| Background and objective:The pathogenesis and etiology of IBD are very complicated. According to thecurrent study and our previous work, dysregulation of TLR signal pathway and Thimmune reaction was thought to play a vital role in IBD. The further finding is thatthere is different at the dysregulations beteen various types of IBD, but the detailedmechanism is unclear. Tim-1is not only crucial to modulate Th immune reaction, butalso connective with TLR signal pathway. However, the role of Tim-1in IBD,especially its effect on Th immune reaction and TLR sigalling, have not been reportedyet. In order to explore the role Tim-1in IBD, we observe the alteration of Tim-1signal pathway, and its relationship with Treg and TLR singal pathway in various ofmouse IBS model. Through this experiment, we offer an innovative access to probeinto the mechanism of IBS, and provide a new taget and direction to prevention andtreatment of IBD.Method：1. Experiment groups①Mice+IgG2a②DSS model+IgG2a③TNBSmodel+IgG2a④Mice+Tim-1-Ab⑤DSS model+Tim-1-Ab⑥TNBS model+Tim-1-Ab2. Establishment of experimental colitis mice model(1) Establishment of DSS-induced colitis, BALB/c mice were given5%DSS ad-libitum for7days. Mice only given normal sodium were used as the control group.(2) Establishment 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. (3) Intervention of Tim-1signal pathway in mice:12h before modeling, inject100μg anti-Fc antibody each mouse intraperitoneally to eliminate unspecificabsorption, then give them each an injection of250μg Tim-1antibodyintraperitoneally.The control mice were given IgG2a of equal dose.3. Observation of the following indexes(1) Observe the weight and disease activity index (DAI) of each group everyday;(2) Observe the macroscopic shape of colon in mice, and the change ofpathology through HE staining;(3) Detect the expression of Tim-1,Foxp3,MyD88,SIGIRR protein throughWestern blot;(4) Detect the protein expression of Foxp3,MyD88,SIGIRR和NF-κB p65through immunohistochemistry staining.Results：1. Weight and DAI score of mice in each groupOn the5th and7th day of the experiment, the percentage of mouse weight loss inall model groups were significantly higher than that in corresponding control groups(P<0.01). In the Tim-1antibody treated groups: the percentage of mouse weight lossin DSS model group was significantly higher than that in TNBS model group(P<0.01). In DSS group with Tim-1antibody is higher than group without Tim-1antibody treatment on Day7th(P<0.05).In the groups with or without Tim-1antibody treatment, There were statisticalsignificance of DAI scores in each group, The model groups were significantly higherthan the control group (P<0.01); The DAI scores in all of the Tim-1antibody treatedgroups were higher than the untreated groups, however difference had no statisticalsignificance (P>0.05).2. General morphology of mice colonic mucosa and pathological changeof colonic tissue(1) Macroscopic shape of colonic mucosa, In the groups with or without Tim-1antibody 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 the symptom in model group with Tim-1 antibody treatment were higher than its corresponding group without Tim-1antibodytreatment.(2)The 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<0.01) inthe groups with or without Tim-1antibody treatment; In DSS group with Tim-1antibody treatment was higher than the group without Tim-1antibody treatment(P<0.05).3. Expression of Foxp3in mice colonic tissue(1) Western blot, in the groups with Tim-1antibody treatment, the expression ofFoxp3were lower in both model groups than in control group (P<0.01). And Tim-1antibody treatment did not affect the expression of Foxp3protein (P>0.05).(2) Immunohistochemistry stain, in the groups without Tim-1antibody treatment,the expression of Foxp3in both DSS and TNBS group were significantly lower thanits corresponding control group (P<0.01). In DSS group the expression of Foxp3wassignificantly lower in group with Tim-1antibody treatment than in group withoutTim-1antibody treatment (P<0.05).4. Expression of SIGIRR in mice colonic tissue(1) Western blot,in the groups with or without Tim-1antibody treatment, theexpression of SIGIRR in DSS group were significantly lower than its correspondingcontrol group(P<0.01). In the groups without Tim-1antibody treatment, TNBS groupwas significantly lower than its control group (P<0.05); And Tim-1antibodytreatment did not affect the expression of SIGIRR protein (P>0.05).(2) Immunohistochemistry staining, in the groups with or without Tim-1antibody treatment, the expression of SIGIRR in both DSS and TNBS group werelower than that in corresponding control group (P<0.01). And Tim-1antibodytreatment did not affect the expression of SIGIRR (P>0.05).5. Expression of Myd88in mice colonic tissue(1) Western blot, in the groups with or without Tim-1antibody treatment, theexpression of Myd88in both DSS and TNBS group were higher than that incorresponding control group (P<0.01), and in DSS groups were higher than that in TNBS groups (P<0.01). And Tim-1antibody treatment did not affect the expressionof Myd88protein (P>0.05).(2) Immunohistochemistry stain, in the groups with or without Tim-1antibodytreatment, the expression of MyD88in both DSS and TNBS groups were higher thanthat in control groups (P<0.01). In the group with Tim-1antibody treatment, theexpression of MyD88was higher in DSS group than that in TNBS group (P<0.01). InDSS group the expression of MyD88was significantly higher in group with Tim-1antibody treatment than in group without Tim-1antibody treatment (P<0.05).6. Tim-1expression in mice colonic tissueWester blotting, in the groups without Tim-1antibody treatment, the expressionof Tim-1in both DSS and TNBS groups were lower than that in control groups(P<0.01). in the groups with Tim-1antibody treatment, the expression of Tim-1inTNBS group was lower than that in control groups (P<0.01), and was higher in DSSgroup than in TNBS group (P<0.05). In DSS and control groups, the expression ofTim-1were significantly higher in group with Tim-1antibody treatment than in groupwithout Tim-1antibody treatment (P<0.01).7. NF-κB p65expression in mice colonic tissueImmunohistochemistry stain, in the groups with or without Tim-1antibodytreatment, the expression of NF-κB p65in both DSS and TNBS groups were higherthan that in control group (P<0.01). In DSS group, the expression of NF-κB p65washigher in group with Tim-1antibody treatment than in group without Tim-1antibodytreatment (P<0.01).Conclusion:1. In this experiment, we had successfully built up acute colitis mice modelwhich is induced by DSS or TNBS. Tim-1antibody treatment can aggravate micecolitis, which suggest that Tim-1pathway may participate in the pathogenesis of IBD.2. Tim-1antibody treatment can decrease expression of Foxp3protein in the thecolonic mucosa of experimental colitis mice, which suggest that Tim-1antibody mayaggravate the inflammation of IBD by down-regulating Foxp3+Treg reaction. 3. Tim-1antibody treatment can increase the expression of MyD88and NF-κBp65, which suggest that Tim-1antibody may aggravate the inflammation of IBD byactivating of TLRs/NF-κB signaling pathway. |
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