Study Of Kv1.3 Channel As New Therapia Target In Multiple Sclerosis

Objective:1. To investigate the change of CCR7 and CD45RA before and after SHK blocked Potassium channel Kv1.3 in myelin special CD4⁺T lymphocyte, and its relation with multiple sclerosis.2. To study the variety of cytokine INF- γ and IL-4 ,and the balance of cytokine network after blocking Kv1.3 Potassium channel.3. To evaluate the expression of Potassium channel Kv1.3mRNA on CD4⁺T cell member in different state. In molecular level, to understand the mechanism of the influence of potassium channel blocker on activation of T lymphocyte in the MS.4. To study the role of potassium channel blocker SHK in the treatment of EAE. Methods:1. Peripheral blood mononuclear cells(PBMCs) were isolated from activated MS. INF- β treated MS and normal control, CD4⁺T lymphocytes were isolated by positive selection method with anti-CD4-coated magnetic beads. To establish culturing MBP- and MOG-reactive CD4⁺T lymphocyte lines.2. The immunofluorescence staining of different group T cell were labeled with CD3, CD4, CCR7, CD45RA fluorescence-antibody or homotype control and analyzed by a four-color flow cytometer.3. The change of Voltage-dependent Kv1.3mRNA in different condition were assessed by reverse transcriptase-polymerase chain reaction(RT-PCR) semi-quantitative analysis.4. CD4⁺T cells in different condition secreting INF- γ or IL-4 were detected by enzyme-linked immunospot assay (ELISPOT) analysis.5. C57BL/6 (H-2b) mice were induced to EAE by injecting subcutaneously MOG_35-55 peptide in CFA and prevented or treated with SHK. Both the clinical symptoms and histopathologic changes of the CNS were observed.Results:1. The proliferation of myelin-special CD4⁺T lymphocyte and the inhibition by SHK in activated MS were significant, the stimulation of MBP and MOG was undifferentiated.2. The most part of phenotype in activated MS was CD4⁺CCR7^-CD45RAT cell and the percentage was increased by myelin antigen stimulating. SHK greatly inhibited CCR7^-CD45RA^- in activated MS.3. CD4⁺T cell secreting INF- γ was the main cell group in the perpherial blood of activated MS. In the myelin special CD4⁺T cell of activated MS, the amount of IFN- γ was high and IL-4 was low, but IFN- γ decreased and IL-4 had no change after adding SHK.4. The expression of Kv1 .3mRNA of CD4⁺T lymphocyte in activated MS was the intensest in all groups , myelin antigen up-regulated Kvl.3mRNA level both in activated MS and INF- β treated MS, but there was no change after adding SHK.5. The mice in the experimental group all developed typical symptoms of EAE, the duration of onset and peak was significantly delayed in the prevention EAE compared with EAE of treated and untreated with SHK. There was no significant difference observed in the duration of onset and peak, clinical course and histopathologic findings in the CNS between the treated and the untreated group.Conclusions:1. Kv1.3 blocker suppressed the proliferation of myelin special CD4⁺T cell in vitro. Voltage-dependent potassium channel Kv1.3 contributed to T lymphocyte activition and proliferation.2. There was strong correlation between Tem phenotype and severity of MS, which may suggest Tem phenotype as the marker to estimate the state of illness and the effect of treatment with potassium channel blocker.3. Blocking Kv1.3 channel in myelin special CD4⁺T cell could maintain the balance of cytokine network, which may speculate that Kv1.3 channel maybe the new target for the treatment of MS.4. The up-regulation of Kv1.3 channel was accomplished at moleculartransference level, there was no significant effect on the expression ofKv1 .3mRNA treated with SHK. 5. Potassium channel blocker SHK had no significant effect on the treatment ofEAE induced by MOG35-55 peptide, but had prevention function in a certaindegree.