基于UPLC-Q-Exactive Orbitrap-MS联合网络药理学探究双黄连解热抗炎的作用机制

Based on UPLC-Q-Exactive Orbitrap-MS combined with network pharmacology to explore the antipyretic and anti-inflammatory mechanism of Shuanghuanglian

目的:基于UPLC-Q-Exactive Orbitrap-MS方法,利用网络药理学和分子对接技术初步探究双黄连解热抗炎的物质基础及作用机制。方法:利用UPLC-Q-Exactive Orbitrap-MS技术鉴定双黄连口服液中的主要化学成分。基于网络药理学,将已定性化合物录入Swiss Target数据库筛选成分靶点;运用GeneCards、TTD等数据库筛选疾病靶点,通过Cytoscape软件构建“中药-成分-靶点-疾病”网络;筛选关键成分,构建PPI网络筛选核心靶点,并对药物疾病共有靶点进行GO和KEGG通路富集。利用Schrodinger软件对关键成分和核心靶点进行分子对接验证。此外,采用腹腔注射脂多糖溶液建立大鼠发热炎症模型,测量体温变化值,ELISA法检测TNF-α、IL-1β、IL-6、cAMP、PGE_(2)含量。结果:双黄连口服液共鉴定出83种化学成分。网络药理学分析得双黄连口服液关键成分包括黄芩素、汉黄芩素、槲皮素、千层纸素A等,10个核心靶点包括GAPDH、TNF、MAPK3等。共有靶点涉及信号转导、转录的正调控、炎症反应等生物过程,通过TNF、PI3K/Akt、Ras、MAPK、TLR、cAMP、花生四烯酸代谢等信号通路发挥治疗作用。对接结果显示较好的结合活性。体内动物实验验证双黄连口服液能够降低发热大鼠体温,减少TNF-α、IL-1β、IL-6、cAMP、PGE2含量。结论:双黄连口服液通过多成分、多靶点、多途径发挥解热抗炎作用,为下一步深入探究其药效物质基础及机制提供参考。

OBJECTIVE Based on UPLC-Q-Exactive Orbitrap-MS,the material basis and mechanism of antipyretic and antiinflammatory of Shuanghuanglian were studied by network pharmacology and molecular docking technology.METHODS The main chemical constituents of Shuanghuanglian oral liquid were identified by UPLC-Q-Exactive Orbitrap-MS.Based on network pharmacology,qualitative compounds were entered into Swiss Target Prediction database to screen component targets,disease targets were screened by GeneCards and TTD databases,"traditional Chinese medicine-component-target-disease"network was constructed by Cytoscape software,key components were screened,PPI network was constructed to screen core targets,and drug disease common targets were enriched by GO and KEGG pathways.Schrodinger software was used to verify the molecular docking of the key components and core targets.In addition,the rat model of fever and inflammation was established by intraperitoneal injection of lipopolysaccharide solution.The changes of anal temperature were measured and the levels of TNF-α,IL-1β,IL-6,cAMP and PGE_(2) were detected by ELISA.RESULTS A total of 83 chemical constituents were identified in Shuanghuanglian oral liquid.Network pharmacological analysis showed that the key components of Shuanghuanglian oral liquid included baicalein,wogonin,quercetin,lycopene A and so on,and 10 core targets included GAPDH,TNF,MAPK3.Common targets were involved in signal transduction,positive regulation of transcription,inflammation and other biological processes,which played antipyretic and anti-inflammatory effects through TNF,PI3K/Akt,Ras,MAPK,TLR,cAMP,arachidonic acid metabolism and other signal pathways.The docking results showed good binding activity.Animal experiments in vivo showed that Shuanghuanglian oral liquid could reduce the anal temperature and the contents of TNF-α,IL-1β,IL-6,cAMP and PGE_(2) in febrile rats.CONCLUSIONS Shuanghuanglian oral liquid plays an antipyretic and anti-inflammatory effect through multi-compo-nents,multi-targets and multi-ways,which provides a reference for further exploration of its pharmacodynamics material basis and mechanism.