| Crohn's disease(CD)is a chronic relapsing inflammatory disorder characterized by chronic inflammation and mucosal tissue damage of the gastrointestinal tract.Although the precise etiology of Crohn's disease remains unclear,research has thus far demonstrated that CD is caused by a combination of genetic,environmental and immunoregulatory factors.Massive numbers of microorganisms reside in the gut lumen,and the intestinal mucosa constitutes an immunologic organ,which tolerates and defends against harmful organisms.Intestinal barriers also play important role in maintaining the homeostasis of mucosal immune.Increasing evidence suggests that an imbalanced mucosal barrier may play an important role in the pathogenesis and disease progression of CD.When the epithelium is intact,intestinal barrier function is largely defined by the construction and expression of TJ as well as intestinal barrier permeability characteristics.In Crohn's disease,the intestinal barrier at the interface between the intestinal microbiome and the lymphoid tissue plays a critical role in shaping the mucosal immune response.In this situation,the intestinal barrier is impaired,and bacterial products and dietary antigens cross the epithelium and enter the lamina propria.Antigen-presenting cells(APCs)take up foreign materials and regulate the differentiation of T cells.For many years,it has been assumed that Crohn's disease is a T helper 1(Th1)-cell-mediated disease,and a novel subset of IL-17-producing CD4+Th cells.Th17 cells,have more recently been implicated in the pathogenesis of CD.In the most appropriate model of human CD,interleukin-10-gene-deficient(Il10-/-)mice also exhibit increases in CD4+Thl and Th17 cells,which secrete a large number of pro-inflammatory mediators such as tumor necrosis factor-?(TNF-?),IL-1?,interferon-?(IFN-?),and IL-17A.Laquinimod(C19H17ClN2O3)is an immunomodulatory drug that has extensively shown its efficacy in inflammatory and autoimmune disorders,especially multiple sclerosis(MS).Recently,phase ? clinical studies reported that 0.6mg/d laquinimod can prevent relapse of MS and delay the disease process.Laquinimod was developed based on its pro-drug Roquinimex,and the drug adverse events of laquinimod were significantly reduced compared to roquinimex.It was reported that roquinimex can inhibited Dextran sodium sulfate(DSS)induced colitis,and mice with DSS induced colitis was one of common model of Crohn's disease.Laquinimod as a small molecule that concentrates in the peripheral immune system as well as in the central nervous system(CNS),and previous studies reported that laquinimod can be used to treat nephritis.Both MS and nephritis are autoimmune disease,and they have similar pathogenesis and partly similar treatment.The mechanism of laquinimod is not clear enough,and laquinimod has shown immunomodulatory effects by downregulating Thl and Th17 cells in animal model of MS.Preclinical data suggest that laquinimod has a direct inhibitory effect on antigen-presenting cells(APCs)and results in anti-inflammatory T cell polarization manifested by a reduction in the frequencies of pro-inflammatory Th1(IFN-?+CD4+)cells and Th17(IL-17A+CD4+)cells.Studies reported that nuclear factor-kappa B(NF-?B)pathway is overactive in both Crohn's disease patients and animal models.Thus,the proposed mechanism of laquinimod may make it ideally suited to reduce gastrointestinal inflammation.However,the effects of laquinimod on colitis have been heretofore unstudied in animal model of Crohn's disease.In this investigation,we determined whether laquinimod ameliorates established spontaneous colitis in Il10-/-mice,and we attempted to explain the potential mechanisms that account for these effects.In this present study,we used the Il10-/-mice as the animal model of Crohn's disease.After 4-week laquinimod treatment,we investigated the changes of clinical manifestation,pathology,NF-?B pathway,subgroup of CD4+T cells,cytokines,the function of intestinal mucosal barrier and the microbacteria.We focus on investigating the function of laquinimod on Crohn's disease animal model,and supporting theory evidence for clinical use of laquinimod.This thesis is divided into three studies:Part 1:The effect and mechanism of laquinimod for ameliorating spontaneous colitis in Il10-/-mice.Objectives:The aim of this study was to investigate the effect and mechanism of laquinimod for ameliorating spontaneous colitis in Il10-/-mice.Methods:Interleukin-10 knockout mice of 16 weeks on a C57BL/6 background were divided into IL-10 KO group and laquinimod group,and C57BL/6 background wild-type mice were divided into WT group.The Il10-/-mice in the