| Inflammatory bowel disease(IBD)including Crohn's disease and ulcerative colitis,is a kind of nonspecific bowel disease that etiology and pathogenesis is still not very clear.The intestinal fibrosis is a common and serious complication of IBD,which will develop an intestinal stricture or obstruction.Some data indicated that,40% above CD has the varying extent intestinal obstruction,80% patient finally need the surgery to treat approximately.Obviously,the intestines fibrosis has become the important question.The CD4~+T cell is in the immune system the most important immunity adjustment cell,which regulates in vivo immunity reply and the maintenance immunity balance stable plays the vital role.The helper t cells(Th)include Th1,Th2 and Th17 which is newly discovered cell subsets.Th1 cells secrete IL-2,IFN-gamma and TNF-alpha and other pro-inflammatory mediator to regulate cellular immune responses and Th17 cells secrete cytokines such as IL-17 and IL-6.The current tendency to Th1,Th17 and cells is primarily involved in the CD.The intestinal fibrosis is a complex and dynamic process,which relates to a dysregulated mucosal immune response leads to alterations in intestinal cytokines,then encourages local collagen depositing.The research has showed that,the activated intestinal fibroblasts(IFBs)and intestinal myofibroblasts(IMFs)by the cytokines multiply and synthetize collagen resulting in an increased deposition of extracellular matrix(ECM)until the lumen begins to narrow and the patient complains of obstructive symptoms.The mesenchymal cells activated by cytokines are regulated by signal transduction.TGF-?1/Smad3 is a key signal transduction in process of fibrosis.The researches in vivo and vitro or in hunman and animal have proved thatTGF-?1/Smad3 is upregulated in process of fibrosis,and meanwhile the fibrosis induced by mesenchymal cells becomes to strengthen.Tumor necrosis factor ligand-related molecule1A(TLl A)is a novel member of TNF-superfamily,which can enhance the activity of Th1 cytokines,and promote the occurrence of inflammatory response and intestinal fibrosis,and induce the occurrence of fibrous stenosis.Unfortunately,there is currently no report focusing on the relation of TL1 A and IL-17,IFN-?.Studies have shown that IL-17 plays a dual inflammatory and protective role in the pathogenesis of inflammatory bowel disease and it may play an important role in fibrosis process.Th1 cells play a role in anti fibrosis by secreting interferon gamma.It has been shown that INF-? can inhibit the proliferation and migration of fibroblasts and it can delay intestinal fibrosis.TL1A is one of IBD susceptibility genes,and TL1 A over-expressed in lymphoid and myeloid cells would lead to intestinal inflammation and fibrosis.These results suggest that TL1 A plays a crucial role in T cells and APCs of the intestinal mucosa in IBD,upregulation of TL1 A expression can promote inflammation and fibrosis of intestinal mucosa.Intestinal fibrosis is a complex and dynamic process.The mechanism of intestinal fibrosis is not clear in inflammatory bowel disease,which may be related to the interaction of inflammatory cells,cytokines and intestinal stromal cells.Objective: To establish experimental colitis induced by DSS for to elucidate the role of TL1 A in colitis associated intestinal fibrosis induced by intrarectal administration of DSS.Methods:1 LCK-CD2-TL1A-GFP transgenic(Tg)mice were identified and selected by RT real-time PCR.2 Chronic colitis was induced by administration of dextran sodium sulfate(DSS)drinking water.WT and lymphoid TL1A-Tg C57BL/6 mice(20-25 g,8-10 weeks old)were grouped as follows: Control group(n=6,DSS group(n=6),The Mice in control group received regular water,the mice in DSS group received 2% DSS drinking water on day 1~5,8~12,15~19,22~26,27 and 28,distilled water during intermission,day-29 was the sacrifice day.3 Severity of colitis was observed by the mental state,body weight(BW)changes,hair gloss,appetite,stool,fecal occult blood(by benzidine method)and hematochezia situation.4 Colon histology changes and pathology score,Myeloperoxidase(MPO)were measured in each group.5 Immunofluorescence technique was used to detect the localization and expression of CD4~+T lymphocytes,IFN-?,and IL-17 in the intestinal tissue of mice,and to evaluate the changes of immune function of CD4~+IFN-?