基于网络药理学及实验验证探讨红花-白芍药对治疗偏头痛的作用机制

Mechanism of Carthami Flos-Paeoniae Raidx Alba herb pair in treatment of migraine based on network pharmacology and experimental verification

目的基于网络药理学及实验验证探讨红花-白芍药对治疗偏头痛的作用机制。方法从中药系统药理学数据库与分析平台(TCMSP)数据库中筛选红花、白芍的有效成分, 使用Swiss Target Prediction预测有效成分的作用靶点;运用GeneCards获取偏头痛的疾病靶点, 利用Venny 2.1获取交集靶点。使用STRING进行蛋白质-蛋白质相互作用(PPI)分析并使用Cytoscape构建网络图, 利用Metascape进行基因本体(GO)和京都基因与基因组百科全书(KEGG)信号通路富集分析。使用Cytoscape软件构建"药物-靶点-通路"网络图。采用硝酸甘油法建立偏头痛大鼠模型, 依据随机数字表法将造模成功的大鼠分为模型组和观察组(每组5只)。观察组灌胃1.8 g/kg红花-白芍溶液, 模型组灌胃等体积0.9%氯化钠溶液, 1次/d, 连续给药2周。治疗后评估症状改善情况及检测大鼠血清核心靶点V-Rel网状内皮增生病毒癌基因同源物A(RELA)、蛋白激酶B1(Akt1)、丝裂原活化蛋白激酶1(MAPK1)、肿瘤坏死因子(TNF)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、白细胞介素-6(IL-6)表达情况。结果红花-白芍药对中的24个有效成分通过多条通路直接作用于178个疾病靶点治疗偏头痛, 其中6-羟基山柰酚、β-胡萝卜素、黄芩苷、β-谷甾醇、山柰酚等是核心成分, RELA、Akt1、MAPK1、TNF、Caspase-3、IL-6是至关重要的靶点。GO分析显示, 细胞组成(CC)有85个, 生物过程(BP)有228个, 分子功能(MF)有119个。KEGG信号通路富集分析提示红花-白芍药对主要参与晚期糖基化终末产物(AGE)-晚期糖基化终末产物受体(RAGE)、缺氧诱导因子-1(HIF-1)、磷脂酰肌醇3-激酶(PI3K)-Akt和MAPK等信号通路。动物实验验证显示, 与模型组比较, 红花-白芍药对在0~30、60~90、120~150 min 3个时间段内评分均降低(均P<0.01), 大鼠机械性刺激和热刺激痛阈均增加(均P<0.01), 血清RELA、Akt1、MAPK1、TNF、Caspase-3和IL-6水平均降低(均P<0.01)。结论红花-白芍药对主要通过调节IL-17、TNF、Toll受体、T细胞受体、AGE-RAGE、HIF-1、PI3K/Akt和MAPK等信号通路的RELA、Akt1、MAPK1、TNF、Caspase-3和IL-6等疾病靶点治疗偏头痛。

ObjectiveTo study the mechanism of Carthami Flos-Paeoniae Raidx Alba herb pair in the treatment of migraine based on network pharmacology and experimental verification.MethodsTCMSP was queried to screen the drug components of Carthami Flos and Paeoniae Raidx Alba,and Swiss Target Prediction was used to predict the drug components.GeneCards was conducted to obtain disease targets for migraine,and Venny 2.1 was used to obtain intersection targets.PPI analysis with STRING and network diagrams with Cytoscape,GO and KEGG analysis with Metascape.The"drug-target-pathway"network was constructed by Cytoscape software.The migraine rat model was established by the nitroglycerin method,and the rats were divided into a model group and an observation group(5 rats in each group)according to the random number table method.The observation group was given 1.8 g/kg Carthami Flos-Paeoniae Raidx Alba solution,while the model group was given 0.9%sodium chloride solution,once a day,for 2 weeks.Symptom improvement was evaluated after treatment,and the core target of serum V-Rel endotheloproliferative virus oncogene homology A(RELA),protein kinase B1(Akt1),mitogen-activated protein kinase 1(MAPK1),tumor necrosis factor(TNF),cysteine aspartic protease-3(Caspase-3),and interleukin-6(IL-6)expression was detected.ResultsIt was found that 24 active ingredients in Carthami Flos-Paeoniae Raidx Alba herb pair directly act on 178 disease targets through multiple pathways to treat migraine.Among them,6-hydroxykaempferol,beta-carotene,baicalin,beta-sitosterol and kaempferol were the core components.RELA,Akt1,MAPK1,TNF,Caspase-3,IL-6 were critical targets.The GO analysis showed 85 cellular component(CC),228 biological process(BP)and 119 molecular function(MF).Enrichment analysis of KEGG signaling pathway suggested that Carthami Flos-Paeoniae Raidx Alba herb pair was mainly involved in advanced glycosylated end-product(AGE)-advanced glycosylated end-product receptor(RAGE),hypoxia-inducing factor-1(HIF-1),phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)and MAPK signaling pathways.The experimental results showed that compared with the model group,the scores of Carthami Flos-Paeoniae Raidx Alba herb pair decreased at 0~30,60~90,and 120~150 min(all P<0.01),and the pain threshold of mechanical and thermal stimuli increased(all P<0.01).Serum levels of RELA,Akt1,MAPK1,TNF,Caspase-3,and IL-6 were decreased(all P<0.01).Conclusions Carthami Flos-Paeoniae Raidx Alba herb pair can treat migraine by regulating RELA,Akt1,MAPK1,TNF,Caspase-3 and IL-6 signaling pathways including IL-17,TNF,Toll receptor,T cell receptor,AGE-RAGE,HIF-1,PI3K/Akt and MAPK.