| Background:Inflammatory bowel disease(IBD) is a modern non-specific inflammatory gastrointestinal disease that mainly reflects in two forms of Crohn's disease(CD)and ulcerative colitis(UC).The number of IBD patients in the United States has exceeded 1 million and in Europe has exceeded 2.5 million up to 2015.The incidence rate of IBD in Europe and the United States is as high as 0.5%,while in newly industrialized countries,such as Asia,Middle East,and South America,it has also been increasing in recent ten years.Due to the sharp increase in the incidence rate and population in newly industrialized countries,the total number of IBD patients will be equal to the total population of western countries by 2025,and IBD will become a new global burden.IBD usually occurs in young adults.The pathogenic factors of IBD remain unclear,which may associate with mutation of the susceptible gene,environmental changes,imbalance of intestinal flora and abnormal immune response.The uncertainty and diversity of pathogenic factors increase the difficulty for treatment of IBD,in which the disorder of immune function and abnormal immune response are considered as the main factors and targets of IBD treatment.Dendritic cells(DC) are the most potently professional antigen presenting cells(APC),which are the "bridge" connecting innate immunity and adaptive immunity.The differentiation of CD4+T cells can be regulated into effector T cells or regulatory T cells(Treg)through the different maturity of DC,which to make the immune system exerts an immune effect or immune tolerance and participate in the occurrence and development of various autoimmune diseases.Many studies suggest that DC is involved in the pathogenesis of IBD,so DC is considered to be one of the effective therapeutic targets of IBD.CD4+T cells,which are mainly composed of Th cells and Treg,play a major role in the pathogenesis of IBD.The colon tissue of IBD patients is often infiltrated by a large number of CD4+T cells.The imbalance of Thl/Th2 differentiation is deemed to be the main pathogenesis of IBD where the excessive activation of Th1 cells is mainly involved in the pathogenesis of CD,while the excessive activation of Th2 leads to the production of UC.Over-activated Th1 cells secrete excessive IFN-r,which results in reduced Th2 cells differentiation and causes Th1/Th2 cell axis to tilt severely,and eventually leads to an increased IBD inflammation.With the detection of Th17 subsets characterized by IL-17 secretion in IBD patients,the pathogenicity of Th17 in IBD has been gradually determined,which complements the theory of Th1/Th2 imbalance pathogenicity in IBD.Th17 is involved in the inflammatory response by secreting IL-17,which mediates severe tissue damage.Its differentiation depends on the activation of IL-6.IL-6 inhibits TGF-? activated Treg,and the insensitive or dysfunctional Treg cells constitute one of the pathogenesis of IBD.Therefore,the imbalance of Th17/Treg differentiation has become a new pathogenesis mechanism of IBD and has been widely explored.The balance of the differentiation of CD4+T cells is an important component of the pathogenesis of IBD and the main route of IBD treatment.At present,the treatment of IBD mainly focuses on drug treatment,mainly including aminosalicylic acid drugs,corticosteroids,immunosuppressants,monoclonal antibodies,and other selective drugs such as antibiotics,probiotics,vitamins and rosiglitazone.More and more pathogenesis and therapeutic targets of IBD have been developed diversely with the quick development of technology of cell molecular biology and immunology.However,it is still difficult to treat the IBD radically,it may outbreak repeatedly and patients may have intolerance or side-effect(like hyperthermia,myelosuppression)to the therapeutic drug.Therefore,we need to develop safe,high-efficiency and fewer side-effect drugs to expand the treatment of IBD.Objecti ves:Inonotus obliquus is an edible fungus that grows in birch that has been used to treat cancer and inflammation for more than four centuries in Russia,Finland and other areas.Inonotus obliquus polysaccharide(IOP)is a bioactive polysaccharide from Inonotus obliquus with medical value,such as