Study On The Immune Mechanism Of Ginkgolide A In The Treatment Of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis?MS?is generally considered to be an autoimmune disease of the central nervous system?CNS?mediated by T cells.Activated T cells infiltrate CNS from the periphery and secrete pro-inflammatory factors to activate macrophages and microglia to expand inflammatory response.However,T cells and macrophages are classified into pro-inflammatory type and anti-inflammatory type due to their different phenotypic functions.Therefore,regulating the phenotype of T cells and macrophages and maintaining the stability of CNS microenvironment are important molecular mechanisms for the treatment of MS.Ginkgolide A?GA?has been shown to have anti-inflammatory effects in A variety of inflammatory and immune diseases.This study MS classic animal model of experimental autoimmune encephalomyelitis?experimental autoimmune encephalomyelitis,EAE?mice as object,through the in vivo and in vitro experiments to explore the diagnosis and treatment of GA potential of EAE and regulation function of T cells and macrophages and mechanism.Part?Study on the immunomodulatory mechanism of ginkgolide A inthe treatment of EAEObjective:To determine the effect of GA on EAE,and to explore the immuneregulation of GA on T cells and macrophages.Methods:Forty-two female C57BL/6 mice were randomly divided into normal saline?NS?group,EAE control group and GA treatment group according to body weight,with14 mice in each group.Mice in EAE group and GA group were immunized with MOG_35-555-55 peptide to prepare EAE model.Each group was gavage from day 10 to day27 after immunization,normal saline was given to NS group and EAE group,and GA treatment group was given GA.Weight and clinical symptom changes of mice in each group were recorded every day.Mice were sacrificed on the 28th day after immunization.Four mice in each group were fixed with 4%paraformaldehyde,and their spinal cords were frozen and stained with HE and LFB.The spleens of 5 mice in each group were taken,and the mononuclear cell suspension was prepared asepsis.The latter part was labeled with CD4+and CD11b+antibodies,and then the phenotypes of T cells and macrophages were analyzed by flow cytometry.The cell suspensions of the other groups were incubated in 37°C carbon dioxide incubator for48h,and cytokine levels were determined by ELISA.Finally,spinal cord proteins were extracted from 5 mice in each group,and Western blot was used to detect markers of T cells and macrophages as well as the expression of inflammatory signaling pathways.The above data is analyzed using Graphpad Prism.Results:1.GA effectively delayed the mean onset time of EAE mice and reduced the maximum clinical score of EAE mice.In addition,compared with the EAE control group,GA can alleviate the severity of EAE symptoms and reduce the degree of weight loss.2.Under pathological examination,a large number of inflammatory cells could be seen in the spinal cord of EAE mice,and the infiltration of inflammatory cells could be effectively reduced after GA treatment?P<0.05?.Myelin loss in the white matter of the spinal cord was also an important pathological feature of EAE mice,and was significantly reduced after GA treatment?P<0.001?.3.GA regulated the balance of Th1/Th17 and Th2/Tregs of peripheral T cell subsets,reducing the expansion of EAE inflammatory response.Compared with the EAE group,GA inhibited the CD4+T cell subsets expressing IL-17 and IFN-??P<0.05?,and up-regulated the percentages of CD4+TGF-?+and CD4+IL-10+T cell subsets?P<0.0001?.4.GA induced M1-type polarization of macrophages to M2-type.The flow cytometry results showed that compared with the EAE group,the GA group could effectively inhibit the expression of CD8a?P<0.0001?,CD16/32?P<0.0001?,CD40?P<0.0001?and IL-12?P<0.01?in the peripheral M1 macrophage subsets.On the contrary,the GA group increased the expression of IL-10 and CD206 in the M2macrophage subgroup?P<0.01,P<0.001?.In addition,GA inhibited COX-2expression of M1-type marker in macrophages in the spinal cord?P<0.05?,significantly increased the expression of M2-type marker Arg-1?P<0.001?,but did not significantly inhibit iNOS.5.After GA intervention,the expression of inflammatory signaling pathways in the spinal cord was inhibited and the release of cytokines was regulated.GA inhibited the inflammatory pathways of TLR-2,MyD88,JNK?P<0.05?and NF-?B?P<0.01?,down-regulated the levels of pro-inflammatory cytokines TNF-?and IL-1??P<0.001?,and increased the levels of anti-inflammatory cytokines IL-4?P<0.01?.Conclusion:GA can reduce the severity of EAE symptoms,effectively reduce spinal cord demyelination and inhibit inflammatory cell infiltration.The mechanism of action is closely related to the regulation of the imbalance of M1/M2 macrophages and Th1/Th17 and Th2/Tregs T cell subsets in the development of EAE.By regulating the stability of various subsets of T cells,GA can reduce the inflammatory infiltration of peripheral immune cells into CNS and further expand the inflammatory response.GA inhibits the inflammatory signaling pathway and promotes the polarization of macrophages from M1 to M2,effectively improving the inflammatory microenvironment.The natural product GA showed its potential in treating EAE by peripheral immune regulation and improving the central inflammatory microenvironment.Part?Ginkgolide A improves the inflammatory response ofmacrophages in vitroObjective:To investigate the effect of GA on macrophage phenotype induced by MOG_35-55in EAE mice in vitro.Methods:After immunizing 20 female C57BL/6 mice,the spleen was aseptic on day 9 of immunization,and mononucleate cell suspension was prepared.Macrophages were separated by CD11b magnetic beads.After counting,the macrophages were divided into 2 groups of DMSO and GA,which were incubated in 37°C incubator for 72h with DMSO and GA,respectively.Macrophages were collected to analyze their phenotypic changes on flow cytometry,and the supernatant was also taken to determine the levels of cytokines IFN-,IL-17,IL-4,IL-6,IL-10,TNF-?and IL-1?by ELISA.Results:The results showed that GA decreased the expression of M1 markers CD8a?P<0.0001?,CCR7?P<0.0001?,CD11c?P<0.01?,CD40?P<0.0001?and MHC?P<0.0001?,but increased the expression of M2 markers CD206?P<0.01?and CD200?P<0.01?.In addition,GA inhibited the levels of IL-1??P<0.05?,IL-17?P<0.05?and TNF-??P<0.05?,but the level of IL-4 was increased?P<0.05?.Conclusion:GA may act directly on macrophages to play an immunomodulatory role without the assistance and involvement of other cells.GA promotes the polarization of macrophages from M1 to M2,and regulates their cytokine secretion.