The Mechanisms Of IL-35 In The DSS-induced Acute Colitis In Mice

We successfully established IBD mice model induced by DSS,and demonstrated the effect of IL-35 on intestinal immune tolerance,and further explored its mechanismPart I The mechanisms of IL-35 in the DSS-induced acute colitis in mice Objective:Inflammatory bowel disease(IBD),including Crohn's disease(CD)and ulcerative colitis(UC),are chronic inflammatory disorders of the gastrointestinal tract that mostoften diagnosed in adolescence and young adulthood.IBD is caused by a dysregulate mucosal immune response to intestinal microflora in genetically predisposed hosts.IBD can present with the classic symptoms of weight loss,abdominal pain,and blood diarrhea.Once IBD is diagnosed,the goal of therapy consist of eliminating symptoms,normalizing quality of life and preventing complications with minimizing the adverse effect of medications.Interleukin 12(IL-12)family of cytokine has emerged as critical regulators of immunity in infectious and autoimmune disease.IL-35 is the newest members of IL-12 family,which involves two subunits of p35 and Ebi3 which also seen in IL-12 and IL-27.It was reported that IL-35 could suppress Teff cell proliferation and Th17 development.Since it can regulate T cells,IL-35 is an important therapeutic target of immunological disorder.However its expression in IBD has not yet explored.The aim was to investigate evaluated the role of IL-35 in mechanism of IBD through evaluate serum and inflamed mucosal levels in IBD rat model.Methods:Female Bal/c mice aged 5-6 weeks were used in this study.They were quarantined for the first 7 days,and then randomized into two groups:DSS group and controls group.All mice were killed in the 7th days.A fasting blood sample was taken from eyes of mice models.The large bowels were cut and flused with saline.Histological examination was performed on paraffin-embedded sections after(H&E)staining.Then through ELISA?RT-PCR?Western Blot,compare the two groups and analyze the expressing of IL-35,STAT1 in mice models.Statistical analysis:the experimental data are presented as mean±standard deviation(mean±SD).statistics comparsion use One-way ANOVA,with P<0.05 as significant difference.SPSS21.0 software is used for statistical analysis.Results:Serum IL-35 concentration was significantly reduced in the IBD model.DSS group had significantly higher Ebi3 and IL-12a gene expression compared with control group(P<0.05).Western blot analysis showed a significant upregulation of STAT1,IL-35(P<0.05)in inflamed mucosa as controls.Conclusions:The over-expressed and enhanced production of IL-35,STATlin colonic mucosa may play a role in the inflammatory process of IBD,IL-35 maybe become one new potential detected and therapeutic cytokine for IBD.However,the results were limited for performed in an efficient mice model,clinical tests needs to be performed in the future.Part II The effect of SHIP1 expression on the proliferation and apoptosis of macrophage systemObjective:By regulating the expression of SHIP 1,we analyzed the proliferation and apoptosis of RAW264.7 and BMDM cell lines,and discussed the role of and SHIP1 in macrophages.Methods:1?The target site was predicted by three databases(miRBase,PicTar and miRanda).2?Construct targets dual luciferase reporter vectors,which are co-transfected to macrophage with miR-155minic(overexpression of miR-155)or inhibitor(inhibiting the expression of miR-155),to verify weather miR-155 is capaple of binding with binding with the 3'-UTR of the SHIP1 gene.3?Make real-time and quantitative PCR and Western blot test to detect the expression on mRNA and protein of SHIP1 under influences of miR-1554?Cell proliferation was analyzed using an MTT assay after interfered by interaction miR-155 and SHIP15?Check expression changes of inflammatory cytokines IL-6,TNF-a,IL-1?.IFN-?with ELISA methods.6?Statistical analysis:the experimental data are presented as mean±standard deviation(mean±SD).statistics comparsion use One-way ANOVA,with P<0.05 as significant difference.SPSS21.0 software is used for statistical analysis.Results:1?SHIP1 are predicted to be miR-155 target genes by bioinformatics software2?By constructing dual luciferanse reporter vector containing the miR-155 targets,miR-155 is confirm to integrate with the predicted binding bite on SHIPlmRNA,miR-155 can significantly inhibit the mRNA and protein levels of SHIP1(P<0.05)3?An MTT assay was performed to test the effects of miR-155 and its target SHIP-1 on the proliferation of raw264.7 cells.Cell proliferation was significantly elevated following the over-expression of miR-155 and was decreased after the up-regulation of SHIP-14?The cells that over-expressed miR-155 exhibited the highest levels of secretion of these factors,while SHIP-1 restoration could inhibit the over-production of IL-6,TNF-?,IL-1?,and IFN-? in these two cell typesConclusion:These results indicate that miR-155 serves its pro-inflammatory function by repressing SHIP-1 expression in macrophages.Part ? Effect of IL-35 on macrophage polarization in IBD mouse model and its mechanismObjective:To investigate the effect of IL-35 on macrophage polarization in IBD mouse model and its mechanismMethods:DSS-induced experimental colitis was established in Balb/c mice to determine therole of IL-35.The mice were divided into groups:control group?DSS group?IL-35-treated group.During the 7-d DSS induction,changes in body weight,occult blood,and gross bleeding were assessed and scored for the determination of DAI scores;evaluated the histological changes in colon sections by HE staining;Macrophage polarization was measured by Flow cytometry;both the RNA and protein levels of SHIP-1 by RT-PCR and Western-blot;PCR analysis revealed that the main pro-inflammatory mediators in colitis,including IL-6,TNF-a,IFN-y,and and anti-inflammatory IL-10.Statistical analysis:the experimental data are presented as mean±standard deviation(mean±SD)statistics comparsion use One-way ANOVA,with P<0.05 as significant differenceResults:DSS-treated groups exhibited higher DAI scores compared to the normal distilled water-treated group.In the IL-3 5-treated groups,it was observed that the mice that received IL-35 injection exhibited significantly lower DAI compared with DSS-treat group and the control group.Additionally,the colon length of DSS-treated mice was markedly shortened compared to controls(4.45 ± 1.29 cm,vs7.23 ± 1.35 cmP<0.05),and the IL-35 treated could partly abate such shortening(6.82 ±1.41 cm vs 7.23 ±1.35 cm,P>0.05)1?The mice co-treated with IL-35 displayed remarkably reduced levels of colon inflammation,including neutrophil infiltration,epithelial damage,depletion of goblet cells,and distortion of crypt architectures,compared to the mice treated with DSS group and control group?2?We found that both the RNA and protein levels of SHIP-1 were significantly increased with IL-35 administration whereas activity of its major functional target,4?IHC analysis showed that SHIP-1 was mainly expressed in lymphocytes,neutrophils,and other hematopoietic cells in the inflamed mucosa5?PCR analysis revealed that the IL-6,TNF-a,IFN-?,,were all suppressed to a large degree after IL-35 treatment;IL-10 demonstrated an opposite trend in expressionConclusion:IL-35 can alleviate the DSS induced IBD mouse model,and may promote the polarization of M2 Mega cells by regulating the expression of SHIP 1.