基于网络药理学和实验验证探究胆南星对癫痫的作用机制

To explore the mechanism of Arisaema cum Bile on epilepsy based on network pharmacology and experimental verification

本研究采用网络药理学方法和膜片钳技术探究胆南星(Arisaema cum Bile)对癫痫(epilepsy)的作用机制。采用TCMSP数据库和文献挖掘获得胆南星的成分及靶点,通过GeneCards和OMIM数据库检索癫痫疾病靶点。应用STRING、DAVID在线平台进行蛋白互作(protein-protein interaction)、KEGG (Kyoto Encyclopedia of Genes and Genomes)信号通路和GO (Gene Ontology)基因功能富集分析。利用Cytoscape 3.7.2软件构建“中药-成分-靶点-通路-疾病”拓扑分析网络。动物实验已获得河北医科大学实验动物福利伦理委员会批准。网络药理学结果发现鹅去氧胆酸(chenodeoxycholic acid, CDCA)、去氧胆酸(deoxycholic acid)和β-谷甾醇(β-sitosterol)等9个有效成分,及其相应的5-羟色胺转运体(5-hydroxytryptamine transporter)、GABA_(A)受体α2亚型(gamma-aminobutyric acid receptor type A subunit alpha2)和乙酰胆碱受体α-7亚型(neuronal acetylcholine receptor subunit alpha-7)等22个关键靶点。信号通路和基因功能富集分析结果涉及5-羟色胺能突触(serotonergic synapse)、GABA能突触(GABAergic synapse)和离子跨膜转运(ion transmembrane transport)等神经元兴奋性调节相关的通路。脑片电生理实验结果证明, β-谷甾醇和CDCA可抑制小鼠海马CA1锥体神经元动作电位(action potential),影响动作电位的基强度(rheobase)、发放延迟(delay)和去极化时程(depolarization duration),联合应用的抑制作用更加显著。本研究从网络药理学的角度结合电生理实验初步探讨了胆南星治疗癫痫的多成分、多靶点和多途径机制,为胆南星的进一步研究提供了思路。

This study aims to explore the mechanism of Arisaema cum Bile on epilepsy using combination of network pharmacology and patch clamp recording. Active ingredients of Arisaema cum Bile were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and literatures.Epilepsy-related targets were identified from GeneCards and OMIM database. STRING platform was employed to perform protein-protein interaction(PPI), and David platform was used for Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway and Gene Ontology(GO) enrichment analysis. The drug-ingredient-target-pathway-disease network was constructed with Cytoscape software. The animal experiments were approved by the Laboratory Animal Ethical and Welfare Committee Hebei Medical University. Nine compounds were detected as the active ingredients of Arisaema cum Bile, including chenodeoxycholic acid(CDCA), deoxycholic acid and β-sitosterol,etc. A total of 22 key potential targets were identified, including 5-hydroxytryptamine transporter(SLC6A4),gamma-aminobutyric acid receptor type A subunit alpha 2(GABRA2) and neuronal acetylcholine receptor subunit alpha-7(CHRNA7). These targets were associated with biological processes of serotonergic synapse, GABAergic synapse and ion transmembrane transport. Brain slice electrophysiology experiments revealed that β-sitosterol and CDCA inhibited the action potential(AP) of CA1 pyramidal neurons in the mouse hippocampus. Both β-sitosterol and CDCA affected the properties of AP, such as rheobase, delay and depolarization duration. In addition, the inhibitory effect of AP was more prominent when the two compounds were given together. Combining with network pharmacology and electrophysiological experiments, our study reveals the potential mechanisms of Arisaema cum Bile for the treatment of epilepsy in a "multi-ingredients, multi-targets and multi-pathways" manner.Our study provides a reference for further studies of Arisaema cum Bile.