anti-inflammatory,anti-neoplastic and immunoregulation.However,the role of it in the prevention and treatment of DSS-induced experimental colitis and related mechanisms is still unclear.Therefore,this study uses the various concentration IOP(100,200,300 mg/mL)to explore its therapeutic effect in the chronic colitis caused by DSS and explore its therapeutic mechanisms associated with DC and CD4+T cells to provide feasibility and theoretical basis for using IOP to treat IBD.It also provides new possibilities for IBD immunocyte treatment by the in vivo transfusion of tolerant bone marrow-derived dendritic cells(BMDC)induced by IOP.Part ? Therapeutic effect of IOP on chronic colitis induced by DSSMethods:IOP was extracted by the hot water extraction method and the polysaccharide composition of IOP was detected by high performance liquid chromatography.BALB/c male mice were treated with 3%DSS three-cycle for 43 days to establish chronic colitis model,the experimental group was treated with three different concentrations of IOP(100,200,300 mg/kg).The weight of mice and whether they have the phenomenon of blood in the stool and diarrhea was monitored every day and conducted disease index(DAI)scores on it every day.The severity of intestinal inflammation in mice was assessed through measuring the length of the colon of the mice.The mice colonic paraffin sections were stained with hematoxylin-eosin(HE)staining and marked the histological score for it.Immunohistochemistry(IHC)was used to detect the expression of tight junction(TJ)protein ZO-1 and Occludin in mice colon tissues and observe the completeness of the colon of the mice and the damage of mucosa.Reverse transcription-polymerase chain reaction(RT-PCR)method was used to detect the gene level expression of specific cytokines and transcription factors associated with Th1,Th2,Th17,and Treg cells in mice colon tissues.IHC and western blot(WB)methods were used to detect expression of JAK-STAT signaling pathway-associated proteins such as STAT1,STAT3,STAT6 in colon tissue associated with the differentiation of CD4+T cell.Results:After testing,the content of mannose,rhamnose,glucose,galactose,xylose and arabinose in the IOP was 9.2%,4.4%,46.6%,11.5%,11.1%,and 4.3%,respectively.The content of polysaccharide was more than 90%,therefore,it was suitable for subsequent research.The changing rate of the weight of colitis mice was small after IOP(100,200,300 mg/kg)treatment,the length of colon was significantly restored,the DAI and histological scores were significantly reduced,the losses of colon tissue TJ protein ZO-1 and Occludin were less when comparing with the model group,which protected the integrity of colonic tissue significantly.IOP(100,200,300 mg/kg)had improved inflammation in colitis mice significantly.According to the result of RT-PCR,IOP(100,200,300 mg/kg)reduced the expression of Thl and Th17-associated proinflammatory cytokines IFN-y,IL-17 and specific transcription factors T-bet and ROR-yt in colon tissue and increased the expression of Th2,Treg-associated anti-inflammatory factors IL-4,IL-10 and specific transcription factors GATA-3,Foxp3.Meanwhile,the results of IHC and WB showed that IOP(100,200,300 mg/kg)had a regulatory effect on the JAK-STAT signaling pathway in the upstream pathway associated with CD4+T cell differentiation.It decreased the phosphorylation level of STAT1 and STAT3 and the amount of nuclear input,and increased the phosphorylation level of STAT6 and the amount of nuclear input.The flow cytometry results showed that IOP(100,200,300 mg/kg)inhibited the differentiation of Thl and Th17 and promoted the differentiation rate of Th2 and Treg in spleen and MLN.IOP maintained the balance of Th1/Th2,Th17/Treg cell axis in colitis mice.Summary:1.IOP(100,200,300 mg/kg)significantly alleviates the inflammation of chronic colitis induced by DSS.2.The therapeutic effect of IOP(100,200,300 mg/kg)on experimental colitis is related to the balance of Th1/Th2 and differentiation of Th17/Treg.3.IOP(100,200,300 mg/kg)regulates the differentiation of CD4+T cell subsets associated with STAT1,STAT3,STAT6.Part ? The influence of IOP on the differentiation of CD4+T cellsMethods:Immunomagnetic beads negative sorting method was used