| Background and objective:Inflammatory Bowel Disease is a group of chronic intestinal inflammatory disease with unknown etiology,including ulcerative colitis and crohn’s Disease.It is more common in western countries,but in recent years,the incidence of IBD rises year by year,and has become a global disease.It is forecasted that in the next 10 years,patients with IBD will occur exponential growth,China will have more than 1.5 million of IBD patients.The etiology and pathogenesis of IBD is unknown,the change of living environment and many other factors are the cause of the rising incidence of IBD,studies have shown that intestinal bacteria and their metabolites act on host susceptible to gene susceptibility,which makes IBD build up immune answer.Therefore,to clarify the onset of IBD immune abnormalities and its regulatory mechanismis extremely important for digging out a novel treatment for IBD.Toll-like receptor 4(TLR4)signaling pathway plays an important role in IBD abnormal immune response,but its specific regulatory mechanism has not been fully elucidated.McIntire discovered a new gene family Tim(T cell Ig domain and mucin domain)family in 2001,Tim proteins play an important role on regulation of T cell responses,and it is one of the most important proteins regulating immune abnormalities found in recent years.Tim is the key protein in adjusting Th immune response,and have close relationship with TLRs signaling pathways.Tim-1 mainly expressed in the Th2 cells,and Tim-4 is the natural ligand,expressed on dendritic cells(DC);Besides,Tim-3 expressed in the Thl cells,and partially expressed on Macrophages(Mφ)and DC cells,Galectin-9(Gal-9)is the ligand of Tim-3.Therefore,in addition to regulating Th immune,because of the express of Tim-4 and Tim-3 proteins on innate immune cells,and TLRs is widely expressed on cells of the innate immune,Tim protein may can regulate the inflammatory response by regulating TLRs signaling pathway.At present,Tim related researches were mainly concentrated in immune related diseases and skin grafts,but very few studies were in IBD.The role of Tim protein and its ligand in the development of IBD,and its relationship with Th immune response and TLRs signaling pathways were still unclear.Therefore,we aimed to study the specific effect of the ligand Tim-4 and Gal-9 in experimental colitis model of mice,and the influence of the ligand on TLR4 signaling pathway in vivo;to illustrate the role of Tim-3 on the LPS activated TLRs signaling pathways in the macrophages in vitro;to elucidate gut microbiota changes in different colitis model and the effect of Gal-9.Based on these studies,we aimed to explore the role of Tim related ligand in UC and CD,and its regulation mechanism,to provide theoretical and experimental basis for the effective prevention and control of IBD.Methods:I.Effect and mechanism of Tim-1 signaling pathway on experimental colitis,and the role of Tim-4 intervention(1)Experimental groups:Fifty Balb/c mice were randomly divided into five groups(n=10 in each)as follows:①Control group②TNBS model group:TNBS model+PBS i.p.③TNBS+Tim-4 group:TNBS model+Tim-4 i.p.④DSS model group:DSS model+PBS i.p.⑤DSS+Tim-4 group:DSS model+Tim-4 i.p.(2)Establishment of experimental colitis models:①Establishment of TNBS-induced colitis:Balb/c mice were fast but drink freely for 24 hours,with ethyl ether anesthesia,and enemaed with 2.0mg TNBS/ethanol(50%)enema.②Establishment of DSS-induced colitis:Balb/c mice were given 5%DSS for drinking ad-libitum for 7 days.(3)Administration of Tim-4 intervention:Third days after modeling,lOug Tim-4/0.4ml PBS i.p.was given to mice for a period of 5 days.The mice in the model group were given 0.4ml PBS i.p.only.(4)Assessment:① Changes of DAI② The degree of pathological changes and inflammation in colon tissues was observed by H&E staining.③ The Tim-1,TLR4 and MyD88 gene expression of colon tissues were measured by Real-time q-PCR④The protein expression of Tim-1,TLR4,MyD88,and NF-κB p65 in colon tissues were detected by immunohistochemistry⑤The protein expression of Tim-1,TLR4,MyD88 in colon tissues were detected by Wes automated western blotting system(ProteinSimple,San Jose,CA)⑥The levels of IL-1β,IFN-y,and IL-6 in serum were determined by multiplex beads enzyme immunoassay.Ⅱ.Effect and mechanism of Tim-3 signaling pathway on experimental colitis,and the role of Gal-9 intervention(1)Experimental groups:Fifty Balb/c mice were randomly divided into five groups(n=10 in each)as follows:①Control group②TNBS model group:TNBS model+deionized water i.p.