【作者】 阳衡；

【导师】 宋治；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 2-DG抗痫作用的评价目的：探讨2-DG抗痫作用的疗效。方法：6-8周成年雄性C57BL/6小鼠进行随机分配为对照组、致痫组、2-DG干预组,建立匹罗卡品致痫模型。对不同组行为学的变化进行监测,观察其自发性痫性发作的癫痫潜伏期、癫痫评分、痫性发作持续时间,同时观察不同组脑电图的变化。结果：1.对不同组(对照组、致痫组、2-DG干预组)行为学监测显示：与对照组相比,匹罗卡品致痫后,致痫组、2-DG干预组41.5%的C57BL/6小鼠存在自发性痫性发作。相对于致痫组,中、高剂量2-DG干预组小鼠潜伏期增加,癫痫发作评分、痫性发作持续时间降低(癫痫潜伏期：15±4分钟VS35±4、33±5分钟；癫痫评分为5.1±0.5VS3.9±0.4、3.8±0.5；痫性发作持续时间为122±7分钟VS35±6分钟、42±7分钟),而且有统计学意义。2.对不同组(对照组、致痫组、2-DG干预组)脑电图监测显示：对照组脑电图是以α、β波为主且波幅较小；匹罗卡品致痫模型组可见大量的尖波、棘波和尖慢波：2-DG干预组脑电图是也主要是以α、β波为主且波幅较小。结论：2-DG在匹罗卡品癫痫模型中具有抗痫作用。2-DG上调KATP亚基Kir6.1、Kir6.2发挥抗痫作用目的：研究糖酵解抑制剂2-DG对ATP依赖性钾离子通道亚基Kir6.1、 Kir6.2mRNA和蛋白表达的影响与抗痫作用的关系。方法：1.6-8周成年雄性C57BL/6小鼠进行随机分配为对照组、致痫组、2-DG干预组,建立匹罗卡品致痫小鼠模型。将造模成功的小鼠(致痫组,2-DG干预组)在癫痫持续状态(SE)后4小时、1天、7天、30天、60天和正常生理盐水对照组(各组n=4-6)的海马组织应用Real-time PCR方法检测使用糖酵解抑制剂2-DG前后各组小鼠海马组织中ATP敏感性钾通道亚基Kir6.1、Kir6.2mRNA的变化。2.将造模成功的小鼠(致痫组、2-DG干预组)在癫痫持续状态(SE)后4小时、1天、7天、30天、60天和对照组(各组n=4-6)的海马组织应用western-blot方法检测糖酵解抑制剂2-DG前后各组小鼠海马组织中ATP敏感性钾通道亚基Kir6.1、Kir6.2mRNA蛋白的变化。结果：1.相对于对照组,在第1、7、30天致痫组小鼠海马组织Kir6.1和Kir6.2mRNAs上调,而且有统计学意义；相对于致痫组,在第1、7、30天中、高剂量2-DG干预组海马组织Kir6.1和Kir6.2mRNAs上调,而且有统计学意义。在其它时间点差异不明显。2.相对于对照组,在第1和30天致痫组小鼠海马组织Kir6.1蛋白上调,而且有统计学意义；在第1、7天致痫组小鼠海马组织Kir6.2蛋白上调,而且有统计学意义；相对于致痫组,在第1天中、高剂量2-DG干预组海马组织Kir6.1蛋白上调,而且有统计学意义。在第1、7、30天中、高剂量2-DG干预组海马组织Kir6.2蛋白上调,而且有统计学意义。在其它时间点差异不明显。结论：糖酵解抑制剂2脱氧葡萄糖上调KATP亚基Kir6.1、Kir6.2mRNA和蛋白表达发挥抗痫作用。2-DG经KATP通道抗痫作用机制体外实验研究目的：探讨2-DG经KATP通道抗痫作用的机制方法：1.在体外海马脑片CA3区,给予10uM荷包牡丹碱、7.5mM高钾模拟痫性发作,诱发动作电位频率增加,应用盲法膜片钳技术监测使用10mM2-DG前后神经元细胞动作电位频率的变化。2.在体外海马脑片CA3上,高频电刺激诱发LTPGlu模拟致痫模型,记录给予2-DG后海马脑片CA3区神经元LTPGlu(谷氨酸介导突触传递的长时程增强)的变化；给予KATP通道激活剂Diazoxide (300nM),LTPGlu的变化以及在KATP通道抑制剂Gliben (20uM)预处理后给予10mM2DG后LTPGlu的变化。结果：1.在体外海马脑片CA3区上,10mM2-DG降低荷包牡丹碱和高钾致痫模型的动作电位频率。2.在体外海马脑片CA3区上,高频电刺激诱发LTPGlu模拟致痫模型,10mM2-DG阻挡高频电刺激诱发LTPGlu。3. Kir6.2通道激动剂Diazoxide (300nM)阻挡高频电刺激诱发海马CA3区神经元LTPGlu, Kir6.2通道阻断剂Gliben (20uM)拮抗2-DG (10mM)阻挡高频电刺激诱发海马CA3区神经元LTPGlu作用。结论：2-DG在体外脑片致痫模型上具有抗痫作用,其机制是经KATP通道的激活而发挥抗痫作用。2-DG抗痫作用的信号通路研究目的：研究糖酵解抑制剂2-DG抗痫作用的信号通路。方法：1.在体内匹罗卡品致痫模型上,采用Elisa技术记录使用糖酵解抑制剂2-DG前后静脉血中DAG的变化。2.在体外海马脑片上,高频电刺激诱发LTPGlu模拟致痫模型,给予PKC激动剂phorbol(PMA)预处理后,应用盲法膜片钳技术记录给予2-DG前后LTPGlu的变化。结果：1.在体内匹罗卡品致痫模型中,相对于对照组,在第1天致痫组小鼠静脉血DAG上调,而且有统计学意义(p<0.05)；在第7、30、60天致痫组小鼠静脉DAG下调,其中第7、30天致痫组小鼠静脉DAG下调有统计学意义(p<0.05)。2.相对于致痫组,在第1天中、高剂量2-DG干预组小鼠静脉DAG上调,而且有统计学意义(p<0.01)；在第7、30、60天中、高剂量2-DG干预组小鼠静脉DAG下调,其中第7、30天致痫组小鼠静脉DAG下调有统计学意义(p<0.05),在其它时间点差异不明显。3.在体外海马脑片上,PKC激动齐(?)phorbol(500nM)拮抗2-DG(10mM)阻挡高频电刺激诱发海马CA3区神经元LTPGlu作用结论：DAG的下降与2-DG的抗痫作用有关；PKC激动剂能抑制2-DG的抗痫作用。更多还原

【Abstract】 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.更多还原