Study On The Role Of SSeCKS And PLZF In The Development Of Central Nervous System Inflammatory Reaction

Neuroinflammation constitutes a fundamental process involved in the progression of several central nervous system(CNS)disorders including Alzheimer's disease,Parkinson's disease and stroke.It is considered a common pathophysiological mechanism in a variety of neurological diseases.The role of astrocyte activation in the neuroinflammatory response is increasingly important,which promotes microglia activation and exacerbates its own activation to exacerbate neuroinflammatory effects in the neuroinflammatory response.Neuroinflammation often can cause neuronal damage.This paper is intended to study both astrocyte activation and neuronal damage leading to neuronal damage.In the early work our group has screened the differential proteins in central nervous system.The expression of SSeCKS and PLZF was found to be significantly different.The preliminary study found that SSeCKS plays a key role in the regulation of astrocyte activation,but its mechanism needs to be further explored.PLZF regulates cell proliferation,cell cycle and apoptosis.However,PLZF in the inflammation caused by neuronal damage in the role is unclear.In this paper,the subject is divided into two parts.The first part analyzes the molecular mechanism of SSeCKS regulation in astrocytes.The second part analyzes the expression of PLZF in neuroinflammation and its role in neuronal injury.The two parts are:Part ?.Effect of SSeCKS Regulated TAK1 Activation on Astrocyte ActivationBackground:Astrocyte activation plays an important role in the neuroinflammatory response.The preliminary study found that SSeCKS played a regulatory role in the activation of astrocytes,whose mechanism is unclear.Objective:To study the role of SSeCKS and TAK1 in astrocyte activation and its regulatory mechanism.Methods:Rat primary astrocytes were isolated and cultured,and LPS was used to stimulate astrocytes to induce cell activation.Constructed SSeCKS and TAK1 expression and interference vector,then transfected into astrocytes.Immunoprecipitation was used to detect the expression of protein,and the co-immunoprecipitation was used to detect the interaction.ELISA was used to detect changes in inflammatory cytokine release.Results:The astrocytes were treated with 0 ng/ml,1 ng/ml,10 ng/ml,100 ng/ml,1?g/ml and 10 ?g/ml LPS for 6h.It was found that the expression of SSeCKS and TAK1 increased with the increase of concentration,and reached the highest at 1?g/ml.The phosphorylation of TAK1 also increased with increasing concentration.The expression of SSeCKS and TAK1 was increased with time after treatment with 1?g/ml LPS for Oh,1h,3h,6h,12h and 24h.The phosphorylation of TAK1 was found to reach the highest at 6 h and then gradually decreased.Immunoprecipitation with SSeCKS antibody(AKAP250)and TAK1 was performed.It was found that the interaction between the two reached the highest at 1 ?g/ml LPS treatment at different concentrations of LPS,and the interaction between them increased with time and reached the highest at 6h.In HEK293T cells,SSeCKS and TAK1 were transfected respectively.Immunoprecipitation assay showed that SSeCKS could bind to TAK1 and that exogenous protein binding also increased after LPS treatment.HEK293T cells were transfected with SSeCKS and TAK1 truncated mutant vectors respectively.The interaction between SSeCKS and TAK1 was found to be a multi-site binding by immunoprecipitation and immunoblotting,and TAK1 was bound to SSeCKS through its N-terminus and C-terminus.The combination of SSeCKS and TAK1 promoted by LPS treatment can be inhibited by PKC inhibitors,and activation of astrocytes is inhibited when PKC inhibitors are treated.Detection of subcellular distribution of SSeCKS and TAK1 by plasma membrane separation assay revealed that LPS treatment resulted in an increase in SSeCKS.Increased SSeCKS is mainly located in the cytoplasm,and the expression and activation of TAK1 is also mainly in the cytoplasm.SSeCKS can be localized through its own cytoplasm,thereby promoting the distribution of cytoplasm and promoting TAK1 activation.In astrocytes,interfering with the expression of SSeCKS and TAK1,blocking the interaction between them,p38 signal of MAPK and AKT signal transduction was inhibited,and cell activation of GFAP and PCNA were also inhibited.In addition,the expression of TAK1-binding protein TRAF6 and TAB1 and the secretion of inflammatory factor TNF-a were also inhibited.Conclusion:The expression of SSeCKS and TAK1 in the activation of astrocytes increased with cell activation,and the interaction of SSeCKS and TAK1 increased with the activation of cells.SSeCKS could promote the phosphorylation of TAK1 and promote the downstream P38 and Akt Signal transduction,thereby promoting astrocyte activation.Part II.Role of PLZF in Neuronal Injury Induced by NeuroinflammationBackground:Neuroinflammation can lead to neuronal damage,but its mechanism is unclear.PLZF regulates cell proliferation,cell cycle and apoptosis.However,PLZF in the inflammation caused by neuronal damage in the role is unclear.Objective:To study the role of PLZF in neuronal injury induced by nerve inflammation and its regulatory mechanism.Methods:The inflammatory model of central nervous system caused by LPS injection was established.The expression of protein was detected by immunoblotting,and the cell distribution was detected by immunohistochemistry.Isolated cultured cortical neurons,constructed inflammatory injury model,siRNA transfected interfering protein expression,and detected neuronal survival.Results:The expression of PLZF was detected at Oh,3h,6h,9h,12h,Id,3d,5d and 7d after LPS injection.The expression of PLZF was increased at 9h and reached its peak on the third day.Immunohistochemical staining showed that PLZF was relatively low in the sham-operated cortex and markedly increased 3 days after LPS injection.Immunofluorescence staining showed that PLZF was associated with NeuN-positive neurons,GFAP-positive astrocytes and CD11b-positive microglia,but increased PLZF was predominantly in neurons.It was further found that the expression of caspase-3,cyclinDl and CDK4 activated by LPS injection was time-dependent and reached the highest level on day 3,which was consistent with PLZF expression.Immunofluorescence showed that activated caspase-3 positive cells were co-located in neurons.The expression of PLZF was up-regulated and the expression of activated caspase-3,cyclinD1 and CDK4 was up-regulated in mixed glial cellsCM-treated neuronal model.The expression of caspase-3,cyclinD1 and CDK4 was decreased by using siRNA to knock down the expression of PLZF in neuronal cells.Intravenous detection of neuronal apoptosis revealed that knockdown of PLZF expression could inhibit neuronal apoptosis.Conclusion:PLZF is up-regulated in brain tissue of neuroinflammatory response.PLZF is mainly located in neurons and is associated with neuronal damage.The expression of PLZF in cortical neurons in vitro can inhibit CM-induced neuronal injury.