Research On The Interaction Between Glial Cells In The Regulation Of Epilepsy By TNFα-TNFR1-NFκB

Objective:To study the activation of BV2 microglia induced by KA,and use it to induce astrocytes to observe the role of TNFα-TNFR1-NFκB in epileptic neuroinflammatory response.Methods:Using microglia epilepsy model induced by KA,CCK8 was used to detect the effects of different concentrations of KA on the viability of microglia,the release of TNF-α,IL-1β,IL-6 was detected by ELISA after KA stimulation for different times.The expression levels of apoptosis related proteins Caspase-3,Caspase-9 and p53 in microglia were detected by Western blot;the changes of intracellular calcium concentration in microglia were detected by cell calcium imaging.The primary mouse astrocytes were extracted and cultured to the third generation.The purity was identified by immunofluorescence.The astrocytes were randomly divided into four groups:microglia culture medium（MCM）group,KA stimulated microglia culture medium（KA-MCM）group and TNF-αNeutralization group and TNFR1antagonism group.The cells in MCM group were treated with MCM for 6 hours;the cells in KA-MCM group were treated with KA-MCM for 6 hours;in TNF-αneutralization group,KA-MCM was pretreated with TNFαneutralization for 2 hours,then treated astrocytes;The astrocyte in TNFR1 antagonist group were treated with TNFR1 antagonist for 2 hours,and then treated with KA-MCM for 6 hours.The expression of NF-κB p65,p-NF-κB p65 and IκBαin astrocytes was detected by Western blot,the nuclear translocation of NF-κB p65 was detected by immunofluorescence.Results:（1）KA induces inflammation and promotes apoptosis of BV2 microglia in vitroCCK8 results showed that the activity of BV2 microglia decreased gradually with the increase of KA concentration.The results of ELISA detection of inflammatory factors showed that TNF-α,IL-1β,IL-6 increased gradually with the extension of KA stimulation time.Western blotting showed that the expressions of apoptosis related proteins Caspase-3,Caspase-9 and p53 in BV2 microglia in KA group were significantly higher than those in control group（P<0.05）.Calcium imaging detected the intracellular Ca²⁺level of BV2 microglia.It was found that the Ca²⁺level of BV2 cells in KA group was significantly higher than that in control group（P<0.0001）.（2）TNFα-TNFR1 regulates the activation of astrocytes by microgliaWestern blotting showed that compared with MCM group,the phosphorylation of NF-κB p65 was higher（P<0.001）and IκBαwas lower（P<0.01）.This change can be weakened by TNFαneutralization（p-NF-κB p65:P<0.05,IκBα:<0.01）and TNFR1inhibition（p-NF-κB p65:P<0.01;IκBα:P<0.001）.The results of cellular immunofluorescence showed that the nuclear translocation of NF-κB p65 increased in KM group,which could be reversed by TNF-αneutralization and TNFR1 antagonism.Conclusion:KA can activate BV2 microglia and promote the apoptosis of BV2 microglia,so as to play a neurotoxic role.Microglia culture medium activated by KA can activate astrocytes and block TNFα-TNFR1 between them can inhibit the cascade inflammatory response between glial cells.