LC-MS结合网络药理学探讨紫锥菊抗猪流行性腹泻病毒的作用机制

doi:10.16431/j.cnki.1671-7236.2025.09.042

摘要/Abstract

摘要： 【目的】采用液相色谱-质谱联用(liquid chromatograph mass spectrometer，LC-MS)和网络药理学探究紫锥菊(Echinacea purpurea，EP)抗猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)的活性成分、靶点及潜在作用机制，为进一步开发新型抗PEDV的治疗药物提供理论依据。【方法】构建PEDV感染IPEC-J2细胞模型，利用不同浓度EP提取物处理细胞模型。采用实时荧光定量PCR检测PEDV N蛋白mRNA转录水平，并分析EP提取物对PEDV复制的影响；采用LC-MS对EP中的化学成分进行定性定量分析；利用TCMSP、SwissTargetPrediction、NCBI数据库检索EP药用活性成分与抗PEDV作用相关的分子靶点，取交集靶点输入STRING数据库构建蛋白相互作用(PPI)网络并筛选关键靶点；构建“EP-活性成分-交集靶点”网络，同时用DAVID数据库对交集靶点进行GO功能和KEGG通路富集分析，预测EP抗PEDV作用的核心成分、关键靶点及潜在机制。利用分子对接验证主要核心成分与关键靶点的结合能力。【结果】体外试验结果表明，与对照组相比，EP提取物显著抑制了PEDV N蛋白mRNA转录水平(P＜0.05)。LC-MS检测发现，EP中含有甜菜碱、鞣花酸、菊苣酸等81种化学成分，分类占比最高的主要为酚类、萜类、黄酮类等。基于网络药理学筛选，EP中鞣花酸、(+)-儿茶素、苦参碱等18种药用活性成分可能作用于144个靶点发挥抗PEDV作用，重要核心成分有山奈酚、漆黄素、木犀草素等，关键靶点包括肿瘤蛋白p53(tumor protein 53，TP53)、白细胞介素-6(interleukin-6，IL-6)、类固醇受体辅激活因子(steroid receptor coactivator,SRC)、丝裂原活化蛋白激酶 1(mitogen-activated protein kinase 1，MAPK1)等，主要涉及Th17细胞分化、NOD样受体信号通路、FoxO信号通路等，参与细胞免疫、细胞凋亡、细胞周期等过程。分子对接结果显示，鞣花酸与TP53、IL-6、SRC、MAPK1结合能均＜－29.308 kJ/mol，且鞣花酸与MAPK1结合力最强。【结论】本研究分析预测EP是基于多成分、多靶点、多途径的协同复杂作用，利用山柰酚、漆黄素、木犀草素等核心成分作用于TP53、IL-6、SRC、MAPK1等靶点，通过调控Th17细胞分化、NOD样受体、FoxO等信号通路发挥抗PEDV作用。

关键词: 猪流行性腹泻病毒(PEDV); 紫锥菊; 液相色谱-质谱联用; 网络药理学

Abstract: 【Objective】 Liquid chromatograph-mass spectrometer (LC-MS) and network pharmacology were used to explore the active ingredients of Echinacea purpurea (EP),targets and potential mechanism of EP against Porcine epidemic diarrhea virus (PEDV),so as to provide a theoretical basis for the further development of new anti-PEDV therapeutic drugs. 【Method】 A PEDV-infected IPEC-J2 cell model was constructed,and the cell model was treated with EP extracts of different concentrations.The mRNA transcription level of PEDV N protein was detected by Real-time quantitative PCR,and the effect of EP extract on PEDV replication was analyzed.The chemical constituents in EP were qualitatively analyzed by LC-MS.TCMSP,SwissTargetPrediction,NCBI databases were used to search the active ingredients of EP and the molecular targets related to anti-PEDV effects.The intersection targets of EP and PEDV were obtained and entered into the STRING database to construct protein-protein interaction (PPI) network and screen the key targets.The "EP-active ingredient-intersection target" network was constructed,and GO function and KEGG signal pathways of intersection targets were analyzed using the DAVID database to predict the core components,key targets and potential mechanisms of the anti-PEDV effect of EP.Finally,molecular docking was used to verify the binding ability of the main core components and key targets. 【Result】 In vitro results showed that compared with control group,EP extract significantly inhibited PEDV N protein mRNA transcription levels.LC-MS detection showed that EP contained 81 chemical components such as betaine,ellagic acid and cichoriic acid,and the highest classification proportion was mainly phenols,terpenoids and flavonoids.Based on network pharmacological screening,18 medicinal active ingredients such as ellagic acid,(+)-catechin and matrine in EP might exert anti-PEDV effects on 144 targets.The important core components of EP against PEDV included kaempferol,urushetin,luteolin,etc.The key targets included tumor protein 53 (TP53),interleukin-6 (IL-6),steroid receptor coactivator (SRC),mitogen-activated protein kinase 1 (MAPK1),etc.,mainly involved in Th17 cell differentiation,NOD-like receptor signaling pathway,FoxO signaling pathway,etc.,which involved in cellular immunity,apoptosis,cell cycle and other processes.The molecular docking results showed that the binding energy of ellagic acid with TP53,IL-6,SRC and MAPK1 was all less than －29.308 kJ/mol,among which ellagic acid had the strongest affinity with MAPK1. 【Conclusion】This study demonstrated that the anti-PEDV effect of EP was based on the synergistic action of multiple components, multiple targets, and multiple pathways. EP mainly used kaempferol, urushetin, luteolin and other core components to act on TP53, IL-6, SRC, MAPK1 and other targets, and regulated signaling pathways such as Th17 cell differentiation, NOD-like receptor and FoxO to treat PEDV.

Key words: Porcine epidemic diarrhea virus (PEDV); Echinacea purpurea; LC-MS; network pharmacology

中图分类号:

毛驰文, 黄明凤, 李淼淼, 陈兰, 罗怡泓, 连凯琪, 马圣明, 朱二鹏. LC-MS结合网络药理学探讨紫锥菊抗猪流行性腹泻病毒的作用机制[J]. 中国畜牧兽医, 2025, 52(9): 4471-4483.

MAO Chiwen, HUANG Mingfeng, LI Miaomiao, CHEN Lan, LUO Yihong, LIAN Kaiqi, MA Shengming, ZHU Erpeng. Exploration of the Mechanism of Echinacea purpurea Against Porcine Epidemic Diarrhea Virus Based on LC-MS and Network Pharmacology[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4471-4483.

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