IL23 and RORγt.2.The DAI scores and histological damage scores in the DSS+C.butyricum +anti-TLR2 group was lower than the DSS group,lightly higher than the DSS+C.butyricum group,(all P<0.05).The expression levels of TLR2 were the highest in the DSS group,decreased in the DSS+C.butyricum groups;after the application of CD282,the expression levels in the DSS+anti-TLR2 group were lower than that in the DSS group,(all P<0.05).In the peripheral blood of mice,the expression of Th17 cells in the control group was the lowest,DSS group was the highest,and which decreased after the application of C.butyricum;after the addition of CD282,the proportion of Th17 cells in the DSS+anti-TLR2 group decreased compared with the DSS group;the proportion of Th17 cells in the DSS+C.butyricum+anti-TLR2 group increased compared with DSS+C.butyricum,but was still significant lower than that in the DSS group,(all P<0.05).The expression levels of My D88,NF-κBp65 and RORγt in the DSS group were higher than those in the control group;after adding C.butyricum,the expression levels of these indicators decreased;when addition of CD282,compared with the DSS group,the expression levels of the above indicators in the DSS+anti-TLR2 group decreased;(all P<0.05).After adding CD282 and C.butyricum,the expression levels of the above indicators in DSS+C.butyricum+anti-TLR2 group were slightly higher than that in the DSS+C.butyricum,but still significantly lower than that in the DSS group,(all P<0.05).3.C.butyricum supernatant downregulated the expression levels of TLR2,My D88,NF-κBp65,phospho-NF-κBp65,and RORγt in HCT-116 cells.Partial blockage of TLR2 by CD282 weakened the inhibitory effects of C.butyricum supernatant on the above pathway components(all P<0.05).After Pam3CSK4 activation of TLR2,the C.butyricum supernatant still significantly inhibited the expression levels of the above pathway components(all P<0.05).Conclusion: 1.C.butyricum can improve DSS-induced acute intestinal inflammation in mice,dose-dependently improve their DAI scores and histological damage scores.C.butyricum can inhibit the expression level of TLR2,down-regulate the proportion of Th17 cells in peripheral blood,and down-regulate the expression levels of IL17,IL23 and RORγt,which has certain protective effect on DSS-induced acute intestinal inflammation in mice.2.C.butyricum can inhibit the expression levels of RORγt and IL23,thereby down-regulate the expression of Th17 cells and affect which to secrete IL17,and then alleviate DSS-induced mouse colitis,by partially inhibiting the TLR2/My D88/NF-κB signaling pathway.3.The C.butyricum supernatant can inhibit the TLR2/My D88/NF-κB signaling pathway and the expression of RORγt in HCT-116 cells.These effects are at least achieved through inhibition of TLR2. |
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