③TNBS+Gal-9group:TNBS model+Gal-9 i.p.④DSS model group:DSS model+deionized water i.p.⑤DSS+Gal-9 group:DSS model+Gal-9 i.p.(2)Establishment of experimental colitis models:As mentioned above in Part I.(3)Administration of Gal-9 intervention:Third days after modeling,10ug Gal-9/0.4ml deionized water i.p.was given to mice for a period of 5 days.The mice in the model group were given 0.4ml deionized water i.p.only.(4)Assessment:①The Tim-3 gene expression of colon tissues were measured by Real-time q-PCR② The protein expression of Tim-3 in colon tissues were detected by immunohistochemistry and Wes automated western blotting system(ProteinSimple,San Jose,CA)③Others were the same as mentioned Part I,but except the assessment of Tim-1 expression.III.The effect of Tim-3 on TLR4 signaling pathway in murine macrophages(1)Experimental groups:①Control group:Wild type RAW264.7 cells②Tim3 silencing group:macrophages with shRNA interference③Wild type RAW264.7 cells+100ng/ml LPS④Tim3 silencing group+100ng/ml LPS(2)The protein expression of Tim-3,TLR4,MyD88,pNF-κB p65 were detected by Wes automated western blotting system(ProteinSimple,San Jose,CA)ⅡⅡ.The changes of gut mircobiota in mice with experimental colitis and the intervention effect of Gal-9(1)Experimental groups:As mentioned in Part Ⅱ(2)Collect the fresh fecal samples of the different groups.Extract the bacterial DNA using the QIAamp DNA kit and then amplify the target 16S rRNA segment.(3)After sample mixture,purification and database establishment,it is ready for computer sequencing.Using the high-throughput sequencing platform Miseq from Illumina theV4 region of the samples were undergone pair-end sequencing.(4)When the sequencing has finished,the bioinformatics methods were applied to analyze the structure of the gut microbiota.Result:I.Effect and mechanism of Tim-1 signaling pathway on experimental colitis,and the role of Tim-4 intervention1.Effect of Tim-4 intervention on DAI in experimental colitisDAI in TNBS and DSS model groups were higher than the normal control group,(p<0.01);DAI in the DSS model group increased gradually after modeling,DAI on the seventh day was highest,significantly higher than the normal group(p<0.01).With Tim-4 intervention,DAI in TNBS model group decreased,while increased in the DSS model group,but there was no significant difference(p>0.05),and there was significant difference comparing with the control group.2.Effect of Tim-4 intervention on the pathological and inflammatory change of colon tissues:The pathological change of colonic mucosa by H&E staining showed that the colonic mucosa in model groups had different degrees of congestion,edema,damage,exudation,and inflammatory cell infiltration;but the normal control group showed no obvious abnormalities.Inflammation and pathology scores showed that,the degree of inflammation,lesion depth and crypt damage in TNBS colitis mice were significantly higher than those in normal control group(p<0.01).With Tim-4 intervention,the degree of colonic damage were improved,with significant difference in the inflammation degree(p<0.05).The degree in DSS group increased with Tim-4 intervention,with significant statistical difference compared with normal control group(p<0.01).3.Effect of Tim-4 intervention on TLR4/NF-κB signaling pathway in colonic tissues:(1)Effect of Tim-4 intervention on change of Tim-1 expression:RT-PCR results showed that Tim-1 mRNA expression in both the TNBS and DSS model groups was up-regulated,with significant difference from the normal control group in DSS group(p<0.01).With Tim-4 intervention,Tim-1 expression was significantly increased in TNBS group,with significant difference compared with the normal group(p<0.05);but without significant decrease in the DSS group.IHC results showed that Tim-lexpression of the inflammatory cells model groups had different degrees in the cytoplasm and on the membrane;and there were negative or weakly positive expression in normal control group.A rapid histological score for the semi-quantitative assessment of colonic mucosa showed that,compared with normal control group,Tim-1 in TNBS and DSS model groups were up-regulated,with significant increase in DSS model group(p<0.01);With Tim-4 intervention,the expression of Tim-1 in TNBS group increased,but the expression of Tim-lprotein in DSS group is decreased(p>0.05).Wes automated western blotting test results showed that the expression of Tim-1 protein in colonic mucosa of both the TNBS model group and DSS group was significantly increased(p<0.01);After Tim-4 intervention,the expression of Tim-1 was up regulated.Compared with the normal group,the expression of Tim-1 protein in colonic mucosa of DSS colitis model group was increased(p<0.05);After Tim-4 intervention,the expression of Tim-1 decreased significantly(p<0.01).