The Therapeutic Effect And Mechanism Of Low Molecular Weight Heparin In A Model Of Experimental Mouse Colitis

The first part:Establishment of Colitis Model that Acute Colitis Progresses to Chronicity in Mice Induced with Dextran Sulfate SodiumBackground:Ulcerrative colitis(UC) and Crohn's disease(CD) are the major chronic inflammatory bowel diseases(IBD) affecting the gastrointestinal tract in humans.The etiology of both CD anduC still remains largelyunclear.However,in recent years,epidemiologic and genetic studies in man and particularly,in IBD-related animal models suggested that a combination of genetic susceptibility factors and altered immune response driven by microbial factors in the enteric environment contributes to the initiation and chronification of these diseases.When chosen appropriately,these models can beused to investigate pathophysiological mechanisms and they are valuable tools to test emerging therapeutic strategies in the preclinical phase.As the onset of inflammation is immediate and the procedure is relatively straightforward,chemically induced models of intestinal inflammation belong to the most commonlyused IBD animalmodels.Although they have,like all othermodels, limitations,they resemble in some important immunological and histopathological aspects IBDs in humans.Aims:To establish a model that acute colitis progresses to chronicity with DSS, and explore its pathological characteristics.Methods:Forty-eight C57BL/6 mice were divided into model group and control group.Model group were fed with 3%DSS solution ad libitum for 5 days followed by distilled water for 3 weeks to induce a colitis model,which progressed from the acute phase to chronic inflammation Control group was only fed with distilled water for 26 days.The disease activity index(DAI) of all the mice was evaluated daily.The mice were killed on 5th,12th,19th and 26th day,macroscopic and histopathological score,inflammatory score of the colon were also evaluated.Results:At the 5th day of the model,watery diarrhea,fecal blood,loss of body weight were seen in model group.At this stage,the histopathological changes in the colon showed multipleulcer foci with predominant neutrophil infiltration into the mucosa and submucosa,marked loss of crypts and reduction of goblet cells.3 weeks after DSS removal,the symptoms as diarrhea and bloody stool diminished,but the colon histopathological analysis showed more localized smallulcer foli,signs of surface epithelium regeneration irregularly,infiltration of more number of inflammation cells.The lesions in the distal colon were more serious than those which in the proximal and middle colon in both acute and chronic phases.Conclusions:C57BL/6 mice fed with 3%DSS solution for 5 days followed by distilled water for 3 weeks may establish an ideal colitis model,of which clinical and pathological changes are similar with human colitis,provides a new model for the further studies of colitis. The second part:The therapeutic effect and mechanism of low molecular weight Heparin in a Model of experimental mouse colitis.Background:Beyond its well-known anticoagulant activity,unfractioned heparin also exhibits a broad spectrum of immunomodulating and anti-inflamm atory properties in animal models and in humans.However,their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated.In inflammatory bowel disease,the normal healing process during restitution can be disrupted by the inflammation.This may be due to loss of growth factors,cell adhesion molecules,or both,resulting in a reduced rate of healing.Heparin treatment aids healing inulcerative colitis.Day et al.tested the hypothesis that syndecan-1 as the predominant epithelial syndecan,could contribute to the healing process through its function as a coreceptor for FGF-2.A marked reduction of syndecan-1 immunostaining was found in reparative epithelium from inflammatory bowel disease patients.In vitro experiments demonstrated that gastrointestinal epithelial cells displayed reduced proliferative response to FGF-2 after enzymatic removal of heparan sulfate.The FGF-2 response could be completely restored by the addition of heparin,giving strong support in favor of the postulated function of syndecan-1.Inflammatory bowel disease is also characterized by intestinal permeability changes and large numbers of neutronphils trafficking through the epithelium(154). Epithelial cells are a source of a number of cytokines and express several cytokine receptors,whereas transepithelial migration of neutrophils can be promoted by chemoattractants such as leukotriene B4,platelet activating factor and N-formyl-peptides.Furuta et al.have suggested that chemokines may associate with epithelial surfaces and activate polymorphonuclear neutrophils(PMN) in hypoxia-induced mucosal disorders.They found that epithelial hypoxia induced surface IL-8 expression and expression of the HSPG perlecan,and possibly other cell-surface HSPGs.Hypoxia-induced IL-8 expression decreased significantly after treatment with heparinaseâ…¢.Disaccharides derived from heparin and heparan sulfate regulate proinflammatory mediator secretion from intestinal epithelial cells.It is tempting to speculate that the changes in syndecan-1 expression occurring in inflammatory bowel disease might contribute to changes in neutrophil adhesion and transmigration via a modification of chemokine action.Aims:To investigated whether a low dose of low-molecular-weight heparin-enoxaparin (Clexane,AVENTIS Pharma Specialites,France)ameliorates the inflammatory response in DSS-induced experimental colitis.To observe the effects of low molecular weight heparin on