| Objective To observe the expression patterns of SSeCKS in rat models of EAE and investigate its possible functions. Understanding the mechanism of SSeCKS in the pathogenesis of EAE may contribute to the development of new therapeutic strategies against EAE and MS.Methods1) The model was preparated in Lewis rat by MBP-induced. The rats were sacrificed at different phase after EAE immunization. Demyelination was depicted by Luxol-fast blue (LFB) staining, and cellular infiltration was showed by H&E staining.2) RT-PCR and Western Blot were used to detect the changes of SSeCKS expression during EAE, and the distribution and localization of SSeCKS in spinal cord of EAE was investigated by immunofluorescence double staining. RT-PCR and immunofluorescence double staining were used to detect the expressions of TNF-αand iNOS in EAE and their relevance to the activation of astrocyte.3) Primary cultures of astrocyte and oligodendrocyte were isolated from postnatal day 0~1 Lewis rats. SSeCKS-siRNA and nonspecific-siRNA plasmids were transfected into astrocytes. Cytokines secreted from astrocyte were detected by sandwich enzyme-linked immunosorbent assay (ELISA) in order to examine the role of SSeCKS in cytokines secreted of astrocyte, induced by LPS or LPS/IFN-γ. The effects of SSeCKS expression in astrocyte on cell viability of oligodendrocyte cultured in CMs were detected by MTT and TUNEL assay. Immunofluorescence double staining was used to detect the relationship between SSeCKS expression in astrocytes and oligodendrocyte apoptosis.Results1) MBP induced a monophasic EAE process. EAE clinical disease in lewis rats had an onset between days 7~10 post immune (PI), peaked at days 13~16 PI and exhibited spontaneous remission by day 30 PI. Lewis rats at peak EAE presented significant inflammatory cell infiltrates and demyelinations, but restored their pathological phenotype at the phase of recovery.2) The expression of SSeCKS exhibited a low level in normal and CFA control spinal cord and markedly increased from E1 stage to remission period with a peak at the E3 stage after EAE induction. SSeCKS were located in many types of cell, such as neuron, astrocyte and oligodendrocyte, but not in microglia. Notably, up-regulated expression of SSeCKS was frequently observed in astrocytes in both the gray matter and white matter in EAE rat spinal cord, compared to control rat spinal cord. The inflammatroy cytokines TNF-α, as well as iNOS were increased in EAE, and their levels in the spinal cord of EAE were correlated with disease severity.3) TNF-αand NO were induced by LPS or LPS/IFN-γin primary astrocytes in vitro. SSeCKS was involved in TNF-αand NO secretion in primary astrocytes. SSeCKS regulated oligodendrocyte apoptosis mediated by conditioned medias from astrocytes. TNF-αand NO were responsible for CM-induced oligodendrocyte injury. SSeCKS possibly regulated oligodendrocyte apoptosis by proinflammatory factor TNF-αand NO induced by activated astrocytes. Conclusions1) In the study, the rat model of EAE was established.2) SSeCKS expression was changed during EAE, which suggested SSeCKS was involved in EAE. SSeCKS was upregulated in astrocytes in spinal cord of EAE rats. The inflammatroy cytokines TNF-α, as well as iNOS was up-regulated in EAE, and their levels in the spinal cord of EAE were correlated with disease severity. These results indicated that SSeCKS was involved in regulation of biological function of astrocytes in EAE.3) The conditioned media from astrocytes treated by LPS or LPS/IFN-γcaused cell death in oligodendrocyte cells. Transient transfection of SSeCKS-siRNA suppressed LPS or LPS/IFN-γ-stimulated TNF-αand NO secretion in astrocytes. Pretreatment astrocytes with neutralizing antibody to TNF-αand inhibition of NO resulted in reduction of oligodendrocyte apoptosis. Collectively, these observations implied that SSeCKS possibly regulated oligodendrocyte apoptosis by proinflammatory factor TNF-αand NO induced by activated astrocytes. |
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