(2)Effect of Tim-4 intervention on change of TLR4 expression:RT-PCR results showed that TLR4 mRNA expression in both the TNBS and DSS model groups was up-regulated,with significant difference from the normal control group(p<0.05,p<0.01).With Tim-4 intervention,TLR4 expression was significantly decreased in TNBS group,without significant difference compared with the normal group(p>0.05);but TLR4 expression in the DSS group still increased,with significant difference from the normal group(p<0.01).IHC results showed that TLR4 expression of the gland cells and inflammatory cells model groups had different degrees in the cytoplasm and on the membrane;and there were negative or weakly positive expression in normal control group.Compared with normal control group,TLR4 in TNBS and DSS model groups were up-regulated(p<0.01);With Tim-4 intervention,the expression of TLR4 in TNBS group decreased(p<0.05),but the expression of TLR4 protein in DSS group is still in an upward trend(p>0.05).Wes automated western blotting test results showed that the expression of TLR4 protein in colonic mucosa of TNBS model group was significantly increased(p<0.01);After Tim-4 intervention,the expression of TLR4 was down-regulated,and there was significant difference between the two groups(p<0.01).Compared with the normal group,the expression of TLR4 protein in colonic mucosa of DSS colitis model group was increased(p<0.05);After Tim-4 intervention,the expression of TLR4 was significantly higher than that of normal control group(p<0.01).(3)Effect of Tim-4 intervention on change of MyD88 expression:RT-PCR results showed that MyD88 mRNA expression in both the TNBS and DSS model groups was up-regulated,with significant difference from the normal control group(p<0.01).With Tim-4 intervention,MyD88expression was significantly decreased in TNBS group,but increased in the DSS group,with significant difference(p<0.05,p<0.01).IHC results showed that MyD88 expression of the gland cells and inflammatory cells model groups had different degrees in the cytoplasm and on the membrane;and there were negative or weakly positive expression in normal control group.MyD88in TNBS and DSS model groups were up-regulated(p<0.01);With Tim-4 intervention,the expression of MyD88in TNBS group decreased(p<0.05),but the expression of MyD88 protein in DSS group is still in an upward trend(p>0.05).Wes automated western blotting test results showed that the expression of MyD88 protein in colonic mucosa of TNBS model group was significantly increased(p<0.01);After Tim-4 intervention,the expression of MyD88 was down-regulated(p>0.05).Compared with the normal group,the expression of MyD88 protein in colonic mucosa of DSS colitis model group was increased(p<0.05);After Tim-4 intervention,the expression of MyD88 was significantly higher than that of normal control group(p<0.05).(4)Effect of Tim-4 intervention on change of NF-κB p65expression:IHC results showed that NF-κB p65 expression of the gland cells and inflammatory cells in model groups had different degrees in the nuclear andcytoplasm;and there were staining the cytoplasmic,but negative or weakly positive expression of nucleus staining in normal control group.Compared with normal control group,NF-κB p65 in TNBS and DSS model groups were up-regulated(p<0.01);With Tim-4 intervention,the expression of NF-κB p65 in TNBS group decreased(p<0.05),but the expression of NF-κB p65 protein in DSS group is still in an upward trend(p>0.05).4.Effect of Tim-4 intervention on the levels of IL-1β,IFN-γ,IL-6,in serum:Compared with the normal control group,the release of IL-1β,IFN-γ,IL-6 in TNBS model and DSS model groups increased;With Tim-4 intervention,cytokine release in TNBS group significantly reduced,and the release level of IFN-y reduced significantly(p<0.01,p<0.05),but the cytokine release in DSS group increased(p>0.05).II.Effect and mechanism of Tim-3 signaling pathway on experimental colitis,and the role of Gal-9 intervention1.Effect of Gal-9 intervention on DAI in experimental colitisDAI in TNBS and DSS model groups were higher than the normal control group,(p<0.01);With Gal-9 intervention,DAI in TNBS model group