laquinimod group received laquinimod(5mg/kg/d diluted in PBS)treatment third times a week for 4 weeks,whereas the mice in WT and IL-10-KO groups received the same volume of PBS.The weight,stools,hair of mice and disease activity index(DAI)were measured every week.Four weeks after administration,the mice were sacrificed by CO2,and proximal colons were collected for investigate the changes of pathology and NF-?B pathway.Remaining colons were collected to measure the level of CD4+CD45+ T lymphocytes,IFN-y,and IL-17A.Results:Il10-/-mice gained less weight than WT mice,and laquinimod treatment revealed to significantly increase the weight of Il10-/-mice(p<0.05).For DAI,the score was higher in Il10-/-mice compared to WT mice.Laquinimod treatment significantly reduced DAI scores of Il10-/-mice(p<0.05).After the mice were sacrificed,we first collected the proximal colon tissues to test the effects of laquinimod on colitis through an H&E-stained microscopic study(n=6?8 in each group).H&E staining revealed inflammatory cell infiltration in the mucosal of Il10-/mice compared with wild type mice.The inflammatory score was significantly higher in Il10-/-mice of the IL-10-KO group.In the mucosa of Il10-/-mice treated with laquinimod,a significant reduction in colonic inflammation was found with reduced inflammatory cells infiltration and a partially restored glandular architecture compared with the IL-10-KO group.Correspondingly,lower mean inflammation scores were found in Il10-/-mice treated with laquinimod than those treated with PBS(p<0.05).To understand the mechanisms underlying the activity of laquinimod,western blotting of the expression of phosphorylated forms of p65 and I?B in mouse colon was performed among the three groups.The expression levels of phospho-I?B were significantly higher in the Il10-/-mice,and treatment with laquinimod significantly reduced the changes of phospho-I?B(p<0.05).Though the change of phospho-p65 was not significantly prevented by laquinimod,the level of phospho-p65 was still decreased in the laquinimod group.Immunofluorescence staining of non-phosphorylated and phosphorylated forms of p65 was also undertaken.Results revealed that the fluorescence strengthened with some enhanced bright spots in the Il10-/-mice compared to control mice.In contrast,the fluorescence was faint and the bright spots were decreased after laquinimod treatment.Compared with wild-type mice,Il10-/-mice with PBS treatment showed more CD4+CD45+T lymphocytes in the lamina propria,while laquinimod treatment significantly decreased the percentage of CD4+CD45+T lymphocytes in colonic mucosa of Il10-/-mice,resulting in reduced intestinal inflammation(p<0.05).In the Interleukin-10-gene-deficient spontaneous colitis model,we evaluated the expression of typical inflammatory cytokines in colonic tissues.The pro-inflammatory cytokines secreted by Thl and Th17 cells,IFN-?,and IL-17A were significantly increased in Il10-/-mice when compared with wild type mice.In laquinimod treated mice,the cytokines were significantly lower than Il10-/-controls(p<0.05)Conclusion:Il10-/-mice have spontaneous colitis,laquinimod can ameliorate colon colitis,reduce syndrome of Il10-/-mice and inflammation scores.The effect of laquinimod on Crohn's disease colitis is associated with NF-?B pathway and the differentiation of CD4+T lymphocytes.Part 2:Laquinimod ameliorates intestinal epithelial barrier function in interleukin-10 knockout miceObjectives:The aim of this study was to investigate the effect of laquinimod on epithelial barrier function in Il10-/-mice.Methods:Interleukin-10 knockout mice of 16 weeks on a C57BL/6 background were divided into IL-10 KO group and laquinimod group,and C57BL/6 background wild-type mice were divided into WT group.The Il10-/-mice in the laquinimod group received laquinimod(5mg/kg/d diluted in PBS)treatment third times a week for 4 weeks,whereas the mice in WT and IL-10-KO groups received the same volume of PBS.Four weeks after administration,the mice were sacrificed by CO2,and proximal colons were collected for experiments.Results:Intestinal permeability of the colon was measured using an Ussing chamber in vitro.Intestinal permeability to mannitol was significantly increased with a corresponding decrease in electrical resistance in Il10-/-mice compared with WT mice.In laquinimod-treated Il10-/-mice,these changes were largely prevented(p<0.05).To identify whether there were changes in the distribution and expression of apical junctions after laquinimod treatment,immunofluorescence and western blotting of occludin and ZO-1 were performed(p<0.05).Immunofluorescence staining revealed that occludin and