The Roles Of Embelin In The Regulation Of Dendritic Cells And Experimental Autoimmune Encephalomyelitis

Embelin is a kind of herbal extracts with anti-cancer and anti-inflammatory properties.However,the anti-inflammatory mechanism of embelin is not fully understood.Dendritic cell(DC)is the most powerful antigen presenting cell(APC)which can activate Naive T cell.Studies showed that DC is closely related to numerous autoimmune diseases.With the development of in vitro DC Cell Cultural Techniques,the studies of DC make a rapid development and become a central issue of immunology.Meanwhile,the role of DC in autoimmune diseases has been discovered gradually.It is found that the antigen process and presentation of DC can affect the disease onset and progression of autoimmune diseases.Multiple sclerosis(MS)is an autoimmune central nervous system(CNS)inflammatory and demyelination disease.The pathogenesis of MS is unknown,but it is generally believe that immune factors are closely related.MS has been considered as one of major healthy problem and can affects the patient’s life quality all the time,which have aroused wide concern in medical field.To investigate MS,many laboratories have made some progress through the establishment of ideal animal model for human MS in vivo studies.Experimental autoimmune encephalomyelitis(EAE),the most extensively studied animal model of autoimmune disease,is considered an ideal animal model for human MS as a result of its symptoms is extremely similar to human MS.The pathological mechanism of EAE/MS has not yet been fully understood,but it is generally believed that EAE is a central nervous system demyelination disease caused by autoimmune inflammation which is mediated by type 1 and type 17 T helper cells(TH1 and TH17).While directly differentiation of different T helper cells depends on different antigenic stimulation from DC.DC uptake and process the foreign or autoantigen,then present the immune signal to T cells and B cells.Meanwhile,according the different signaling derived from DC,naive T cells directly differentiate into different T cell subsets which have different immune function.Nowadays,the regulatory roles of embelin on DC and its therapeutic effect on MS/EAE are not clear,which have been studied in this thesis.Objective:It is generally believed that MS,atherosclerosis,diabetes,cancer,liver fibrosis,lung fibrosis and other diseases are related to chronic inflammation.Embelin is a kind of herbal extracts with anti-cancer and anti-inflammatory effect.However,the anti-inflammatory mechanism of embelin is not fully understood..Inflammation is complex physiological responses of body to prevent pathogen infection and tissue damage,in which the immune response is an important mediator.Long-term chronic inflammation,too long or too strong stimulus by inflammatory factors will induce the nonresolving inflammation.Then the malignant transformation of non-resolving inflammation will cause a variety of inflammatory diseases.Therefore,this study starts from DC which is the frontier cell of immune system and inflammatory response.In order to explore the effect of embelin in the regulation of immune response,we conducted the studies on the differentiation,mature,antigen-presenting of DC,as well as the proliferation of T cells and the secretion of cytokines and inflammatory factors of different T cell subsets mediated by DC in response to embelin in vitro.To further explore the in vivo regulatory role of embelin in immune response,the T cells mediated EAE was used in this study.Overall,we investigate the roles and anti-inflammatory mechanism of embelin by DC in vitro and EAE in vivo.The immune suppression and anti-inflammatory physiological processes of embelin may associated with a variety of inflammatory diseases,which will provide a new way for prevention and treatment of MS and other inflammatory diseases.Methods:1 In vitro studies:The CD4+ monocytes from blood of healthy human were used for in vitro DC differentiation.Meanwhile,the embelin was present during DC differentiation and maturation.To determine the effects of embelin on DC differentiation and maturation,the cytokine expression was detected by real-time PCR and the expression of DC surface markers CD80,CD83,CD86 and HLA-DR were measured by flow cytometry.Besides these,the effect of embelin on antigen uptake of immature DC was assayed by endocytosis Assay.The capacity of mature DC stimulated T cells proliferation was determined by CSFE,and capacity of mature DC stimulated different T cell subsets differentiation were assayed by cytokine production.2 In vivo study:The C57BL/6 mice were used for EAE induction by using MOG35-55 peptides.The EAE mice were treated with embelin by gavage method.The disease incidence and clinical score were measured to evaluate the preventive and therapeutic effects of embelin on mouse EAE.Results:1,Embelin significantly inhibit DC differentiation by inhibit the expression of DC surface markers such as CD80,CD83,CD86 and HLA-DR.2,Embelin inhibit LPS-induced DC maturation by inhibit the expression of DC surface markers such as CD80,CD83,CD86 and HLA-DR.3,Embelin inhibit the endocytosis capacity of immature DC.4,Embelin inhibit the capability of mature DC-stimulated allogeneic T cells proliferation.5,Embelin not only significantly inhibited the expression of inflammatory cytokines TNF-α,but also decreased the gene expression of IL-12,IFN-y and IL-6,IL-23 which essential for differentiation of TH1 and TH17 respectively.6,The clinical score of embelin treatment group is reduced(embelin 20 mg/kg/day and 50 mg/kg/day).Embelin treatment significantly decrease disease score of mouse EAE,embelin also reduced the highest clinical score and the total clinical scores in treated EAE mice.Therefore,Embelin obviously improved the incidence and disease progression of mouse EAE.Conclusions:Embelin can inhibit the differentiation and maturation of DC,the endocytosis of iDC and the capability of mDC stimulated T cells proliferation.Besides,embelin can significantly decrease the gene expression of inflammatory cytokines as well as the gene expression of cytokines of T helper cells(TH1 and TH17).We first demonstrated embelin can ameliorates the development of EAE.These results indicate that embelin has immunosuppressive effects and play a anti-inflammatory role on autoimmune central nervous system diseases of EAE.Further more,we first discovered embelin can inhibit DC function in vitro and with therapeautic effects on EAE.The anti-inflammatory effects of embelin may mediate,at least partialy,by inhibiting the function of DC and the differentiation and proliferation of T cells which is mediated by DC.Our studies may provide a theoretical basis and new therapeutic way for the treatment of MS and other immune inflammatory diseases.