天麻钩藤饮对癫痫小鼠海马区miRNA-9表达的影响及对神经损伤的保护机制
Analysis on the effects of Tianma Gouteng Decoction on miRNA-9 expression in hippocampus of epileptic mice and its protective mechanism against nerve injury
陈宏 1冯大庆 1张明浩 1李可心 1邓琳千 1李淼 1李贵星 1宋倩男1
作者信息
- 1. 黑龙江中医药大学附属第一医院儿科一科,哈尔滨 150040
- 折叠
摘要
目的:探讨天麻钩藤饮干预后癫痫小鼠脑电图的变化、小鼠海马区miRNA-9表达水平的变化以及对神经损伤的保护机制.方法:50只雄性昆明小鼠,随机分成对照组、模型组、卡马西平组、中药高剂量组和中药低剂量组,每组10只.造模前1周,对照组和模型组均予20 mL·kg-1·d-1 0.9％氯化钠溶液进行灌胃;卡马西平给予30 mg·kg-1·d-1卡马西平混悬液灌胃,中药高、低剂量组分别予天麻钩藤饮按30、10 mL·kg-1·d-1灌胃.通过盐酸匹罗卡品构建小鼠急性癫痫模型.对各组小鼠进行行为学和脑电图观察.采用实时荧光定量PCR技术检测小鼠海马区miRNA-9表达水平的变化;并且通过免疫组化检测神经元特异核蛋白(NeuN)标记的神经元核抗原、FJB标记损伤的神经元与人血小板内皮细胞黏附分子-1(PECAM-1)标记的血小板内皮细胞黏附分子、紧密连接蛋白-1(ZO-1)标记的胞质紧密连接蛋白1,蛋白印迹法测定ZO-1/ZONAB的水平.结果:与对照组比较,模型组小鼠癫痫发作的频率多、持续时间长、发作级别高(P＜0.01),小鼠脑电图尖波、棘波、棘慢波明显增多,海马区miRNA-9的表达明显下调(P＜0.01);与模型组比较,中药高、低剂量组小鼠癫痫发作频率、持续时间及发作级别减少(P＜0.05),小鼠脑电图痫性波形减少,小鼠海马区miRNA-9的表达升高(P＜0.01).与模型组比较,3个用药组NeuN细胞显著增多(P＜0.05),损伤的神经元细胞显著减少(P＜0.05),中药高、低剂量组海马CA3区PECAM-1免疫反应显著降低(P＜0.05),ZO-1蛋白水平显著降低(P＜0.05),而ZONAB蛋白水平显著升高(P＜0.05).结论:天麻钩藤饮可抑制癫痫诱导的神经元损伤,并对维持血脑屏障稳定性有保护作用,其可能通过ZO-1/ZONAB信号通路所介导,从而发挥抗癫痫的作用.
Abstract
Objective:To investigate the changes of electroencephalogram(EEG),expression level of miRNA-9 in hippocampus of mice and protective mechanism against nerve injury after Tianma Gouteng Decoction intervention in epileptic mice.Methods:Fifty male Kunming mice were randomly divided into control group,model group,Carbamazepine group,TCM high-dose group and TCM low-dose group,with 10 mice in each group.One week before modeling,both control group and model group were given 20 mL·kg1·d-1 normal saline for intragastric administration;Carbamazepine was given 30 mg·kg-1·d-1 Carbamazepine suspension by intragastric administration.Tianma Gouteng Decoction was used to administer 30 mL·kg 1·d-1 and 10 mL·kg-1·d-1 to high dose group and low dose group,respectively.The acute epilepsy model mice was established by pilocarpine hydrochloride.Behavior and EEG were observed in each group.The expression of miRNA-9 in the hippocampus of mice was detected by real-time fluorescence quantitative PCR.Immunohistochemistry was used to detect neuron specific nuclear protein(NeuN)-labeled neuron nuclear antigen,FJB marks damaged neurons and platelet endothelial cell adhesion molecule-1(PECAM-1)-labeled platelet endothelial cell adhesion molecule-1,tight connexin-1(ZO-1)-labeled cytoplasmic tight adhesive protein 1.The level of ZO-1/ZONAB was determined by Western Blot.Results:Compared with the control group,model group mice seizure frequency,duration and seizure level were high(P＜0.01),electroencephalogram in mice sharp wave,spike wave,spine slow wave obviously increased,the expression of the hippocampus of miRNA-9 significantly lowered(P＜0.01);Compared with model group,high dose group and low dose group mice seizure frequency,duration and seizure level were reduced(P＜0.05),mice EEG epileptiform wave decreases,the expression of miRNA-9 was increased in the hippocampus of mice(P＜0.01).Compared with model group,NeuN cells in Carbamazepine group,high-dose group and low-dose group were significantly increased(P＜0.05),and damaged neurons were significantly decreased(P＜0.05).Pecam-1 immune response in hippocampal CA3 region was significantly decreased in high-dose group and low-dose group(P＜0.01).ZO-1 protein level was significantly decreased(P＜0.05),while ZONAB protein level was significantly increased(P＜0.05).Conclusion:Tianma Gouteng Decoction can inhibit neuronal damage induced by epilepsy and has a protective effect on the stability of blood-brain barrier,which may be mediated by ZO-1/ZONAB signaling pathway,so as to play an antiepileptic role.
关键词
天麻钩藤饮/癫痫小鼠/海马区/miRNA-9/神经损伤Key words
Tianma Gouteng Decoction/Epileptic mice/Hippocampus/miRNA-9/Nerve injury引用本文复制引用
基金项目
中国博士后科学基金特别资助项目(第十三批)(2020T130178)
出版年
2024
NETL
NSTL
万方数据