+ and CD4~+IL-17+in the process of intestinal fibrosis.6 Cells were extracted from MLN and spleen then counted one by one.And the percentage of CD4~+IFN-?+ and CD4~+IL-17+cells was measured by flow cytometry.7 The concentrations of CD4~+IFN-?+ and CD4~+IL-17+ from LPMC,MLN monocytes,spleen monocytes supernatant and serum were tested by enzyme-linked immune sorbent assay(ELISA).8 Masson staining and Sirius red staining was used to detect the thickness of collagen fibers in colon tissue and evaluate the degree of fibrosis.9 The expressions of collagen I,collagen III,Vimentin,?-SMA and TGF-?1/Smad3 were dected by immunohistochemistry.Results1 The TL1 A transgenic mice were identified by PCR.The TL1 A DNA located at 192 bp in Tg mice,while it did not express at 192 bp in WT mice.2 The results of severity of chronic colitis showed that compared to that in the control group,body weight had distinctly greater loss,DAI score were higher,colon length became shorter,histological score(WT mice:11.75±0.50 vs 00.00±0.00,P<0.05;Tg mice: 14.00±1.05 vs 0.00±0.00,P<0.05)and MPO activity were much higher in DSS group(P<0.05).Compared to WT mice,the colitis bowel inflammation degree of Tg mice was higher(P<0.05).3 Compared to the control group,the average optical density of DSS in WT and Tg mice was significantly higher than that in IL-17 group(WT mice:84.528 U/g±11.967 U/g vs 75.493 U/g±15.095 U/g,P<0.05;Tg mice: 126.426U/g±15.939 U/g vs 81.950 U/g±14.655 U/g,P<0.05).Compared to the control group,the average optical density of DSS in WT and Tg mice was significantly higher than that in IFN-? group(WT mice: 81.978 U/g±11.967U/g vs 61.248 U/g±10.908 U/g,P<0.05;Tg mice: 115.428 U/g±13.933 U/g vs94.562 U/g±17.030 U/g,P<0.05).4 Compared to the control group,WT and Tg mice had more collagen deposition and more severe fibrosis in DSS group(WT mice: 0.36±0.016 vs0.27±0.009,P<0.05;Tg mice: 0.47±0.05 vs 0.28±0.009,P<0.05).5 Compared to the control group,the percentage of CD4~+IFN-?+ and CD4~+IL-17+ cells in total CD4~+T cells raised in DSS group(MLN:CD4~+IFN-?+: 8.04%±0.44% vs 6.71%±0.26%,P<0.05;MLN: CD4~+IL-17+:5.61%±0.19% vs 2.47%±0.28%,P<0.05);Spleen: CD4~+IFN-?+:10.45%±0.66% vs 7.29%±0.58%,P<0.05;Spleen: CD4~+IL-17+: 3.33%±0.33% vs 2.10%±0.36%,P<0.05).6 Inflammatory markers of IFN-?,IL-17 in LPMC,monocytes cells from MLN and spleen culture supernatants and serum were tested by ELISA.Compared to the control group,the IFN-?,IL-17 level raised in DSS group(WT mice: the concentrations of IFN-? from LPMC: 1,277.484±9.211 pg/m L vs 56.994±4.960 pg/m L,P<0.05;the concentrations of IL-17 from LPMC:31.628±1.055 pg/m L vs 30.800 ±5.714 pg/m L,P<0.05;Tg mice: the concentrations of IFN-? from LPMC: 1,382.315±152.343 pg/m L vs120.444±35.144 pg/m L,P<0.05;the concentrations of IL-17 from LPMC:36.143±8.630 pg/m L vs 35.744±1.446 pg/m L,P<0.05).7 The thickness and collagen deposition of colon were increased in the Tg group,which were much more severity in WT group(0.47±0.05 vs0.36±0.016,P<0.05;0.40±0.01 vs 0.32±0.015,P<0.05).The data detected by immunohistochemistry showed tha expressions of Vimentin,?-SMA,collagen I,and collagen III were increased.Conclusion:1 TL1 A promoted the production of IFN-?,IL-17 and promoted thechronic experimental colitis and intestinal fibrosis.2 TL1 A promotes the the proliferation and differentiation of fibroblasts and I,III Collagen synthesis,and then aggravates the intestinal fibrosis degree of chronic experimental colitis mice.3 TL1 A promoted the intestinal fibrosis by upregulating the levels of TGF-?1and Smad3. |
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