Diacylglycerol Kidnaseζ Is Involved In The Process Of Rat Experimental Autoimmune Encephalomyelitis

Objective:Diacylglycerol kinase ζ (DGKζ) is an enzyme which phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA) to regulate physical and pathological fuctions. Proinflammatory cytokines produced by microglias, such as tumor necrosis factor a (TNFa), contribute to the inflammatory reaction. To investigate the function of DGKζ in experimental autoimmune encephalomyelitis (EAE) rat and provide new method for clinical cure of multiple sclerosis (MS), we performed the EAE rat model and detected the protein levels, intracellular localization and distribution of DGKζ and TNFa in brainstem and spinal cord.Methods:EAE were induced in Wistar rats by guinea pig spinal cord homogenate. The protein levels, cellular localization and distribution of DGKζ and TNFa in EAE rat brainstem and spinal cord was detected by western blot, immunohistochemical staining and immuno fluorescent double-labeling staining.Results:1. Western blot showed that in both brainstem and spinal cord the expression of DGKζ and TNFα in score1-2group was significantly lower than that in normal group (p<0.05), then increased significantly in score3(p<0.05) and score4group. But there was no significant difference between score3and score4group (p>0.05);2.(1) Immunohistochemical and immunofluorescent double-labeling staining showed that DGKζ was expressed in nucleus of neuron in normal rat brainstem and spinal cord. The number of DGKζ immuno reactive cells greatly decreased in score1-2group. In score3and4group, DGKζ was expressed in neuron and activated microglia. In normal group and each EAE groups, the expression of DGKζ was weak in the fibers of brainstem, but strong in white matter of spinal cord.(2) Immunohistochemical and immunofluorescent double-labeling staining showed that in brainstem TNFa was expressed in cytoplasm in normal group, fibrous structures were TNFa positive. In score1-2group TNFa was expressed in perinuclear area and the number of TNFa immunoreactive cells decreased. In score3and score4group, TNFa was expressed strongly in nucleus and the number of TNFa immuno reactive cells increased obviously. Fibrous structures were TNFa positive. In normal group and each EAE groups, TNFa was expressed in white matter, cytoplasm and synaptic bouton of neurons in gray matter of spinal cord.(3) Immunofluorescent double-labeling staining showed that DGKCζ and TNFa was co-expressed in neurons and fibers.Conclusion:1. The expression of DGKCζ decreased significantly in score1-2group and then increased significantly in score3, suggesting that DGKζ might take part in the pathological changes of EAE in score3phase.2. The expression changes of DGKζ in neuron and activated microglia suggest that DGKζ play important role in the EAE pathological process.3. The protein level of DGKζ showed similar changes to that of TNFa and DGKζ co-expressed with TNFa in neurons and fibers, indicating functional interaction between them.