to obtain the CD4+T cells in the spleen of C57BL/6 male mice to explore the influence of IOP on the differentiation process of Th1,Th2,Th17 and Treg cells.The cells which were stained with CFSE have been divided into five groups:CD4+T group,control group,and IOP(L,M,H)groups.The cells that are not in CD4+T group were all treated with anti-mouse CD3? antibody(5 ?g/mL)and anti-mouse CD28 antibody(3 ?g/mL)for 3 days.The influences of IOP(L,M,H)on CD25,CD69,the proliferation rate,and the proliferation index of CD4+T cell activated by antibodies were detected by flow cytometry.The gene level expression of specific cytokines IFN-y,IL-4,IL-17,IL-10 and transcription factors T-bet,GATA-3,ROR-yt,Foxp3 associated with Th1,Th2,Th17,and Treg cells in mice colon tissues were detected by RT-PCR.The expressions of IFN-?,L-4,IL-17,IL-10 were detected by enzyme linked immunosorbent assay(ELISA).The influences of IOP(L,M,H)on the differentiation of Th1,Th17 and Treg cells were detected by flow cytomety.Results:The results of flow cytometry showed that IOP(L,M,H)didn't affect the expression of CD69 and CD25 in the early and late activation phase of activated CD4+T cells and it also didn't affect the proliferation rate and proliferation index of CD4+T cells compared with control group.The results of RT-PCR and showed that IOP(L,M,H)significantly decreased the expression levels of Thl and Th17-associated proinflammatory IFN-y,IL-17 and specific transcription factors T-bet and ROR-yt in activated CD4+T cells and increased the expression of Treg-associated anti-inflammatory cytokines IL-10 and specific transcription factor Foxp3.However,the effects of IOP(L,M,H)on Th2 related cytokine IL-4 and transcription factor GATA-3 were limited.The results of directed differentiation of CD4+T cells showed that IOP(L,M,H)significantly decreased the differentiation of Thl and Th17 and increased the differentiation rate of Treg.Summary:1.IOP(L,M,H)doesn't participate in the activation and proliferation of the activated CD4+T cells,but participate in the differentiation stage of CD4+T cells.2.IOP(L,M,H)inhibits the differentiation of Thl and Th17,and promotes the differentiation of Treg.However,the influence of IOP(L,M,H)on Th2 polarization is limited.Part ? The influence of IOP on the mature and function of BMDCMethods:DC is the most important APC that stimulates the differentiation of CD4+T cells,which determined by the expression of MHC-II,co-stimulatory molecules,and the cytokines from DC.The ability to mediate immune tolerance or immune activation of DC is determined by its maturity.So,we investigated the influence of IOP on BMDC maturity and the ability to induce tolerance.The bone marrow cells of C57BL/6 male mice that are 6-8 weeks old were treated with the rmGM-CSF(20 ng/mL)rmIL-4(20 ng/mL)for 6 days in 6 well plate,than the suspended and loose adherent cells collected(which is also called as BMDC).Part of BMDCs were stimulated with 100 ng/mL lipopolysaccharides(LPS)for 24 h(which is also called as BMDCLPS)in absence or presence IOP(10,20,40 mg/mL)condition.The indicators(MHC-?,CD40,CD86)related to the mature of BMDC and the secretion of cytokine related to the differentiation of CD4+T cells were detected by flow cytometry and ELISA methods.BMDC and CD4+T cells were cultured in the ratio of 1:5 to establish mixed lymphatic reaction(MLR)system.The differentiation of Th1,Th17 and Treg were detected by flow cytometry,and the cytokines such as IL-12,IL-6,TGF-? were detected by ELISA.Results:The expression levels of MHC-II,CD40 and CD86 are the standard for judging the maturation of BMDC,which are also the first and second signal for the activation of CD4+T cells.The results showed that the expressions of MHC-II,CD40 and CD86 on the surface of BMDCLPS were higher than BMDC obviously.Results of ELISA and flow cytometry analysis showed that BMDCLPS secreted lots of IL-12 and IL-6,which promoted the CD4+T cells differentiation into Thl and Th17.However,IOPL,M,H-BMDCLPS expressed low MHC-II,CD40 and CD86,and kept low maturity.Expression of TGF-? secreted from IOPL,M,H-BMDCLPS was increased,which promoted CD4+T cells differentiation into Treg and kept the tolerance in MLR system.Summary:1.IOP(10,20,40 