decreased(p<0.05),while increased in the DSS model group(p>0.05).2.Effect of Gal-9 intervention on the pathological and inflammatory change of colon tissues:Inflammation and pathology scores showed that,the degree of inflammation,lesion depth and crypt damage in TNBS and DSS colitis mice were significantly higher than those in normal control group(p<0.01).With Gal-9 intervention,the degree of colonic damage were improved(p<0.05).The degree in DSS group increased with Gal-9 intervention,with significant statistical difference compared with normal control group(p<0.01).3.Effect of Gal-9 intervention on TLR4/NF-κB signaling pathway in colonic tissues:(1)Effect of Gal-9 intervention on change of Tim-3 expression:RT-PCR results showed that Tim-3 mRNA expression in the TNBS model group was down-regulated,WithGal-9 intervention,Tim-3expression was significantly increased(p<0.01);The expression of Tim-3 in the DSS model group was not significantly different from that of the normal control group,and the expression of Tim-3 increased with Gal-9 intervention,and significantly higher than that in the control group(p<0.05).IHC results showed that Tim-lexpression of the inflammatory cells inmodel groups had different degrees in the cytoplasm and on the membrane;and there were negative or weakly positive expression in normal control group.Compared with normal control group,Tim-3in TNBS model groups were down-regulated(p<0.01),but without significant change in DSS model group;WithGal-9 intervention,the expression of Tim-1 in both the TNBS and DSS group increased,with significant difference in the TNBS group(p<0.01).Wes automated western blotting test results showed that the Tim-3 expression in TNBS model group decreased,with the intervention of Gal-9,Tim-3 was up-regulated(p<0.05);Tim-3 expression in DSS group had no obvious change,but the expression increased with the intervention of Gal-9,and significantly different from the normal control group(p<0.05).(2)Effect of Gal-9 intervention on change of TLR4 expression:RT-PCR results showed that TLR4 mRNA expression in both the TNBS and DSS model groups was up-regulated,with significant difference from the normal control group(p<0.05).WithGal-9 intervention,TLR4 expression was significantly decreased in TNBS group(p<0.05);but TLR4 expression in the DSS group still increased,with significant difference from the normal group(p<0.01).IHC results showed that compared with normal control group,TLR4 in TNBS and DSS model groups were up-regulated(p<0.01);WithGal-9 intervention,the expression of TLR4 in TNBS group decreased(p<0.05),but the expression of TLR4 protein in DSS group is still in an upward trend(p>0.05).Wes automated western blotting test results showed that the expression of TLR4 protein in colonic mucosa of TNBS model group was significantly increased(p<0.01);After Gal-9 intervention,the expression of TLR4 was down-regulated(p<0.05).Compared with the normal group,the expression of TLR4 protein in colonic mucosa of DSS colitis model group was increased(p<0.05);After Gal-9intervention,the expression of TLR4 was significantly higher than that of normal control group(p<0.01).(3)Effect of Gal-9 intervention on change of MyD88 expression:RT-PCR results showed that MyD88 mRNA expression in both the TNBS and DSS model groups was up-regulated,with significant difference from the normal control group(p<0.01,p<0.05).WithGal-9 intervention,MyD88expression was significantly decreased in TNBS group,but increased in the DSS group.IHC results showed that,compared with normal control group,MyD88in TNBS and DSS model groups were up-regulated(p<0.01);WithGal-9 intervention,the expression of MyD88in TNBS group decreased(p<0.05),but the expression of MyD88 protein in DSS group is still in an upward trend(p>0.05).Wes automated western blotting test results showed that the expression of MyD88 protein in colonic mucosa of TNBS and DSS model group was significantly increased(p<0.05);After Gal-9 intervention,the expression of MyD88 was down-regulated in TNBS group,but was significantly higher in the DSS group.(4)Effect of Gal-9 intervention on change of NF-κB p65expression:IHC results showed that compared with normal control group,NF-κB p65 in TNBS and DSS model groups were up-regulated(p<0.01);WithGal-9 intervention,the expression of NF-κB p65 in TNBS group decreased(p<0.01),but the expression of NF-κB p65 protein in DSS group is still in an upward trend(p>0.05).4.Effect of Gal-9 intervention on the levels of IL-1β,IFN-γ,IL-6 in serum:Compared with the normal control group,the release of IL-1β,IFN-γ,IL-6 in TNBS model and DSS model groups increased;With Gal-9 intervention,cytokine release in TNBS group significantly reduced,but the cytokine release in DSS group increased,with significant increase in IL-6.