摘要
目的:癫痫是诸多原因导致的以大脑神经元细胞异常放电为主要特征的中枢神经功能失常综合征。寻找新的癫痫药物治疗靶点一直是神经病学研究的重点和热点。2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)通过介导K_(ATP)通道亚基Kir6.1、Kir6.2 mRNA和蛋白质的上调而发挥抗癫痫作用。通过使用TargetScan和miRBase的数据库对miRNA及相关靶基因的序列进行互补配对分析推测miR-194可能是K_(ATP)通道的上游信号分子。本研究旨在探讨2-DG通过miR-194调节K_(ATP)通道亚基Kir6.1、Kir6.2发挥抗癫痫作用的机制。方法:建立无镁癫痫细胞模型,将细胞随机分为对照组、癫痫组(EP组)、EP+2-DG组、miR-194干预组(包括EP+miR-194 mimic、EP+miR-194 mimic+2-DG、EP+mimic control、EP+miR-194 inhibitor、EP+miR-194 inhibitor+2-DG及EP+miR-194 inhibitor control共6组)。使用2-DG对miR-194模拟物进行干预,采用膜片钳方法检测自发性复发性癫痫样放电,应用real-time PCR检测神经元miR194、Kir6.1、Kir6.2的mRNA表达,蛋白质印迹法检测Kir6.1、Kir6.2的蛋白质表达水平。结果:与对照组相比,EP组自发放电电位幅度差异无统计学意义(P>0.05),自发放电频率增加(P<0.05)。与EP组相比,EP+2-DG组自发放电频率降低(P<0.05)。与EP+miR-194 mimic control组相比,EP+miR-194 mimic组Kir6.1、Kir6.2的mRNA和蛋白质表达均下调(均P<0.05)。与EP+miR-194 inhibitor control组对比,EP+miR-194 inhibitor组Kir6.1、Kir6.2的mRNA和蛋白质表达均上调(均P<0.05)。经miR-194模拟物预处理后,K_(ATP)通道亚基Kir6.1、Kir6.2 mRNA及蛋白质表达水平降低。与EP+2-DG组相比,EP+miR-194 mimic+2-DG组Kir6.1、Kir6.2的mRNA和蛋白质表达均下调(均P<0.05);EP+miR194 inhibitor+2-DG组Kir6.1、Kir6.2的mRNA和蛋白质表达均上调(均P<0.05)。结论:2-DG可能通过miR-194上调K_(ATP)通道亚基Kir6.1、Kir6.2的表达发挥抗癫痫作用。
Objective:Epilepsy is a syndrome of central nervous system dysfunction caused by many reasons,which is mainly characterized by abnormal discharge of neurons in the brain.Therefore,finding new targets for epilepsy therapy has always been the focus and hotspot in neurological research field.Studies have found that 2-deoxy-D-glucose(2-DG)exerts anti-epileptic effect by up-regulation of K_(ATP) channel subunit Kir6.1,Kir6.2 mRNA and protein.By using the database of TargetScan and miRBase to perform complementary pairing analysis on the sequences of miRNA and related target genes,it predicted that miR194 might be the upstream signaling molecule of K_(ATP) channel.This study aims to explore the mechanism by which 2-DG exerts its anti-epileptic effect by regulating K_(ATP) channel subunits Kir6.1 and Kir6.2 via miR-194.Methods:A magnesium-free epilepsy model was established and randomly divided into a control group,an epilepsy group(EP group),an EP+2-DG group,and miR-194 groups(including EP+miR-194 mimic,EP+miR-194 mimic+2-DG,EP+miR-194 mimic control,EP+miR-194 inhibitor,EP+miR-194 inhibitor+2-DG,and EP+miR-194 inhibitor control groups).The 2-DG was used to intervene miR-194 mimics,patch-clamp method was used to detect the spontaneous recurrent epileptiform discharges,real-time PCR was used to detect neuronal miR-194,Kir6.1,and Kir6.2 expressions,and the protein levels of Kir6.1 and Kir6.2 were detected by Western blotting.Results:Compared with the control group,there was no significant difference in the amplitude of spontaneous discharge potential in the EP group(P>0.05),but the frequency of spontaneous discharge was increased(P<0.05).Compared with the EP group,the frequency of spontaneous discharge was decreased(P<0.05).Compared with the EP+miR194 mimic control group,the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 mimic group were down-regulated(all P<0.05).Compared with the EP+miR194 inhibitor control group,the mRNA and protein expressions of Kir6.1 and Kir6.2 in the EP+miR-194 inhibitor group
作者
阳衡
张忱
YANG Heng;ZHANG Chen(Department of Neurology,Third Xiangya Hospital,Central South University,Changsha 410013,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2022年第8期1099-1107,共9页
Journal of Central South University ：Medical Science
基金
国家自然科学基金(81801295)。
