The Role And Mechanism Of MIF Small Molecule Inhibitor IMG-122 In The Treatment Of Experimental Autoimmune Encephalomyelitis

Part 1:The effect of MIF small molecule inhibitor IMG-122 on nervous function and inflammatory response in EAE animal modelObjectives:To investigate the therapeutic effect of IMG-122,the independent research and development of new type of MIF small molecule inhibitors,we observe the change of the function of motorius movement in EAE(experimental autoimmune encephalomyelitis)mice treated with it.In addition,the degree of cell activation and infiltration and the content of inflammatory cytokines related to the lumbar intumescence spinal cord inflammation in the incidence peak mice were detected to explore its regulatory mechanisms of immune microenvironment in EAE.Methods:The EAE was induced in 40 SPF C57BL/6 mice with MOG35-55 and the animals were divided into the vehicle group(N=20)and IMP-122 treatment group(N=20)as per weight.Clinical score of EAE was graded by the international commonly used Kono's scoring method to monitor neural function in mice.All mice of the treatment group were injected(i.p.)with IMG-122 at 20mg/kg twice a day(8 hours dosing interval)since 7 days to 35 days after immunization.At the peak period of EAE,the spinal cord lumbar enlargement tissues of 8 mice in each group were sliced(8?m),the activation and infiltration of related inflammatory cells were detected by immunofluorescence,and the expression of inflammatory cytokines IL-6,IL-1?,IL-17 and IL-10 in spinal cord tissues were detected by fluorescence quantitative PCR.Results:The onset of EAE of mice treated with IMG-122 started later than the vehicle group,and the neurological function score of the treatment groups was also lower than the other group with statistical significance(P<0.05).At the peak of the disease,more significant activation of astrocytes and microglia in the spinal cord lumbago of vehicle group were observed among two groups.In addition,compared with the vehicle group this period(P<0.05),the expression of inflammatory cytokines of IMG-122 treatment group was significantly lower.Conclusion:1.IMG-122 small molecule inhibitor can reduce the neurological function score of EAE mice.2.IMG-122 small molecule inhibitor can effectively inhibit the activation of astrocytes.3.IMG-122 small molecule inhibitor can inhibit the release of inflammatory factors.Part 2:The effect of IMG-122 small molecule inhibitor on themigration and activation of macrophages/microglia in EAE animal modelObjectives:Treating EAE animal model with IMG-122,lumbar intumescence spinal cord tissue of mice in peak morbidity were processed for immunohistochemical staining.Through detection of peripheral macrophages infiltration to the central nervous systems and activation of spinal cord microglia,we explore the migration and activation of macrophages/microglia to figure out the moderating effect of the small molecule inhibitor IMG-122.Methods:The EAE was induced in 40 SPF C57BL/6 mice with MOG35-55 and the animals were divided into the vehicle group(N=20)and IMG-122 treatment group(N=20)as per weight.Clinical score of EAE was graded by the international commonly used Kono's scoring method to monitor neural function in mice.All mice of the treatment group were injected(i.p.)with IMG-122 at 20mg/kg twice a day(8 hours dosing interval)since 7 days after immunization.Lumbar intumescence spinal cord tissue of 8 mice in peak morbidity of each group were sliced(8pm)to detect the infiltration degree of macrophages and the activation degree of microglia.Results:In vehicle group,we observed a large number of macrophages infiltrated into the white matter of the central nervous system at the spinal cord enlargement,and all the infiltrating macrophages could express MIF.Moreover,the microglia activation in central nervous system remarkably increased,and the infiltration and activation in posterior horn of spinal cord was to a serious extent.With the treatment of IMG-122,the amount of macrophage infiltration to white matter of the central nervous system declined,and the difference between the two groups have statistical significance(P<0.05).This phenomenon indicated IMG-122 could effectively suppress macrophage migration and the activation of microglia.Conclusion:1.MIF small molecule inhibitor IMG-122 is capable of reducing the neurological function score of EAE mice.2.MIF small molecule inhibitor IMG-122 can effectively inhibit the migration of macrophages.3.MIF small molecule inhibitor IMG-122 can effectively inhibit the activation of macrophages/microglia cells.Part3:The effect of MIF small molecule inhibitor IMG-122 on theproliferation and differentiation of T cells in EAE animal modelObjectives:Through the induction of EAE animal model and treatment of small molecule inhibitor IMG-122,examine the therapeutic effects to explore the regulatory effect of it on the proliferation and differentiation of T cells.Methods:The EAE was induced in 20 SPF C57BL/6 mice with MOG35-55 and the animals were divided into the vehicle group(N=10)and IMG-122 treatment group(N=10)as per weight.Clinical score of EAE was graded by the international commonly used Kono's scoring method to monitor neural function in mice.All mice of the treatment group were injected(i.p.)with IMG-122 at twice a day with an interval of 8 hours since 7 days after immunization to the peak period of EAE(the 20th day).Meanwhile,20 SPF C57BL/6 mice(not induced by MOG35-55)were given intraperitoneal injection of normal saline twice a day as the normal control.At the peak period of incidence,spleen tissues of each mice group were exteriorized to detect the proportion of Thl and Th17 cells in CD4+T cells by flow cytometry.Results:The proportion of Thl and Thl7 in CD4+T cells in the spleen both of the vehicle group and IMG-122 treatment group was significantly higher than that of the normal control mice group with statistical difference.(P<0.05)In addition,the proportion of Thl cells in IMG-122 treatment group increased and that of Th17 cells decreased compared with vehicle group,but showing no statistical difference.Conclusion:1.Massive activation and proliferation of T cells occur in mouse spleen after EAE inducement.2.MIF small molecule inhibitor IMG-122 could inhibit the proliferation and differentiation of Th17 cells to some extent,and promote the proliferation and differentiation of Thl cells.