Ⅲ.Effects of Tim-3 silencing on TLR4 signaling pathway in macrophage RAW264.71.The influence of LPS on TLRs signaling and Tim-3 in macrophage cell strain Raw264.7When exposed to LPS,TLR4 and MyD88 mRNA increased,so LPS stimulation can activate TLR4 signaling pathway in macrophages.According to the experimental results,TLR4/NF-κB signaling pathway proteins were detected after stimulated by 100ng/ml LPS for 24h.2.The influence of Tim-3 silencing on the TLR4 signaling pathway in macrophage cell strain Raw264.7LPS stimulation increased the protein expressions of TLR4,MyD88 and pNF-κ Bp65 in wild type macrophages(p<0.05);and the expression of TLR4 and pNF-KBp65increased significantly in shRNA silencing macrophage cells(p<0.01),with slightly increased MyD88 protein expression(p>0.05).In the absence of LPS,the protein expressions of TLR4,MyD88 and pNF-κBp65 protein between the shRNA Tim-3 silencing group and wild type macrophages had no significant difference(p>0.05);But with LPS stimulation,the protein TLR4 and pNF-KBp65 expressions in the Tim-3 silencing cell was significantly higher than that of wild type macrophages(p<0.01),without significant difference about MyD88 protein expression(p>0.05);In addition,in the presence of LPS,the expression of Tim-3 was significantly decreased in wild-type macrophages(p<0.01).ⅡⅡ.Change of gut microbiotain expremental colitis and the intervention effect of Gal-9 with gut microbiota1.Change of gut microbiotain expremental colitisExperimental colitis had a great impact on gut microbiota.The PCoA analysis revealed that thestructure of gut microbiota was significantly different between the normal control group and model groups(the contribution of PC1 was24.59%and the contribution of PC2 was 11.55%).The a-diversity index was different between the groups,with lower chao and shannon index and with higher simpson index in the model groups.The Alpha diversity box showed that the TNBS and DSS models were different from those in the normal group,and the difference between the DSS model and the control group was more significant.At the phylum level,the proportion of sequences assigned to Firmicutes was significantly decreased in the DSS group,but increased in TNBS group,besides,Deferribacteres increased in the DSS group.At the genus level,the proportion of sequences assigned to Rurminococcus and Coprococcus were increased while the reads assigned to Bilophila,Butyricimonas and Paraprevotella were decreased in the TNBS group compared to the normal control group.The proportion of sequences assigned to Bacteroides,Mucispirillu,m,Helicobacter.and Paraprevotella were increased,while the reads assigned to Parabacteroides,Bilophila,Butyricimonas,and Anaeroplasma were decreased in the DSS group compared to the normal control group.There were changes of intestinal flora in mice with experimental colitis.The increase of the genus Ruminococcus is specific flora imbalance in CD.2.Effects of Gal-9 on gut microbiota in mice with experimental colitisAfter Gal-9 intervention,the diversity index of intestinal flora in TNBS and DSS mice was significantly improved,including Chao index and Shannon index,while the Simpson index decreased.At the phylum level,no significant difference was found in bacterial abundance before and after Gal-9 intervention(p>0.05);But in the genus level,the relative abundance of Odoribacter in TNBS model was higher in the untreated group(1.98%vs 0.51%,p<0.05);the relative abundance of Oscillospira was higher in DSS model after Gal-9 intervention(6.42%vs 4.04%).Conclusion:1.Tim-1 ligand Tim-4 and Tim-3 ligand Gal-9 could affect the severity of intestinal inflammation in experimental colitis,and have different effects on different colitis models.2.Tim-1 ligand Tim-4 and Tim-3 ligand Gal-9 could regulate the TLR4/NF-κB signaling pathway in experimental colitis,and have different effects on different colitis models.3.Down regulation of Tim-3 on macrophage may exacerbate the inflammatory response induced by LPS,possibly by activating the TLR4/NF-κB signaling pathway.4.The gut microbiota changed in the colitis model.Gal-9 could significantly improve the intestinal flora of TNBS induced colitis model. |
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