Effects Of Inflammatory Cytokines On The Activation Of Astrocytes And The Expression Of SPLA2-?A

Background and Objective: Different degrees of inflammatory response occur in various central nervous system diseases,such as head injury,stroke,epilepsy and degenerative diseases.Inflammatory cytokines promote the occurrence and development of inflammatory response,and inflammatory cytokines are produced during inflammatory response.Astrocytes are the largest group of cells in the brain,which play an important role in inflammatory response.Secreted phospholipase A2 belongs to a class of phospholipid hydrolase family,which is closely related to inflammation.The purpose of this study is exploring the methods of isolation,purification and culture of astrocytes from rat cerebral cortex.Providing the basis for cytology of further research.To study the changes of the activity of astrocytes and the expression of sPLA2-?A,and to explore the possible mechanism.Methods: On the basis of primary culture of rat cerebral cortical astrocytes in vitro,the fourth generation of cultured cells were used for the experiment.The cells were randomly divided into control group and experimental group.The experimental group were: 10 ng/ml IL-1? group,100 ng/ml IL-1? group,10 ng/ml TNF-? Group,100 ng/ml TNF-? Group,10 ng/ml IL-1? plus 10 ng/ml TNF-? Group,100 ng/ml IL-1? plus 100 ng/ml TNF-? group.24 hours later,cell viability was detected by CCK-8 assay.Then choose 10 ng/ml IL-1? plus 10 ng/ml TNF-? group to continue the experiment.Detection the expressin changes of IKK?,IKK?,NF-?B,p65 and sPLA2-?A by real-time quantitative PCR and ELISA.Results: Through the separation and purification,the typical rat cortical astrocytes were obtained.The morphology of astrocytes was irregular,the neurites were longer and radial.GFAP staining showed that the purity of astrocytes was over 95%.The activity of astrocytes in the experimental group was higher than that in the control group.Among them,the activity of astrocytes in 100 ng/ml IL-1? group,100 ng/ml TNF-? group,10 ng/ml IL-1? plus 10 ng/ml TNF-? group and 100 ng/ml IL-1? plus 100 ng/ml TNF-? group were significantly higher than the control group,with statistical significance(P<0.05,P<0.001,P<0.001,P<0.001).10 ng/ml IL-1? and 10 ng/ml TNF-? group compared with the control group,the expression of sPLA2-?A at mRNA and protein levels were significantly increased,with statistical significance(P<0.05).The expression of NF-?B at mRNA and protein levels in 10 ng/ml IL-1? and 10 ng/ml TNF-? group was significantly higher than the control group,with statistical significance(P<0.05).Compared with the control group,the expression of 10 ng/ml IL-1? and 10 ng/ml TNF-? group IKK?,IKK? and p65 were increased,with statistical significance(P<0.05,P<0.05,P<0.05).Conclusion: When stimulated by inflammatory cytokines,astrocytes actived meanwhile the expression of sPLA2-?A increased,which may be produced by the IKK?/IKK?-NF-?B-sPLA2-?A pathway.