mg/mL)maintains BMDCLPS in immature state.2.BMDCLPS treated with IOP(10,20,40 mg/mL)can promote the differentiation of CD4+T cells into Treg and has the function of maintaining immune tolerance.Part ? Therapeutic effect of IOPL-BMDC on acute colitis induced by DSSMethods:Various of tolerant DC with stable function have been proved to have effective treatment function for IBD model.We studied the therapeutical effect of BMDC treated with IOP on colitis induced by DSS,and explored the treatment mechanism associated with CD4+ T cells.C57BL/6 mice were randomly divided into 4 groups(n=6):control group,model group,BMDC group,and IOPL-BMDC group.Colitis mice were given with 3%DSS in drinking water,control groups were given with normal drinking water for 6 days.Before 3 days(-day3)administration of 3%DSS and the third day(day3)after treated with 3%DSS,the BMDC group mice and the IOPL-BMDC group mice were intraperitoneally injected with 2 × 106/mouse of BMDC or IOPL-BMDC,respectively.On day 7,the mice were sacrificed by cervical dislocation,length of colon was measured and the DAI score evaluated everyday.Paraffin sections of colon tissues were analyzed with HE stain,and the histological scores were assessed.IHC method was used to measure colonic epithelial barrier damage in colitis mice,and the expressions of tight junction proteins ZO-1 and Occludin in colon tissues were examined.Flow cytometry was used to analyse the polarization rate of Th1,Th17 and Treg in spleen and MLN.ELISA method was used to detect the expression of IL-12,IL-6,TGF-?,IFN-y,IL-17,and IL-10 in colon tissue.Results:The results showed that the changing rate of the weight and DAI sore were decreased significantly both in the BMDC group and IOPL-BMDC group.Meanwhile,the colon length and the survival rate were improved in this two groups.The results of HE and IHC showed that the integrity of the tissues of colitis mice in both BMDC and IOPL-BMDC groups were well,the loss of TJ protein ZO-1 and occludin were inhibited,and the damage of mucosa was decreased obviously.Meanwhile,the anti-inflammation effect of IOPL-BMDC was better than BMDC.The results of ELISA showed that IOPL-BMDC significantly inhibited the secretion of proinflammatory cytokines IL-12,IFN-?,IL-6,IL-17 associated with Thl and Th17 differentiation and improved the secretion of anti-inflammatory cytokines TGF-? and IL-10 related to Treg differentiation.Flow cytometry results showed that polarization rate of Th17 was few in spleen and MLN either in control mice or in colitis mice.The polarization rate of pro-inflammatory Th1 was increased and the anti-inflammatory Treg was decreased in model group.Conversely,the polarization rate of pro-inflammatory Th1 was down-regulated and the anti-inflammatory Treg was up-regulated in both BMDC and IOPL-BMDC group,and IOPL-BMDC group mice have shown more effective regulation ability for differentiation of CD4+T cells than BMDC group.Summary:1.IOPL-BMDC alleviates DSS induced acute colitis through regulating the CD4+T cells differentiation.2.IOPL-BMDC provide a new possibilities for cell therapy of IBD.Conclusion:IOP(100,200,300 mg/kg)can alleviate inflammation condition of DSS induced chronic colitis,and regulate Th1/Th2,Th17/Treg cell balance in spleen,MLN and colon tissue of colitis mice,which mechanism may associate with JAK-STAT pathway.IOP(10,20,40 mg/mL)have no influence in CD4+T cells activation and proliferation in vitro,but can regulate CD4+T cells differentiation directly.IOP(10,20,40 mg/mL)inhibit differentiation of Th1 cell and Th17 cell,and promote differentiation of Treg cell,but have limited influence in differentiation of Th2 cell.IOPL,M,H can maintain BMDC lower maturity in LPS(100 ng/mL)induced inflammatory condition.Meanwhile,IOPL,M,H can promote the polarization of Treg cell,and inhibit polarization of Th1 cell and Th17 cell in MLR system.IOPL-BMDC can alleviate inflammation of DSS induced acute colitis,and regulate differentiation of CD4+T cell in spleen and MLN in colitis mice.IOPL-BMDC inhibit the polarization of Th1 cell and improve the polarization of Treg cell,but it have limited influence in polarization of Th17 cell. |
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