interleukin-1β,interleukin-10 and Sydecan-1 in a model that acute colitis progresses to chronicity induced with DSS in C57BL/6 mice, and define the mechanisms of its anti-inflammatory effects.Methods:1.Fifty-four C57BL/6 mice were divided randomly into three groups. Model group and treatment group were fed with 3%DSS solution ad libitum for 5 days followed by distilled water for 3 weeks to induce a colitis model,which progressed from the acute phase to chronic inflammation.Control group was only fed with distilled water for 26 days.Eighteen mice in treatment group were given enoxaparin at the dose of 5μg / mice through subcutaneous injection once a day,for 20 day.Thirty-six mice in model group and control group received the same volume of saline instead of enoxaparin once aday.2.The disease activity index(DAI) of all the mice,including body weight loss, stool consistency and blood in feces were examed daily.The mice were killed on 5, 12th,19th,and 26th day,colonic tissue of mice were collected,macroscopic and histopathological score,inflammatory score of the colon were also evaluated.3.The mRNA expression of IL-1β,IL-10 and Syndecan-1 in colonic mucosa were assessed by RT-PCR.Taking appropriate mouse colon tissue,adding 1 ml Trizol on ice after homogenating,after standing 5 minutes at room temperature, adding 0.2 mL chloroform,concussion,five minutes after standing at room temperature,12000 g 4℃centrifuging for 15 minutes.Supernatant was moved to a new centrifuge tube,adding the same volume of isopropanol with the supernatant, shocking,standing for 10 minutes at room temperature.12000 g 4℃centrifuging for 10 minutes,disposing supernatant,adding to the precipitation 1 mL 75%ethanol precipitation cleansing,12000 g 4℃centrifuging five minutes,the precipitation was put at room temperature to dry.By adding DEPC treated water 10uL～20uL to dissolve RNA.OD₂₆₀/OD₂₈₀ absorbance was measured by the ratio of RNA concentration. Counting the treatment group,the model group and the normal control group total RNA by RT-PCR.Mixed appropriate PCR product with 5×loading buffer, taking a electrophoresis run at a 1.5%agarose,80 v voltage.4.The expression of Syndecan-1 protein in colonic mucosa were also evaluated by immunohistochemistry.Dewaxing paraffin sections to water,high-pressure paraffin sections antigen retrievaling,dropping 3%H₂O₂,and incubated at room temperature for 10 minutes to eliminate endogenous peroxidase activity.Dropping the first antibody,incubated 2 hours at room temperature.Dropping the second antibody with modest biotin-labeled,37℃incubated 30 minutes.DAB coloring,fully tap water rinsing,staining,dehydrating,transparenting,and mounting.Results:1.Compared with the control group,inflammation score of the model group and the treatment group was significantly increased,while the treatment group inflammation score was lower than the model group after the molecular weight heparin treatment.An independent sample t-test compared the means of the two independent groups,at the 20th day,the difference was significant(t = 2.712,P = 0.022).Compared with the control group,histologic score of the model group and the treatment group was significantly higher,at different time points an independent sample t-test compared the means of two groups respectively,the treatment group score after the heparin treatment at all time points was lower than the model group, the difference is significant,at the 5th,12th,20th day,tvalues were 2.390,2.907,3.101,P values were 0.038,0.016,0.011.2.Compared with the control group,the IL-1β,IL-10 mRNA expression of the model group and the treatment group was significantly increased,and the IL-1β, IL-10 mRNA expression of the model group after heparin treatment was significantly decreased,and had interaction effects between the time factor and the treatment factor (F 54.796,19.601 respectively,P = 0.000),At different time points an independent sample t-test compared the means of the two independent groups respectively,the difference is statistically significant.The t values of IL-1βmRNA expression' t test at the 5th day,12th day,20th day were 2.522,20.511,25.175 respectively,P values were 0.030,0.000,0.000 respectively;at the 5th day,12th day,20th day the t values of IL-10 mRNA expression' t test were 2.964,14.532,10.730,P values were 0.014,0.000,0.000.3.Compared with the control group,Sydecan-1 mRNA and protein expression of the model group and the treatment group were lower at the 5th day(P = 0.000); but At the 12th day,20th day,Sydecan-1 mRNA and protein expression of the treatment group were higer than the model group after treatment with heparin,and had interaction effects between the treatment factor and the time factor(F values were 7.782,5.313,P values were 0.000,0.001),at different time points among the three groups were done One-Way ANOVA,the difference was significant.Done an One-Way ANOVA to compare the Sydecan-1 mRNA expression of the three independent groups,at the 5th day,12th day,20th day the F value were 36.046,46.343,13.259,P value were 0.000;at the 5th day,12th day,20th day,as a result of Sydecan- 1 protein expression'One-Way ANOVA,the F value were 16.270, 38.074,16.462 respectively,P value were 0.000.Conclusions:In the process of acute colitis induced by DSS progressed to chronicity in mice,low molecular weight heparin had anti-inflammatory effects mighe be through down-regulated expressions of IL-1β.The application of low-molecular-weight heparin could reduce intestinal mucosal injury,and might been adopted to replace lost Syndecan-1 from the cell surface,thus speedingup restoration process,and promoting healing of DSS-induced colitis in mice.