基于网络药理学和实验验证探究香青兰调节TLR4/MyD88通路抗脑缺血再灌注损伤作用机制

Mechanism of Dracocephalum moldavica L.in Regulating TLR4/MyD88 Pathway to Treat Cerebral Ischemia-Reperfusion Injury Based on Network Pharmacology and Experimental Validation

目的:借助网络药理学和分子对接等手段,结合大鼠脑缺血再灌注损伤实验验证,探究香青兰抗脑缺血再灌注损伤的作用机制。方法:通过本草组鉴数据库(HERB)筛选出香青兰的主要成分,通过人类基因的综合数据库(GeneCards)、在线人类孟德尔遗传数据库(OMIM)、遗传药理学与药物基因组学数据库(PharmGKB)、治疗靶点数据库(TTD)等数据库筛选出脑缺血再灌注损伤的疾病靶点。将香青兰主要活性成分靶点和脑缺血再灌注损伤疾病靶点在在线维恩图中得到交集靶点,通过蛋白质相互作用分析数据库(STRING)建立香青兰主要成分-脑缺血再灌注损伤疾病靶点的PPI网络图,利用R语言的工具包进行GO和KEGG的富集分析。从有机小分子生物活性数据库(PubChem)获取主要活性成分的构象图,利用AutoDock软件对香青兰主要成分与脑缺血再灌注损伤疾病靶点进行分子对接。采用大脑中动脉栓塞(MCAO)模型制备大鼠脑缺血再灌注损伤模型,造模前后灌胃给予香青兰提取物,进行大鼠神经功能评分,大脑切片TTC染色,ELISA法检测血清中TNF-α和脑组织中IL-6、IL-1β的含量,蛋白质印迹法检测脑组织TLR4(TOLL样受体4)、MyD88(髓分化因子88)、TNF-α蛋白表达。结果:筛选得到香青兰的主要活性成分2,5-二羟基苯甲酸、田蓟苷、草甘磷、百里香酚、等16个,活性成分的作用靶点419个,脑缺血再灌注损伤疾病靶点1566个,香青兰活性成分和疾病靶点的交集靶点共183个;Cyto-scape软件分析得到TLR4、TNF、AKT1、SRC等核心靶点,GO富集分析得到生物过程包括丝裂原活化蛋白激酶级联的正向调节、对氧化应激的反应、对外部刺激反应的正向调节、等,细胞组分包括膜筏、膜微区、突触前膜的组成部分、等,分子功能包括蛋白质丝氨酸/苏氨酸/酪氨酸激酶活性、蛋白酪氨酸激酶活性、蛋白质丝氨酸/苏氨酸激酶活性、等,KEGG富集分析得到涉及的主要信号通路有AGE-RAGE信号通路、PI3K-AKT信号通路、Ras信号通路、等;分子对接的结果显示香青兰主要活性成分和脑缺血再灌注损伤疾病核心靶点有良好的结合能力。在大鼠脑缺血再灌注损伤模型中,香青兰提取物处理能够明显减少梗死面积,改善神经功能评分,降低炎症因子血清TNF-α,脑组织IL-6、IL-1β的含量,明显下调TLR4、MyD88和TNF-α蛋白表达。结论:香青兰能够多成分、多靶点、多通路地发挥抗脑缺血再灌注损伤的作用,其中抑制TLR4/MyD88介导的炎症反应扮演重要角色。

Objective:To explore the mechanism of Dracocephalum moldavica L.in treating cerebral ischemia-reperfusion injury based on network pharmacology,molecular docking,and experimental verification in rats.Methods:The main components of Dracocephalum moldavica L.were retrieved from HERB database,and the targets of cerebral ischemia-reperfusion injury from GeneCards,OMIM,PharmGKB,TTD and other databases.The common targets shared by the active components of D.moldavica and cerebral ischemia-reperfusion injury were obtained via the online Venn diagram,and the protein-protein interaction(PPI)network of the common targets was established by STRING.The R language toolkit was used to perform GO and KEGG enrichment analysis.The conformational maps of the major active components were obtained from PubChem,and AutoDock was used for the molecular docking between the major active components and the disease targets.The rat model of cerebral ischemia-reperfusion injury was established by the middle cerebral artery obstruction(MCAO)method.The rats were administrated with the extract of D.moldavica by gavage before and after modeling.The neurological deficit score was recorded,and the brain sections were stainned with 3,5-triphenyltetrazoliumchloride(TTC).Enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor-alpha(TNF-α)in the serum and interleukin-6(IL-6)and IL-1β in the brain tissue.Western blotting was employed to determine the protein levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88)and TNF-α in the brain tissue.Results:D.moldavica contained 16 active components including 2,5-dihydroxybenzoic acid,tilianin,glyphosate,and thymol,which corresponded to 419 targets.A total of 1566 targets of cerebral ischemia-reperfusion injury were screened out,and 183 common targets were shared by the active components and the disease.Cyto-scape predicted TLR4,TNF,AKT1,SRC and other core targets.The core targets were mainly annotated to the GO terms of biological processes(positive regulation of MAPK cascade,response to oxidative stress,and positive regulation of response to external stimuli),cell components(membrane raft,membrane microdomain,and integral component of synaptic membrane),and molecular functions(protein serine/threonine/tyrosine kinase activity,protein tyrosine kinase activity,protein serine/threonine kinase activity).The KEGG enrichment analysis showed that the core targets were mainly enriched in the AGE-RAGE,PI3K-Akt,and Ras signaling pathways.The results of molecular docking showed that the main active components of D.moldavica had strong binding with the core targets of cerebral ischemia-reperfusion injury.In the rat model of cerebral ischemia-reperfusion injury,the extract of D.moldavica significantly reduced the infarct size,improved the neurological function,and lowered the levels of TNF-α,IL-6,and IL-1β,and down-regulated the protein levels of TLR4,MyD88,and TNF-α.Conclusion:D.moldavica L.can treat cerebral ischemia-reperfusion injury in a multi-component,multi-target,and multi-pathway manner by inhibiting the TLR4/MyD88-mediated inflammatory response.