基于网络药理学及实验验证探讨金水六君煎治疗支气管哮喘的作用机制

Mechanism of Jinshui Liujun Decoction(金水六君煎)on the Treatment of Bronchial Asthma:Based on Network Pharmacology and Experimental Verification

目的:金水六君煎是治疗哮喘肺肾不足证的临床常用方。本研究旨在通过网络药理学结合动物实验探讨金水六君煎治疗哮喘的物质基础及分子生物学基础。方法:在中药系统药理学分析平台(TCMSP)筛选金水六君煎化合物成分,从OMIM、TTD等数据库获取哮喘的相关靶点。根据韦恩图获取金水六君煎与哮喘的共同作用靶点并构建化合物-疾病共同靶点的蛋白互作分析(PPI)网络。用David数据库进行基因本体(GO)分析和京都基因与基因组百科全书(KEGG)通路富集分析。以分子对接形式模拟金水六君煎活性化合物与关键靶点间的相互作用。在动物实验中,以鸡卵清蛋白(OVA)诱导哮喘小鼠模型,经金水六君煎灌胃处理后,HE染色观察小鼠肺组织病理学变化,PAS染色观察气道上皮杯状细胞增生情况,ELISA法检测小鼠血清白细胞介素-6(IL-6)、IL-1β、IL-8的含量。结果:共筛选出金水六君煎活性成分312种,潜在作用靶点77个,关键有效成分包括豆甾醇、槲皮素、β-谷甾醇等,关键靶点包括IL-6、丝裂原活化蛋白激酶3(MAPK3)、CXCL8等。GO分析结果显示金水六君煎发挥治疗哮喘的生物学过程主要与参与RNA聚合酶II启动子转录的正调控,调控炎症反应,参与细胞因子活性有关,KEGG分析显示TNF肿瘤坏死因子信号通路、Toll样受体信号通路、T细胞受体信号通路、NOD样受体信号通路可能在哮喘的治疗中起重要作用,分子对接结果显示槲皮素与IL-6结合良好。动物实验结果证实,与正常对照组相比,模型对照组小鼠气道炎性改变明显,PAS评分增加(P<0.01),血清IL-6、IL-1β含量升高(P<0.05);与模型对照组比较,泼尼松13.9 mg/kg组、金水六君煎5、10、20 g/kg组小鼠气道炎症减轻,泼尼松13.9 mg/kg组、金水六君煎5 g/kg组PAS评分显著降低(P<0.01),血清IL-6、IL-1β含量明显降低(P<0.05);金水六君煎10 g/kg组PAS评分降低(P<0.05),血清IL-6含量减少(P<0.05);金水六君煎20 g/kg组血清IL-6、IL-1β含量明显降低(P<0.05)。结论:金水六君煎可能通过豆甾醇、槲皮素、β-谷甾醇等关键活性成分作用于以IL-6、MAPK3、IL-8为代表的关键靶点,从而调控炎症反应,减少以IL-6、IL-8为代表的Th2型细胞因子分泌从而治疗哮喘。

Objective:Jinshui Liujun Decoction(金水六君煎)is widely used in clinical treatment of asthma with lung and kidney insufficiency syndrome.This study aims to explore the material and molecular biological basis of Jinshui Liujun Decoction in the treatment of asthma through network pharmacology combined with animal experiment.Methods:Firstly,the components of Jinshui Liujun Decoction were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and the related targets of asthma from Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),etc.Then,according to the Venn diagram,the common targets of the decoction and asthma were yielded and the protein-protein interaction(PPI)network was constructed,followed by Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Finally,the interaction between the active compounds of the decoction and the key targets was simulated in the form of molecular docking.As for the animal experiment,we used chicken ovalbumin(OVA)to induce asthma in mice and then the asthma mice were treated(i.g.)with Jinshui Liujun Decoction.Afterwards,the histopathological changes of mouse lung were observed based on hematoxylin-eosin(HE)staining,and the proliferation of airway epithelial goblet cells based on periodic acid-Schiff(PAS)staining.The expression of interleukin-6(IL-6),mitogen-activated protein kinase 3(MAPK3),and interleukin-8(IL-8)in serum was detected by enzyme-linked immunosorbent assay(ELISA).Results:A total of 312 active components and 77 potential targets of Jinshui Liujun Decoction were screened out.The key active components included stigmasterol,quercetin,andβ-sitosterol,and the core targets were IL-6,MAPK3,and IL-8.The anti-asthma targets of Jinshui Liujun Decoction mainly involved in the GO terms of positive regulation of RNA polymerase II promoter transcription,regulation of inflammatory response,and the activity of cytokines,and the KEGG pathways of tumor necrosis factor(TNF)signaling pathway,Toll-like receptor signaling pathway,T-cell receptor signaling pathway,and NOD-like receptor signaling pathway.Molecular docking results suggested that quercetin had high binding affinity to IL-6.According to the animal experiment,compared with the normal control group,the model group demonstrated significant changes in airway inflammation and rise of PAS(P<0.01)and levels of IL-6 and IL-1β(P<0.05).Compared with the model control group,13.9 mg/kg prednisone and 5 g/kg,10 g/kg,and 20 g/kg Jinshui Liujun Decoction alleviated the airway inflammation.Moreover,the PAS(P<0.01)and content of IL-6 and IL-1β(P<0.05)in 13.9 mg/kg prednisone group and 5 g/kg Jinshui Liujun Decoction group decreased.In 10 g/kg Jinshui Liujun Decoction group,PAS decreased(P<0.05)and the content of IL-6 reduced(P<0.05).The content of IL-6 and IL-1βin 20 g/kg Jinshui Liujun Decoction group decreased(P<0.05).Conclusion:The key components of Jinshui Liujun Decoction,such as stigmasterol,quercetin,andβ-sitosterol can interact with MAPK3,IL-6 and IL-8 to regulate inflammation and reduce the secretion of Th2 cytokines such as IL-6 and IL-8,thereby alleviating asthma.