The Effect Of Tim-3Signaling Pathway In The Experimental Colitis Of Mice

Background and objective:The etiology and pathogenesis of the inflammatory bowel disease （IBD） is stillunclear, but evidence has accumulated to suggest that abnomal Th immunne responseand overactivation of the Toll-like receptor （TLRs） pathway play an important role inthe pathogenesis of IBD. These abnormity is different in various type of IBD （UC andCD）, and the specific regulation mechanism behind these differences remains to beexplored. Recently, Tim-3was found as an important regulator in Th immuneresponse, and had a close relationship with Toll like receptor （TLR） signalingpathway and regulatory T cells （Treg） function. Therefore, in this article we willfocused on the Tim-3pathway, TLRs pathway and Treg in the different types of IBD,And obseve the changes of the TLRs pathways and Treg after neutralization of Tim-3activity in the different types of IBD mouse model. These datas suggest that theTim-3signaling pathway is a promising target for treatment of IBD.Method:1. Groups: Nine mice in each group.①Mice+IgG1②DSS model+IgG1③TNBSmodel+IgG1④Mice+Tim-3-Ab⑤DSS model+Tim-3-Ab⑥TNBS model+Tim-3-Ab2. Establishment of IBD models①Establishment of DSS-induced colitis,BALB/c mice were given5%DSS ad-libitum for7days. Mice only given normal sodium were used as the control group.②Establishment of TNBS-induced colitis, BALB/c mice were fast but drinkfreely24hours firstly, ethyl ether anesthesia,and enemaed with2.0mg TNBS/ethanol（50%） enema. Mice only given normal sodium were used as the control group.③Intervention of Tim-3pathways in mice,BALB/c mice received one i.p. injection of100μg of anti-Fc to reduce nonspecific binding and either100μg ofTim-3-Ab or control IgG1on day0of establishment of IBD mouse model.3. Observation and detection of each index①Observe the weight and disease activity index （DAI） of each group everyday;②Observe the macroscopic shape of colon in mice, and the change ofpathology through HE staining;③Detect the expression of Tim-3、Tim-1、Foxp3、MyD88、TLR4、SIGIRRprotein through Western blot;④Detect the protein expression of Foxp3、MyD88、SIGIRR�'�NF-κB p65through immunohistochemistry staining.Result:（1） Weight and DAI score of mice:①In the groups with or without Tim-3antibody treatment, the weights of micein all model groups begin to decline, to be lowest on the5^thday, and then rosegradually in the following days.The weight descending percentages of both modelgroups were higher than the control group on both5^thand7^thday（P<0.01）.In TNBSgroup with Tim-3antibody is lower than group without Tim-3antibody treatment onDay7^th（P<0.05）.②In the groups with or without Tim-3antibody treatment, there were significantdeviation of DAI scores in each group on the5^th,7^thday, the DAI scores of anycontrol group was significantly lower than that in the corresponding model groups（P<0.01）, no statistical difference between DSS and TNBS group （P>0.05）; In TNBSgroup with Tim-3antibody is lower than group without Tim-3antibody treatment onDay7^th（P<0.05）.（2） Pathological change of colon①Macroscopic shape of colonic mucosa, In the groups with or without Tim-3antibody treatment, there was no congestion, edema and erosion in colonic mucosa incontrol group; while congestion, edema and erosion in colonic mucosa in DSS andTNBS model groups were obvious, and these symptom in model group with Tim-3antibody treatment were lower than its corresponding group without Tim-3antibodytreatment. ②The pathohistological injury score of colon characterized by the degrees ofcolonic inflammation, pathological depth and crypt destruction, in control group weresignificantly lower than that in the corresponding DSS and TNBS groups（P<0.05） inthe groups with or without Tim-3antibody treatment; In model group with Tim-3antibody treatment was lower than the group without Tim-3antibody treatment（P<0.01）.（3） Expression of Tim-3protein in colonic mucosaIn the groups with or without Tim-3antibody treatment, the expression of Tim-3protein had no statistical difference among all groups （P>0.05）; And Tim-3antibodytreatment did not affect the expression of Tim-3protein （P>0.05）.