艾地苯醌对癫痫大鼠模型神经...量代谢及抗氧化作用机制研究_崔小丽.pdf

44现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023艾地苯醌对癫痫大鼠模型神经元线粒体功能、能量代谢及抗氧化作用机制研究崔小丽，孙宁，赵瑞，马妮，贾瑞华(陕西省人民医院神经内一科，西安 710068）摘要：目的建立癫痫模型，研究艾地苯醌（Idebenone）的抗癫痫作用机制。方法30 只 SD 大鼠，分为空白对照组（正常大鼠）、建模组和建模+艾地苯醌干预组（每组 10 只），建模组和干预组均采用经典氯化锂-匹罗卡品的癫痫诱导方法建立癫痫大鼠模型，艾地苯醌干预 30 天后检测大鼠血清、海马体组织中超氧化物歧化酶活性（superoxide dismutase,SOD）和丙二醛（malondialdehyde,MDA）含量的变化，通过 Nissl 染色评估艾地苯醌对神经元的保护作用。利用 CCK-8 明确艾地苯醌对癫痫细胞模型的最低适用浓度。在无镁诱导的癫痫细胞模型中检测 ATP 生成和线粒体膜电位，评价艾地苯醌对神经元线粒体产能功能的影响。结果建模组大鼠血清 SOD 活性为 190.2518.17 U/ml,较对照组（467.2223.43 U/ml）降低，建模组海马组织中 SOD 活性（107.349.33 U/ml）较对照组（298.7715.32 U/ml）降低，差异均具有统计学意义（t=-22.10，-16.30，均 P 0.05）；艾地苯醌干预后大鼠血清和海马组织中 SOD 活性有所恢复（384.7929.21 U/ml，212.0824.32 U/ml），与建模组相比升高，差异具有统计学意义（t=21.06,13.62，均 P 0.05）。MDA 的检测结果表明，与对照组相比（7.330.87 nmol/L），建模组大鼠血清中 MDA 含量增加（14.010.93 nmol/L），海马组织中 MDA 含量（23.471.89 nmol/L）也较对照组（11.031.28 nmol/L）升高，差异具有统计学意义（t=5.72，9.19，均 P 0.05）；艾地苯醌干预后癫痫鼠血清与海马组织中 MDA 含量均下降（9.350.83 nmol/L,13.771.34 nmol/L），与造模组相比差异具有统计学意义（t=-17.68，-22.87，均 P 0.05）。Nissl 染色提示艾地苯醌干预后活性神经元数目增加（1 977200 个/mm2）,与造模组(1 387146 个/mm2)相比差异具有统计学意义（t=3.32，P 0.050）。细胞学实验表明，艾地苯醌干预能够提高线粒体 ATP 产能（0.920.14 vs 0.580.04），差异具有统计学意义（t=3.75,P=0.000）；四甲基罗丹明甲酯（TMRM）荧光强度检测表明艾地苯醌干预后线粒体膜电位显著提高（0.970.1 vs 0.480.06）,差异具有统计学意义（t=6.59,P=0.000）。结论适量艾地苯醌可能通过保护线粒体功能，降低氧化应激对神经元的损伤发挥抗癫痫作用。关键词：癫痫；艾地苯醌；氧化应激；神经元；线粒体中图分类号：R446.19文献标识码：A文章编号:1671-7414（2023）01-044-06doi:10.3969/j.issn.1671-7414.2023.01.009Study on the Mechanism of Idebenone on Mitochondrial Function,Energy Metabolism and Antioxidation in Epileptic Rats ModelCUI Xiao-li，SUN Ning，ZHAO Rui，MA Ni，JIA Rui-hua(Department of Neurology，Shaanxi Provincial Peoples Hospital，Xian 710068，China)Abstract:ObjectiveTo study the function of idebenone on neurons and the underlying mechanism in epileptic models.Methods30 SD rats were divided into control group(normal rats),modeling group and modeling+idebenone intervention group(n=10 rats in each group).The classic lithium chloride-pilocarpine induced epilepsy model was established to detect the effects of idebenone on superoxide dismutase（SOD）activity and malondialdehyde（MDA）level in serum and hippocampus after 30 days of intervention.Function of idebenone on neurons was detected by Nissl staining.CCK-8 was used to determine the minimum appropriate concentration of idebenone for epilepsy cell models.ATP production and mitochondrial membrane potential were detected in the Mg-free epilepsy cell mode.ResultsThe serum SOD activity in the modeling group was 190.2518.17 