Insulin Attenuates Seizure-Induced Oxidative Stress Via Increasing Zinc-α2-Glycoprotein In Primary Cultured Cortical Neurons

Objective:Our previous study found that ZAG expression was decreased in epilepsy patients and rat models,and ZAG could inhibit epilepsy and epileptiform discharges.Either insulin or overexpressing ZAG suppresses seizure and epileptic discharges,while ZAG is known to influence insulin sensitivity of tissues.But whether insulin regulates ZAG is unknown.Oxidative stress plays an important role in epilepsy,while insulin and ZAG are associated with oxidative stress in various diseases.However,it is unclear whether ZAG presents anti-oxidative stress in epilepsy.This study aimed to investigate the effect of insulin on the expression of ZAG in primary cultured cortical neurons and its mechanism,and to explore the role of insulin and ZAG in seizure-induced oxidative stress.Methods:1.Rat primary cortical neurons were extracted and cultured,and the expression of ZAG in neurons was detected by double-labeled immunofluorescence;2.Different concentrations of insulin（0nM、10nM、50nM、100nM、200nM、500nM）were used to treat cortical neurons.MTS analysis of neuronal viability in different insulin concentrations was performed.RT-qPCR and Western blot were used to detect the changes of AZGP1mRNA and ZAG expression in neurons under different concentrations of insulin.And further explored its mechanism by treatment with AXL1717（IGF-1R antagonist）and BMS-754807（IR and IGF-1R antagonist）;3.Seizure model in cultured neurons was established by Mg²⁺-free extracellular fluid treatment,and the changes of AZGP1 mRNA and ZAG expression were determined by RT-qPCR and Western blot;4.Verification of the transfection effect of LV:primary cortical neurons were divided into 4 groups and transfected with LV:overexpression vector group（Vector）,overexpression group（LV-AZGP1）,low expression vector group（LV-RNAi-Vector）,low expression group（LV-RNAi）.Fluorescence scanning,RT-qPCR and western blot were used to detect the transfection effect;5.Exploration of the mechanism of insulin and ZAG in seizure-induced oxidative stress:cultured neurons were randomly divided into 8 groups and given different treatment factors:Control,Seizure,Vector,LV-AZGP1,LV-RNAi-Vector,LV-RNAi,Insulin and Insulin+LV-RNAi.Observation the levels of O^2-and statistics under Fluorescence scanning.Result:1.ZAG was identified in primary cortical neurons;2.MTS analysis showed that insulin treatment below 200nM had no effect on neuronal activity,but 500nM insulin reduced neuronal activity（P<0.05）.The level of AZGP1 mRNA and ZAG were increased in neurons treated with insulin ranged from 0 to 200nM,and the effect was most obvious at 200nM.Either AXL1717 or BMS-754807 treatment inhibited insulin-induced ZAG elevation;3.The level of AZGP1 mRNA and ZAG was significantly decreased in seizure model compared to controls（P<0.01）;4.The GFP-positive cells in primary neurons were visualized.The expression of AZGP1 mRNA and ZAG was significantly increased in LV-AZGP1 group compared to Vector（P<0.01）.The expression of AZGP1mRNA and ZAG was significantly decreased in LV-RNAi group compared to LV-RNAi-Vector（P<0.01）;5.The production of O^2-was increased in seizure group.Overexpression of AZGP1 or insulin treatment could reduce production of O^2-compared to seizure group,while knock-down of AZGP1 increased O^2-production.The production of O^2-in Insulin+LV-RNAi group was increased compared to insulin group.Conclusion:Insulin-induced ZAG increase in neurons was mainly related to activation of IGF-1R.Insulin presented its antioxidative-stress effect in neuronal seizure models via increasing ZAG.