基于风痰交互响应理论研究癫痫发作-缓解期大鼠海马miRNA表达谱及熄风化痰法的干预作用

Epilepsy is a clinical frequent stubborn disease which has high recurrence rate. There wasn't a specific effective therapeutic treatment method of Modern Medicine to cure the epilepsy. Epilepsy's pathogenesis is mainly 'Wind' during seizure, and 'Phlegm' during paracme according to the theory of Traditional Chinese Medicine. Interactive response of 'Wind' and 'Phlegm' cause the epilepsy. So we will study the epilepsy rats on account of the theory of 'Wind Phlegm Interactive Response' basing on accomplished works. We will use miRNA gene chips to pick up the differential expression microRNAs of rats hippocampuses during seizure and paracme of epilepsy, and use bioinformatics software to predict the target genes of the microRNAs, and use Go-analysis and Pathway-analysis to find the biological functions of the target genes and the relevant biological signal passageways, and use the technology of RT-PCR and Western Blot and Laser Confocal Immunofluorescence Double-labeling method to validate the target genes and the relevant biological signal passageways, and explain the correlation among the signal passageways through analyzing the 'crosstalk', and use banxia baizhu tianma decoction which has the power of subduing wind and dissipating phlegm to cure the epilepsy. The study will reveal the pathogenesis of epilepsy during seizure and paracme on level of albumen and mRNA and on point of view of relevant signal passageways, and explain the mechanism of action of the interference effects of banxia baizhu tianma decoction to the epilepsy.

癫痫是临床常见的疑难病症、复发率高且西医尚无特效治疗手段。中医认为癫痫病机在发作期主要以风为先导，缓解期主要以痰为根本，风痰交互响应，交错致病。故本研究以"风痰交互响应"理论为指导并结合前期工作，以癫痫大鼠为研究对象，利用miRNA表达芯片筛选癫痫发作期和缓解期大鼠海马组织中差异表达miRNA，结合生物信息学对靶基因进行预测，通过Go-analysis和Pathway-analysis发现靶基因的生物功能和可能参与的生物信号通路；并运用RT-PCR、Western Blot技术及激光共聚焦免疫荧光双标法，对靶基因和相关信号通路进行验证，分析信号通路间的串话及其相关性，并应用以熄风化痰法为治则的半夏白术天麻汤进行干预。从蛋白、mRNA水平及相关信号通路角度，揭示癫痫发作期和缓解期的发病机制及"半夏白术天麻汤"对癫痫干预效应的作用机制。

课题以癫痫的“风痰交互响应”病机理论为指导，通过miRNA表达谱研究，探寻与癫痫发作期和缓解期相关的靶基因及其生物功能和生物信号通路，从而阐释癫痫发作期和缓解期的发病机制，以及熄风化痰的经典方剂----半夏白术天麻汤对癫痫发作期和缓解期干预效应的作用机制。.（1）文献研究：应用“中医传承大数据平台”收集、整理《中医方剂大辞典》治疗癫痫的方剂，对筛选出的532首方剂、895味中药进行频次、apriori算法及药物间关联规则，以及Canopy算法层次聚类，并进行方剂匹配得到与半夏白术天麻汤相匹配的3首方剂。说明选择半夏白术天麻汤作为此次研究干预方剂的合理性。.（2）实验研究：运用氯化锂-匹鲁卡品诱导癫痫发作，通过各组大鼠一般行为学改变并结合缓解期大鼠脑电图以及海马HE染色，从大鼠行为学、神经元细胞病理形态学和脑电方面提示造模成功和半夏白术天麻汤的治疗效果。应用miRNA微阵列芯片技术，将各组癫痫大鼠海马miRNA表达谱进行比对，筛选出差异miRNA，并运用生物信息学软件对差异表达最明显的miRNA进行靶基因预测，再对靶基因进行Gene Ontology及KEGG的功能及pathway富集，结果显示癫痫发作期和缓解期模型组与正常对照组比较：有10个重叠出现的差异基因；GO 富集有87个重叠的GO term，最显著的是蛋白质丝氨酸/苏氨酸磷酸酶复合体；pathway富集有16个重叠的信号通路，富集最显著的是糖胺聚糖的生物合成、长时程增强效应、赖氨酸降解，该两项富集分析结果均提示可能与癫痫的发生发展密切相关。以缓解期各组间miRNA-146a-5p为例，RT-PCR验证芯片结果，并采用miRDB进行靶基因预测，miRTarBase、DAVID进行GO功能和KEGG信号通路富集分析，研究发现miRNA-146a-5p与癫痫发生后的炎症反应密切相关，半夏白术天麻汤则有可能通过控制癫痫发生后的炎症反应从而起到抗癫痫的效果。.通过此次研究为进一步细化深入研究癫痫发病机制和半夏白术天麻汤的治疗机制打开从基因到信号通路多个研究切入点。