（4）Expression of Tim-1protein in colonic mucosaIn the groups without Tim-3antibody treatment, Tim-1protein expression inTNBS group was higher than that in control group （P<0.05）. In TNBS group withTim-3antibody treatment was lower than its corresponding group without Tim-3antibody treatment （P<0.05）.（5） Expression of Foxp3in colonic mucosa①Western blot, in the groups with or without Tim-3antibody treatment, theexpression of Foxp3protein had no statistical difference among all groups （P>0.05）.And Tim-3antibody treatment did not affect the expression of Foxp3protein（P>0.05）.②Immunohistochemistry stain, in the groups without Tim-3antibody treatment,the expression of Foxp3protein in both DSS and TNBS group were significantlylower than its corresponding control group（P<0.01）.In TNBS group with Tim-3antibody treatment was significantly higher than the corresponding group withoutTim-3antibody treatment（P<0.01）.（6） Expression of SIGIRR in colonic mucosa①Western blot,in the groups with or without Tim-3antibody treatment，theexpression of SIGIRR protein had no statistical difference among all groups （P>0.05）.And Tim-3antibody treatment did not affect the expression of SIGIRR protein（P>0.05）.②Immunohistochemistry staining, in the groups without Tim-3antibody treatment, the expression of SIGIRR in both DSS and TNBS group were lower thanthat in corresponding control group （P<0.05）. In the groups with Tim-3antibody,there were no statistical difference among all groups （P>0.05）. In both DSS andTNBS groups with Tim-3antibody treatment were higher than the correspondinggroups without Tim-3antibody treatment （P<0.05）.（7） Expression of MyD88in colonic mucosa①Western blot,in the groups without Tim-3antibody treatment, the expressionof MyD88protein in TNBS group was significantly higher than that in control group（P<0.01）.In the groups with Tim-3antibody treatment, the expression of MyD88protein in both DSS and TNBS groups were higher than that in control group（P<0.05）.In TNBS group with Tim-3antibody treatment was lower than thecorresponding group without Tim-3antibody treatment （P<0.05）②Immunohistochemistry stain, in the groups with or without Tim-3antibodytreatment, the expression of MyD88protein in both DSS and TNBS groups werehigher than that in corresponding control groups （P<0.05）. Both in DSS and TNBSgroups with Tim-3antibody treatment were lower than the corresponding groupwithout Tim-3antibody treatment （P<0.05）.（8） Expression of TLR4protein in colonic mucosaWestern blot, in the groups without Tim-3antibody treatment, the expression ofTLR4protein in TNBS group was higher than that in control group and DSS（P<0.01）.In the groups with Tim-3antibody treatment, the expression of TLR4protein in DSS group was higher than that in control group and TNBS group（P<0.05）.In DSS group with Tim-3antibody treatment was higher than that in thecorresponding group without Tim-3antibody treatment （P<0.05）.（9） Expression of NF-κB p65in colonicic mucosaImmunohistochemistry stain, in the groups with or without Tim-3antibodytreatment, the expression of NF-κB p65protein in both DSS and TNBS groups werehigher than that in corresponding control group （P<0.01）. In both DSS and TNBSgroups with Tim-3antibody treatment were lower than group without Tim-3antibodytreatment （P<0.05）.Conclusion: 1. In this experiment, we had successfully established acute colitis mice modelwhich is induced by DSS or TNBS. Tim-3antibody treatment can alleviate micecolitis, which suggest that Tim-3pathway may participate in the pathogenesis of IBD.2. Tim-3antibody treatment can change expression of Tim-3and Tim-1in thethe colonic mucosa of experimental colitis mice, which suggest that Tim-1and Tim-3may participate in the pathogenesis of IBD.3. Tim-3antibody treatment can increase expression of Foxp3protein in the thecolonic mucosa of experimental colitis mice, which suggest that Tim-3antibody mayalleviate the inflammation of IBD by up-regulating Foxp3+Treg reaction.4. Tim-3antibody treatment can decrease the expression of MyD88and NF-κBp65, and increase the expression of SIGIRR, which suggest that Tim-3antibody mayalleviate the inflammation of IBD by inactivating of TLRs/NF-κB signaling pathway.