U/ml,which was lower than that in the control group(467.2223.43 U/ml),and the SOD activity in the hippocampus of the modeling group（107.349.33 U/ml）was lower than that in the control group(298.7715.32 U/ml).The differences were statistically significant(t=-22.10,-16.30,all P0.05).SOD activity in serum and hippocampal tissue of rats recovered after intervention of idebenone,(384.7929.21 U/ml,212.0824.32 U/ml),which was higher than that of the modeling group,with statistical difference(t=21.06,13.62,all P 0.05).Compared with the control group(7.330.87 基金项目：陕西省自然科学基础研究计划(2021JQ-903)：艾地苯醌对癫痫持续状态大鼠学习记忆的影响及其机制的研究。作者简介：崔小丽（1983-），女，硕士，副主任医师，研究方向：癫痫和神经重症，E-mail：。通讯作者：贾瑞华（1986-），男，博士，主治医师，研究方向：癫痫，E-mail:。45现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023nmol/L),the content of MDA in serum in the modeling group was increased(14.010.93 nmol/L).MDA content in hippocampus(23.471.89 nmol/L)was higher than that in control group(11.031.28 nmol/L),and the difference was statistically significant(t=5.72,9.19,all P 0.05).MDA in serum and hippocampal tissue of epileptic rats decreased(9.350.83 nmol/L,13.771.34 nmol/L)after idebenone intervention,and the difference was statistically significant compared with the model group(t=-17.68,-22.87,P0.05).Nissl staining indicated that the number of active neurons increased after idebenone intervention(1 977200 cells/mm2),and the difference was statistically significant compared with the model group(1 387146 cells/mm2)(t=3.32,P0.050).Idebenone intervention could increase mitochondrial ATP productivity(0.920.14 vs 0.580.04),and the difference was statistically significant(t=3.75,P=0.000).TMRM fluorescence intensity assay showed that the mitochondrial membrane potential was significantly increased after the intervention of idebenone(0.970.1 vs 0.480.06),the difference was statistically significant(t=6.59,P=0.000).ConclusionIdebenone can inhibit the oxidative stress induced neuronal damage in epileptic seizures by protecting the capacity of mitochondria and play an antiepileptic role.Keywords:epilepsy；idebenone；oxidative stress；neurons；mitochondria 线粒体是细胞重要的产能结构，神经元线粒体功能对于维持神经元稳态和生理功能具有重要意 义1-4。癫痫常伴随有神经元异常激活5-8，导致神经系统对能量的需求增加9-12，线粒体是活性氧产生的主要位点，极易发生氧化应激相关的损伤，导致神经元缺少 ATP,离子稳态失衡，进一步加剧氧化应激，因此癫痫的进展常伴随有氧化应激平衡的失调和线粒体功能损伤8,13。最新研究表明，神经系统能量代谢稳态的恢复对于改善药物难治性癫痫患者的治疗现状具有重要作用14-17。因此以线粒体为靶标的神经系统疾病的治疗越来越受到人们重 视18。我们前期的研究表明艾地苯醌可减少癫痫持续状态大鼠自发发作的次数及频率，发挥抗癫痫作用19，而作为一种可溶性辅酶 Q10 的结构类似物，艾地苯醌在癫痫发作时对线粒体功能、抗氧化的影响仍有待研究20-21。本研究拟从线粒体功能和能量代谢的角度研究艾地苯醌的抗癫痫作用，为寻找新的抗癫痫药物提供理论依据。1材