The Study Of The Relationship Between Syndecan-1 And Inflammatory Bowel Disease

Background&objectivesSyndecan-1(CD138) which mainly expressed on the surface of epithelial cells physiologically is a member of heparan sulfate proteoglycan family.It is composed of transmembrane core protein,heparan sulfate(HS) chains and chondroitin sulfatase (CS) chains which both bind with extracellular region of Syndecan-1 core protein. The core protein is composed of intracellular region,transmembrane domain and extracellular region.Intact HS chains which are main parts of Syndecan-1 to act effectively can bind with cytokines,growth factors,chemotatic factors,extracellular matrix and heparin binding proteins on the surface of bacterial.They play important roles in maintaining cell morphous,promoting tissue repair,regulating immune function and so on.Transmembrane domain and intracellular region show high conservatism,while the extracellular region is variable because of its different binding sites with which different HS chains and CS chains bind on the core protein.The transmembrane domain participates in the formation of Syndecan-1 dimer and Syndecan-1 polymer.The intracellular region,characterized by its binding with cystoskeleton actin,participates in the formation of cystoskeleton.The core protein and HS chains are important composition of tight junction of epithelial cells and play important roles in maintaining the function of mucosa barrier.Physiologically,the majority of Syndecans-1 are expressed on the cell membrane,only a few of them cut by some excisionase(such as matrix metalloproteinase-7 and so on) shed and become free Syndecan-1 functional extracellular domain.Meanwhile,new Syndecans-1 are synthesized to replenish ones cut by excisionase.IL-1 which secreted by mononuclear cells for example can enhance the expression of matrix metalloproteinase-7.The free Syndecans-1 contain intact HS chains by which Syndecan-1 to effect.Syndecan-1 plays important roles in many steps of inflammatory reaction such as enhancing mitochysis mediated by FGF-2,regulating the interaction between leukocytes and endothelial cells,binding with chemotatic factors to induce neutrophils coming to inflammatory sites, regulating the immigration of T cells,enhancing the virulence of bacterial pathogen and so on.While,Syndecan-1 binding with cellular membrane is an important composition of enteric mucosa barrier.It can prevent inflammatory factors effecting on the intestinal.Inflammatory bowel disease(IBD) including ulcerative colitis(UC) and Crohn's disease(CD) belongs to colon inflammation resulting from nonspecific immune reaction,but the exact etiopathogenisis of IBD has not been known.IBD may be associated with environmental agents effecting on genetic susceptible population. Studies show that the disorganization of tight junction of enteric epithelium cells causes increaced intestinal tract permeability,then environmental agents effect on intestinal tract and activate innate immunity cells of intestinal tract,which induce immunity cells to secrete inflammatory factors such as IL-1 and so on,thus lead to inflammation of intestinal tract ultimately.At present,there is not specific index in diagnosis and monitoring of IBD and also be lack of effective medicine to cure it. Scholars coming from overseas have studied the relationship between Syndecan-1 and common inflammation,but there are few reports about the relationship between Syndecan-1 and IBD.We presume that Syndecan-1 may play important roles in development of IBD in light of the intimate relationship between inflammation and Syndecan-1 as well as Syndecan-1 being an important composition of enteric mucosa barrier.Because IL-1 can enhance the synthesis of matrix metalloproteinase-7 which can dissect Syndecan-1 resulting in shedding of Syndecan-1,we presume that IL-1 may regulate effects of Syndecan-1 in IBD.Mice colitis induced by dextran sulfate sodium(DSS) and trinitro-benzene-sulfonic acid(TNBS) are regarded as animal models for the study of IBD generally.Colitis induced by DSS and TNBS are similar to human ulcerative colitis and Crohn's disease respectively.As the background showed above,we approached the changes of Syndecan-1 and its significance in two kinds of models.1.Inducing two different kinds of IBD in mice by DSS and TNBS respectively.2.Detecting the changes of Syndecan-1 protein expressed in colon mucosa and free Syndecan-1 protein in serum of two different kinds of IBD mice.3.Detecting the changes of Syndecan-1,IL-1,MMP-7 mRNA expressed in two different kinds of IBD mice.Methods1.Preparation of IBD by DSS in mice::A total of 40 BALB/c mice were divided into experimental group(including model group 1,2,3) and control(10 mice per group). Mice in control were given normal diet and distilled water.Mice in model groups were given 4%DSS solution(prepared by distilled water) for 7 days,then distilled water for 10 days followed by 4%DSS solution(prepared by distilled water) for 7 days again.Mice in model group 1,2,3 were executed at the 8th,18th and 25th day respectively,but mice in control were executed at the 8th day.General and morphology changes of colon were observed.2.Preparation of IBD by TNBS in mice: A total of 50 BALB/c mice were divided into into experimental group(including model group 1,2,3,4) and control(10 mice per group).100μl TNBS-ethanol solution was administered rectally to each mice in model groups and 100μl