| Multiple sclerosis(MS) is an autoimmune disease mainly involving white matter in central nervous system(CNS). The exact etiology of MS is not clear, but it has relationship with heredity, environment, diet, hormone excretion etc. Some antigens that have similar structure with components of myelin can activate na?ve CD4 +T cell. The activated CD4+ T cells differentiate to effector T cells such as Th1 and Th17 cells and then attack the myelin and axon, leading to neural dysfunction. MS patients can present varieties of symptoms: fatigue, numbness, limb weakness, impaired vision and dysfunction of autonomic nervous system. Because MS mainly affects young adults and always results in disability, the family and society have a heavy burden.Therapies in acute phase of MS include glucocorticoid, immunoglobulin and plasma exchange; therapies in chronic phase include Inteferon-β and some disease modifying therapies. The above therapies can slow the progression of disease, but fail in reversing disease development. So it is important to find a therapy that can halt disease progression and restore the impaired neural function. The aim of this research is to test new treatments on the animal model of MS: experimental autoimmune encephalomyelitis(EAE), contributing to the cure of MS.In the first part of this research, we injected bone marrow-derived neural stem cell(BM-NSC) and BM-NSC overexpressing TGF-β1(TGF-β1-BM-NSC) into EAE mice and compared the difference in their abilities to treat EAE. In the second part of this research, we tested the therapeutic effect of an inhibitor of hypoxia induced factor-1(HIF-1): BAY 87-2243 on EAE.PART 1: The therapeutic effect of TGF-β1-BM-NSC on EAEMethods:(1) culturing BM-NSC; infecting BM-NSC with retrovirus containing TGF-β1 gene;(2) inducing EAE model with MOG35-55; injecting 1×106 BM-NSC or TGF-β1-BM-NSC or same dose of NS into EAE via tail vein on the day of immunization or at the peak of disease development;(3) evaluating the clinical score and body weight of EAE mice every day;(4) sacrificing EAE mice and extracting the infiltrating CD4+ T cells in CNS; analyzing the percentage of Th1, Th17 and Treg cells in CNS using FACS;(5) analyzing the percentage of Th1, Th17 and Treg cells in peripheral immune system using FACS; testing the MOG35-55-specific proliferative ability of lymphocyte; testing the MOG35-55-specific cytokine production of lymphocyte;(6) performing HE and LFB staining on spinal cord tissue;(7) staining frozen section of CNS tissue with antibodies to detect neurons, oligodendrocytes and astrocytes differentiated from BM-NSC and TGF-β1-BM-NSC.Results:(1) Both BM-NSC and TGF-β1-BM-NSC can form typical neurosphere and differentiate into neurons, oligodendrocytes and astrocytes; TGF-β1 was hardly detectable in the supernatant of BM-NSC culture, while the concentration of TGF-β1 in the supernatant of TGF-β1-BM-NSC culture was much higher(488 pg/ml);(2) both BM-NSC and TGF-β1-BM-NSC injection delayed the disease onset and alleviated the symptoms of EAE mice;(3) both BM-NSC and TGF-β1-BM-NSC reduced the number of infiltrating lymphocytes in CNS and attenuated demyelination;(4) compared to EAE mice in NS group, EAE mice in BM-NSC and TGF-β1-BM-NSC groups had reduced percentage and number of CD4+ T cells in CNS, reduced percentage and number of Th1 cells, reduced number of Th17 and Treg cells;(5) compared to EAE mice in NS group, EAE mice in BM-NSC and TGF-β1-BM-NSC groups had reduced percentage of Th1 and Th17 cells but increased percentage of Treg cells in peripheral immune system; injection of BM-NSC and TGF-β1-BM-NSC inhibited MOG-specific proliferative ability of lymphocyte and the production of proinflammatory cytokines;(6) part of the injected BM-NSC and TGF-β1-BM-NSC differentiated into neurons, oligodendrocytes and astrocytes in CNS;(7) the capacity of treating EAE and regulating immune reactions of TGF-β1-BM-NSC was stronger than that of BM-NSC.Discussion: TGF-β1 alone can induce Foxp3- T cell to