ZO-1 were continuously distributed with bright spots along the membrane of the colonic mucosa in WT mice.In the Il10-/-group,the fluorescence was faint and discontinuous,especially in regions with infiltrations of inflammatory cells.Biotin staining ectopic to the lamina propria or deep into the epithelial surface were found,and some of the surface of villi lacked focused staining.By contrast,the distribution and fluorescence intensity of the staining were significantly improved by laquinimod treatment.Western blotting revealed that occludin and ZO-1 protein levels were reduced in Il10-/-mice when compared to WT mice,and laquinimod treatment partly restored occludin and ZO-1 protein levels.Conclusion:Laquinimod can ameliorate the intestinal epithelial barrier of Il10-/-mice,and the effect of laquinimod on Il10-/-mice was associated with intestinal epithelial barrier.Part 3:The effect and mechanism of laquinimod for ameliorating spontaneous colitis of Il10-/-mice in conventional environment.Objectives:The aim of this study was to investigate the effect and mechanism of laquinimod for ameliorating spontaneous colitis of Il10-/-mice in conventional environment.Methods:Interleukin-10 knockout mice of 9 weeks on a C57BL/6 background were divided into control group,conventional environment(CE)group and laquinimod group(n=6?8 in each group).In CE group and laquinimod group,mice were moved from SPF environment to CE environment.The Il10-/-mice in the laquinimod group received laquinimod(5mg/kg/d diluted in PBS)treatment third times a week for 4 weeks,whereas the mice in WT and IL-10-KO groups received the same volume of PBS.The weight,stools,hair of mice and disease activity index(DAI)were measured every week.Four weeks after administration,the mice were sacrificed by CO2,and proximal colons were collected for experiments.In sterile environment,liver,spleen,kidney and mesenteric lymph nodes were collected to conduct LB(Luria-Bertani)bacterial culture,and proximal colons were collected for Fluorescence in situ hybridization(FISH)experiments.In anaerobic environment,adjacent colons were collected to culture bacteria on Man Rogosa Sharpe(MRS)plate and Brain Heart infusion Agar(BHIA)plate,and in aerobic environment,adjacent colons were collected to culture bacteria on blood plate.Results:Il10-/-mice in CE group gained less weight than control mice,and laquinimod treatment revealed to significantly increase the weight of Il10-/-mice in conventional environment(p<0.05).For DAI,the score was higher in Il10-/-mice of CE group compared to control group,laquinimod treatment significantly reduced DAI scores of Il10-/-mice(p<0.05).After the mice were sacrificed,we first collected the proximal colon tissues to test the effects of laquinimod on colitis through an H&E-stained microscopic study.H&E staining revealed inflammatory cell infiltration in the mucosal of mice in CE group compared with control group.The inflammatory score was significantly higher in Il10-/-mice of the CE group.In the mucosa of mice treated with laquinimod,a significant reduction in colonic inflammation was found with reduced inflammatory cells infiltration and a partially restored glandular architecture compared with the CE group.Correspondingly,lower mean inflammation scores were found in Il10-/-mice treated with laquinimod than those treated with PBS(p<0.05).In CE group,the bacteria from liver,spleen,kidney and mesenteric lymph nodes were more than in control group.After laquinimod treatment,bacteria were decreased in Il10-/-mice(p<0.05).More bacteria invade the intestinal barrier more in Il10-/-mice in CE group than in control mice,and more bacteria were found in the upper and lower crypt.Laquinimod treatment revealed to significantly reduced bacteria in the upper and lower crypt of Il10-/-mice in conventional environment(p<0.05).For culture of colon bacteria,the bacteria on BHIA plate and blood plate were increased significantly in mice of the CE group compared to the control group,and the bacteria on MRS plate were decreased significantly in the CE group.Laquinimod treatment significantly reduced bacteria on BHIA plate and blood plate,and increased bacteria on MRS plate(p<0.05).Conclusion:The spontaneous colitis of Il10-/-mice occurred in advance and was severer in conventional environment than in SPF environment.Laquinimod can ameliorate colon colitis,reduce syndrome of Il10-/-mice and inflammation scores.Bacteria translocation and dysbiosis of Il10-/-mice were severer after moved from SPF environment to conventional environment.Laquinimod can prevent the changes in Il10-/-mice. |
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