physiological salt was administered rectally to each mice in control.Mice in model group 1,2,3,4 were executed at the 3th,7th,14th and 21th day respectively and mice in normal control were executed at the 3th day.General and morphology changes of colon were observed.3.Detecting Syndecan-1 protein expressed in colon mucosa of mice by immunohistochemistry.4.Detecting Syndecan-1,IL-1,MMP-7 mRNA expressed in colon mucosa of mice by RT-PCR.5.Detecting free Syndecan-1 protein in serum of mice by Dot-Blot.6.Data analyses:All the data were analyzed by SPSS 13.0.Data are expressed as mean±SD if they obeyed normality distribution and expressed as median if they did not obey normality distribution.Statistical significance was tested using One-way ANOVA,Two Independent-Samples T Test,K Independent-Samples Nonparametric Test and Two-Independent Samples Nonparametric Test.Statistical significance was set at P values less than.05.Results1.Disease activity index(DAI) scores of mice in DSS and TNBS group were higher than that in control(χ~2=35.058,P＜0.001;χ~2=43.053,P＜0.001 ).Mice executed at the 8th day after DSS administration and the 3th day after TNBS administration obtained the highest scores,and scores gradually decreased with time.Toxic megacolon appeared in a few mice in DSS group.2.Histological scores of colon in DSS and TNBS models were obviously higher than those of control(χ~2=36.828, P＜0.001;χ~2=43.969,P＜0.001).Mice executed at the 8th day after DSS administration and the 3th day after TNBS administration obtained the highest scores,and scores gradually decreased with time.3.Syndecan-1 protein expressed in colon mucosa of mice in DSS and TNBS models were obviously lower than that of control(Welch =1893.280,P＜0.001;Welch=2328.127,P＜0.001).Mice executed at the 8th day after DSS administration and the 3th day after TNBS administration displayed the lowest expression levels of Syndecan-1 protein in colon mucosa,and the expression levels of Syndecan-1 protein gradually incraesed with time.4.Syndecan-1 mRNA expressed in colon mucosa of mice in DSS and TNBS models had no statistically significances compared with those of control(F=0.809 P＞0.05;F=0.815,P＞0.05).IL-1 mRNAs expressed in colon mucosa of mice in DSS and TNBS models were higher than those of control group(F=103.833 P＜0.001;F=88.015 P＜0.001).Mice executed at the 8th day after DSS administration and the 3th day after TNBS administration displayed the highest IL-1 mRNA expression levels,and those gradually decreased with time.The expression levels of MMP-7 mRNA in DSS and TNBS models were higher than those of control group(t=-5.098,P＜0.05;t=-6.153,P＜0.05).5.The levels of free Syndecan-1 protein in serum of mice in DSS and TNBS models were higher than those of control(F=32.399 P＜0.001;F=27.881,P＜0.001) Mice executed at the 8th day after DSS administration and the 3th day after TNBS administration displayed the highest protein level of free Syndecan-1 protein in serum, and the levels of that gradually decreased with time.Conclusions1.DSS and TNBS can induce two kinds of IBD mice successfully.Colitis induced by DSS and TNBS are similar to human ulcerative colitis and Crohn's disease respectively.2.Disease activity and pathological change level of colon in DSS (highest at day 8 after administration) and TNBS(highest at day 3 after administration) induced IBD mice gradually lower with time.3.The decreased protein level of Syndecan-1 in colon mucosa has close relationship with the severity of IBD.Syndecan-1 protein levels of colon mucosa in DSS and TNBS-induced IBD mice group are lower than that in control.Syndecan-1 protein level is the lowest when IBD colitis is the most serious(8 days after DSS administration and 3 days after TNBS administration).Then protein levels of Syndecan-1 gradually rise with the lessening of colitis.4.The decreased protein level of Syndecan-1 in colon mucosa do not result from decreased expression level of Syndecan-1 mRNA but from the increased Syndecan-1 shedding in the colon mucosa.Syndecan-1 protein levels in colon mucosa of the DSS and TNBS-induced IBD mice reduced,while the free Syndecan-1 protein levels in the mice serum increased.Meanwile Syndecan-1 mRNA levels in colon mucosa were not significantly different in both groups.The protein levels of free Syndecan-1 in mouse serum are the highest when them in colon mucosa is the lowest and them in mouse serum gradually lessened with them in colon mucosa gradually reducing.5.The increase of Syndecan-1 shedding is associated with the increased mRNA expression of IL-1 and MMP-7.Some studies have confirmed that the expression of MMP-7 can be induced by IL-1,while Syndecan-1 binding with cellular membrane can be dissected by MMP-7 and shed.In this study,when the expression levels of IL-1 in colon mucosa was high,the ones of MMP-7 was also high.At this point,syndecan-1 levels in colon mucosa lowered while free Syndecan-1 levels in the serum increased.These shows that one of the mechanisms of the increased Syndecan-1 shedding is:increased MMP-7 level promotes more Syndecan-1 being cut from cellular membrane by MMP-7,so Syndecan-1 shedding increases.The high expression of MMP-7 may be associated with increased expression of IL-1.6.Free Syndecan-1 in serum may be an index in diagnosis and prognosis of IBD.When IBD was the most serious,the free Syndecan-1 levels in mice serum were the highest;When the former was improving,the latter gradually lowered.