半夏白术天麻汤对癫痫模型大鼠急性期和慢性期海马神经元miRNA表达谱及生物功能的影响

Effects of Banxia Baizhu Tianma Decoction (半夏白术天麻汤) on miRNA Expression Profile and Biological Function of Hippocampal Neurons in Acute and Chronic Epileptic Rats

目的探讨癫痫急性期和慢性期发病机制,以及半夏白术天麻汤对癫痫干预的效应机制。方法将50只Wistar大鼠随机分为正常对照组、急性期模型组、急性期给药组、慢性期模型组、慢性期给药组,每组10只。除正常对照组外,其余各组大鼠采用氯化锂-匹鲁卡品诱导癫痫发作大鼠模型,造模后1~7天为急性期,8~30天为慢性期。造模成功1天后急性期给药组和慢性期给药组大鼠给予浓度为1. 548 g/ml半夏白术天麻汤2 ml灌胃,急性期给药组灌胃7天,慢性期给药组灌胃30天。其余3组给予2 ml蒸馏水灌胃,均每日1次。正常对照组大鼠5只和急性期模型组、急性期给药组大鼠10只一般行为学观察7天并取脑组织;正常对照组大鼠剩余5只和慢性期模型组、慢性期给药组10只一般行为学观察30天并取脑组织。运用miRNA芯片分析检测各组大鼠海马神经元细胞miRNA表达谱,生物信息学软件预测靶基因及KEGG富集分析,并采用实时荧光定量PCR验证各组间大鼠海马组织中部分差异基因表达,HE染色观察海马神经元细胞病理形态学改变。结果一般行为学观察和HE染色结果显示,半夏白术天麻汤具有抑制癫痫发作程度和频率的作用,并具有减轻和改善癫痫大鼠大脑海马神经元细胞损伤的作用,慢性给药组病理改善更好。急性期模型组和慢性期模型组与正常对照组比较得到10个重叠差异基因。两模型组前10位的通路中有3条重叠,即糖胺聚糖生物合成、长时程增强、赖氨酸降解。选取4个重叠miRNA通过实时荧光定量PCR验证:与急性期模型组比较,急性期给药组4个重叠miRNA差异无统计学意义(P> 0. 05)。与慢性期模型组比较,慢性期给药组rno-miR-146a-5p下调,rno-miR-218a-5p上调(P <0. 05)。结论糖胺聚糖的生物合成、长时程增强效应、赖氨酸降解均可能与癫痫的发生发展密切相关。半夏白术天麻汤有可能是通过调节相关miRNA及其靶基因和信号通路,改善相关生物功能从而发挥其抗癫痫作用,并且对慢性期效果更好。

Objective To investigate the pathogenesis of epilepsy in acute and chronic phase,and the mechanism of Banxia Baizhu Tianma Decoction(半夏白术天麻汤)on epilepsy intervention.Methods A total of 50 Wistar rats were randomly divided into a normal control group,an acute phase model group,an acute phase treatment group,a chronic phase model group and a chronic phase treatment group,with 10 rats in each group.Rats were given lithium chloride pilocarpine to induce the rat model of epilepsy except the normal control group.The acute phase was 1 to 7 days after modeling and the chronic phase was 8 to 30 days.One day after the successful model establishment,the acute phase treatment group and the chronic phase treatment group were given a concentration of 1.548 g/ml Banxia Baizhu Tianma Decoction 2 ml by gastrical perfusion.The acute phase treatment group was administered for 7 days,and the chronic phase treatment group was administered for 30 days.The other 3 groups were given 2 mL of distilled water for gavage,once a day.Five rats in the normal control group and 10 rats in the acute phase model group and the acute phase treatment group were observed for 7 days and the brain tissue was taken out.The remaining 5 rats in the normal control group and 10 in the chronic phase model group and the chronic phase treatment group were performed general behavioral observations for 30 days and brain tissue was then taken out.Analysis of miRNA microarray was used to detect miRNA expression profiles in hippocampal neurons of each group.Bioinformatics software predicted target gene and KEGG enrichment analysis,and real-time quantitative polymerase chain reaction(PCR)was used to verify the expression of some differential genes in hippocampus of rats.HE staining was used to observe the pathological changes of hippocampal neurons.Results The results of general behavioral observation and HE staining showed that Banxia Baizhu Tianma Decoction had the effect of inhibiting the degree and frequency of epilepsy,and had the effect of alleviating and improving the damage of hippocampal neurons in the brain of rats with epilepsy.The pathological improvement in the chronic treatment group was better.Ten overlapping differential genes were obtained in the acute phase model group and the chronic phase model group compared with the normal control group.Three of the top ten pathways in the two model groups overlapped,namely glycosaminoglycan biosynthesis,long-term potentiation,and lysine degradation.Four overlapping miRNAs were selected by real-time fluorescent quantitative PCR for verification.Compared with the acute phase model group,there were no significant differences in the 4 overlapping miRNAs in the acute phase(P>0.05).Compared with the chronic phase model group,rno-miR-146 a-5 p was down-regulated and rnomiR-218 a-5 p was up-regulated in the chronic phase(P<0.05).Conclusion The biosynthesis,long-term potentiation effect and lysine degradation of glycosaminoglycan may be closely related to the occurrence and development of epilepsy.Banxia Baizhu Tianma Decoction may play an anti-epileptic effect by regulating related miRNAs and their target genes as well as signaling pathways,improving related biological functions,and has a better effect on the chronic phase.