Foxp3+ Treg cell, so we produced the TGF-β1 overexpressing-BM-NSC to increase BM-NSC’s immunomodulatory capacity. We can see from the results that TGF-β1-BM-NSC increased the percentage of Treg cells in peripheral immune system and reduced the percentage of Th1 and Th17 cells. Furthermore, TGF-β1-BM-NSC also inhibited MOG-specific lymphocyte proliferation and proinflammatory cytokine production. We suppose that the changes in immune system result from the increased Treg cells and the stronger inhibition of autoimmune reactions. All above, both BM-NSC and TGF-β1-BM-NSC can alleviate EAE symptoms and the latter has a stronger therapeutic effect because of increased Treg-induction capacity.Part 2: The therapeutic effect of HIF-1 inhibitor BAY 87-2243 on EAEMethods:(1) testing the effect of BAY 87-2243 on the number and viability of CD4+ T cells using trypan blue and MTS kit;(2) testing the effect of BAY 87-2243 on the proliferation and apoptosis of CD4+ T cells using Brd U kit and Annexin V-PI double staining;(3) testing the effect of BAY 87-2243 on the differentiation of CD4+ T cells to Th1,Th2,Th17 and Treg cells;(4) testing the effect of BAY 87-2243 on the proliferation and differentiation of oligodendrocyte precursor cell(OPC);(5) inducing EAE model with MOG35-55; applying 4 mg/kg BAY 87-2243 or same dose of control solution to EAE by oral on the day of immunization or at the peak of disease development;(6) see methods(4),(5) and(6) in Part 1.Result:(1) BAY 87-2243(1-1000 n Mol) did not affect the number or viability of CD4+ T cells in vitro;(2) BAY 87-2243(1-1000 n Mol) did not affect the apoptosis of CD4+ T cells in vitro; 1-100 n Mol BAY 87-2243 did not affect the proliferation of CD4+ T cells, but 500-1000 n Mol BAY 87-2243 inhibited the proliferation of CD4+ T cells;(3) the addition of BAY 87-2243 promoted CD4+ T cells to differentiate to Treg cells but inhibited the development of Th1 and Th17 cells;(4) BAY 87-2243 promoted the proliferation of OPC under hypo-nutrition condition, but had no effect on OPC differentiation under normal-nutrition or hypo-nutrition conditions;(5) treatment of BAY 87-2243 by oral delayed the disease onset and alleviated the symptoms of EAE;(6) treatment of BAY 87-2243 reduced the number of Th1 and Th17 cells in CNS of EAE mice;(7) BAY 87-2243 reduced the percentage of Th1 and Th17 cells but increased percentage of Treg cells in peripheral immune system; BAY 87-2243 also inhibited MOG35-55-specific proliferation of lymphocyte and proinflammatory cytokine production;(8) the treatment of BAY 87-2243 inhibited the expression of HIF-1α in the CNS of EAE mice.Discussion: HIF-1 plays a critical role in the Th17/Treg balance: it promotes CD4+ T cells to differentiate to Th17 cells but inhibits the differentiation of Treg cells. So we suppose that if the function of HIF-1 is inhibited in EAE, more Treg cells will be produced and the disability of EAE mice could be alleviated, so we applied HIF-1 ihhibitor: BAY 87-2243 to EAE mice. Our results show that the expression of HIF-1α in CNS was inhibited by BAY 87-2243 and more Treg cells were differentiated from CD4+ T cells in EAE mice. Increased Treg cells led to suppression of lymphocyte proliferation, proinflammatory cytokine production and less demyelination in CNS. All the above changes in immune system contribute to the function recovery in EAE mice.SummaryIn this study we evaluate the therapeutic effect of TGF-β1-BM-NSC and BAY 87-2243 on EAE. We found that both TGF-β1-BM-NSC and BAY 87-2243 can delay disease onset and alleviate disability of EAE mice. TGF-β1-BM-NSC and BAY 87-2243 mainly rely on their immune-regulating ability to treat EAE. In addition, TGF-β1-BM-NSC can directly differentiate to neuron and oligodendrocyte; BAY 87-2243 can promote OPC proliferation under hypo-nutrition condition. In brief, TGF-β1-BM-NSC and BAY 87-2243 are promising MS therapies and this study provide more choices in MS treatment. |
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