Study On The Protective Effect And Mechanism Of Over-expressed GLT-1 On Magnesium-free Epilepsy Co-cultured Cortical Neurons

Objective:To study the protective effect and mechanism of glutamate transporter-1（GLT-1）on neurons in magnesium-free extracellular fluid-induced cytoepilepsy models,and to provide a theoretical basis for further revealing that GLT-1 may be an important target for the treatment of epilepsy.Methods:In order to explore the protective effect of overexpressed GLT-1 on neurons in magnesium-free extracellular fluid-induced cytoepilepsy models,healthy SD lactating mouse cerebral cortex within 24 hours of neonatal freshness was selected to perform primary culture of astrocyte（Ast）and neurons,and the purity of cultured cells was identified by immunofluorescence.The purified Ast was lentivirus transfected to overexpress GLT-1;After transfection,Ast was co-cultured with neurons until day 10,and magnesium-free extracellular fluid induced the establishment of a co-cultured cell epilepsy model.Subsequent experiments were divided into normal control group,magnesium-free group,magnesium-free+GLT-1 overexpression lentivirus group（LV-GLT-1）,and magnesium-free+empty vector lentivirus group（Vector-GLT-1）.Flow cytometry was used to detect the concentration of calcium ions([Ca²⁺]i),reactive oxygen species（ROS）levels and apoptosis rate of neurons in neurons.The microplate method detected the content of Glu in the culture supernatant of the co-culture system.In order to further explore the protective mechanism of overexpressed GLT-1 on neurons in magnesium-free extracellular fluid-induced cytoepilepsy models,the expression of GS,SN1,CX43 and SNAT1 in neuronal cells in astrocytes was detected by q RT-PCR and Western Blot,respectively.Results:1.The purity of astrocytes and neuronal cells was above 95%.2.In the magnesium-free extracellular fluid-induced cell epilepsy model,the expression of GLT-1 in astrocytes decreased significantly compared to the control group,neuronal[Ca²⁺]i,ROS level,apoptosis rate of neurons and Glu level in cultured supernatant were significantly more significant than those in the control group,and overexpression of GLT-1 could significantly reverse the above changes.3.In the magnesium-free extracellular fluid-induced cell epilepsy model,the m RNA and protein expression of GS,SN1 and SNAT1 were significantly lower than those in the control group,and the expression of m RNA and protein of CX43 was significantly higher than that in the control group,and overexpression of GLT-1 could significantly reverse the above change process.Conclusions:1.Astrocyte GLT-1 dysregulation may lead to Glu-mediated neuronal damage.2.Overexpression of GLT-1 can accelerate the metabolism of Glu uptake by astrocytes by promoting glutamate and glutamine circulation and affecting the opening of hemichannels,thereby alleviating the neuronal damage induced by magnesium-free extracellular fluid.