The Mechanism Of Antiepileptic Effect Of Glycolytic Inhibitor2-Deoxy-D-Glucose

Evaluation of the efficacy of antiepileptic effect of2-DGObjective:Explore the efficacy of antiepileptic effect of2-DGMethods:C57BL/6adult mice of6-8weeks were divided randomly into controls,seizure group and2-DG control group, and establish the seizure model of mice induced by pilocarpine. Observe the behavioral changes in different groups. Observe the latent period of seizure、score for seizure and time of duration in seizure group and2-DG intervention group. Meanwhile. Observe the EEG changes in different groups.Results:1.Different group (Control group, seizure group,2-DG intervention group) behavioral monitoring:Compared with Control group,41.5%of seizure group and2-DG intervention group mice have spontaneous seizures induced by pilocarpine. Compared with seizure group, the latent period of seizure prolonged and score for seizure and time duration of seizure shortened in middle and high dose2-DG control groups with statistical significance (the latent period of seizure:15±4min VS35±4、33±5min; score for seizure5.1±0.5VS3.9±0.4、3.8±0.5; time duration of seizure122±7minVS35±6、42±7min)2.Different group (Control group, seizure group,2-DG intervention group) EEG monitoring:the result of normal control group EEG is mainly alpha, beta-wave. and the amplitude is relatively lower. the result of seizure group is a large number of sharp、spikes and sharp slow-wave;And the result of2-DG intervention group EEG is mainly alpha, beta-wave. and the amplitude is relatively lower.Conclusion:In pilocarpine epilepsy model.2-DG play antiepileptic effect. The antiepileptic effect of2-Deoxy-D-Glucose is mediated by up-regulation of ATP-sensitive potassium channel Subunits Kir6.1and Kir6.2Objective:Investigate the relationship between the antiepileptic effect of glycolysis inhibitors2-deoxyglucose and expressions of Kir6.1and Kir6.2subunits mRNA and protein of ATP-sensitive potassium channel (KATP channel).Methods:1. C57BL/6adult mice of6-8weeks were divided randomly into controls, seizure group and2-DG control group, and establish the seizure model of mice induced by pilocarpine.Apply Real-time PCR to detect expressions of Kir6.1and Kir6.2mRNA of ATP-sensitive potassium channel in vivo before and after treatment of glycolysis inhibitors2-DG in4hours,1day,7days,30days,60days of successfully modeling mice in seizure group and2-DG experiment group, and compare with the controls(n=10for each group).2. Apply western-blot to to detect change of Kir6.1and Kir6.2protein in vivo before and after treatment of glycolysis inhibitors2-DG in4hours,1day,7days,30days,60days of seizure group,2-DG experiment group and controls.Results:1. Compared with control group, Kir6.1and Kir6.2mRNA were up-regulated in hippocamal tissue of1、7、30days seizure groups with statistical significance; Compared with seizure group, Kir6.1and Kir6.2mRNA were up-regulated in hippocamal tissue in1、7、30days high does2-DG experiment group significantly, and insignificantly in other time points.2.Compared with control group, Kir6.1protein up-regulated in hippocamal tissue of1and30days seizure groups significantly; Kir6.2protein up-regulated in1and7days seizure groups significantly; Compared with seizure group, Kir6.1protein up-regulated in the first day of high does2-DG group significantly; in1,7,30days high does2-DG groups, Kir6.2protein up-regulated significantly, and insignificantly in other time points.Conclusion:Glycolysis inhibitors2-deoxy glucose plays antiepileptic effect through mediating up-regulation of ATP-sensitive potassium channel subunits Kir6.1and Kir6.2. The study of2-DG plays antiepileptic effect through mediating the ATP-sensitive potassium channel in vitroObjective:Explore the mechanism of2-DG play antiepileptic effect through mediating ATP-sensitive potassium channel.Methods:1.Give lOuM bicuculine,7.5mM high potassium stimulate seizure and induce the increase of the frequency of action potential in vitro CA3region of hippocamal slice. apply blinded clamp patch to record change of neural action potential frequency before and after intervention of10mM2-DG.2. establish high frequency stimulation induce long-term potentiation (LTPGlu) which simulate model of epilepsy in vitro CA3region of hippocamal slice, apply blinded clamp patch to record change of glutamic acid mediated LTPGlu of synaptic transmission before and after intervention of2-DG、the activator of KATP Diazoxide (300nM) and inhibitor of KATP Gliben (20uM)Results:1.2-DG lowers neural discharge frequency in CA3region neurons of hippocamal slice in bicuculine and high potassium epilepsy models.2.2-DG blockades LTPGlu in high frequency stimulation epilepsy model in CA3region neurons of hippocamal slice3. Kir6.2channel activator Diazoxide lowers LTPGlu in CA3region neurons of hippocamal slice in high frequency stimulation epilepsy model, while Kir6.2channel inhibitor Gliben presents inhibition in the antiepileptic effect of2-DGConclusion:2-DG exhibits antiepileptic effect in vitro brain slices epileptogenic models. And the mechanism is2-DG play antiepileptic effect through mediating the activation of ATP-sensitive potassium channel. The study of the signaling pathway of antiepileptic effect of2-DGObjective：Study signaling pathway of antiepileptic effect of2-DG.Methods:1. In vivo pilocarpine epilepsy model, apply Elisa to detect venous blood DAG before and after use of2-DG.2.Establish high frequency stimulation models in vitro hippocamal slice, apply blinded clamp patch to record change of LTPGlu pretreated by2-DG after intervention of PKC activator phorbol(PMA).Results:1. In vivo pilocarpine epilepsy model, compared with control group, venous blood DAG of1day seizure group up-regulated significantly (p<0.05); DAG in7,30and60days seizure groups down-regulates, significantly in7and30days groups (p<0.05).2. Compared with seizure group, DAG of1day high-does2-DG experiment group up-regulated significantly (p<0.05), and down-regulates in7,30and60days high-does2-DG experiment groups, significantly in7and30days groups (p<0.05) and insignificantly in other time points.3. In vitro hippocamal slice, PKC activator phorbol (500nM) shows to inhibit the2-DG effect of lowering neuron LTPGlu.Conclusion:There is a relationship between the reduce of DAG and the anti-epileptic effect of2-DG. PKC agonists can inhibit the anti-epileptic effect of2-DG’s.