摘要
目的联合液相-质谱(liquid-mass spectrometry,LC-MS)分析和网络药理学方法探究由清热活血方防治动脉粥样硬化(atherosclerosis,AS)的潜在靶点。方法首先,采用LC-MS定性分析对清热活血方化学成分进行鉴定,并将得到的成分输入Swiss Target Prediction数据库进行目标靶点预测,并采用Cytoscape 3.7.1构建清热活血方活性成分-核心靶点网络;其次,从疾病数据库中收集AS相关靶点;将AS相关靶点与清热活血方靶点取交集,得到清热活血方抗AS靶点,并制作靶标蛋白相互作用网,然后进行GO(Gene Ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析。之后,随机将50只雄性ApoE^(-/-)小鼠分为空白组、模型组、清热活血方低、中、高剂量组,并通过高脂饮食的方式建立ApoE^(-/-)小鼠AS模型,清热活血方组给予低、中、高剂量药物干预,16周末取主动脉组织,对筛选出的通路进行基因和蛋白水平的验证。最后,将LC-MS定量分析筛选出含量最高的两个成分,与筛选出的通路基因进行分子对接,将靶基因进行二次筛选。结果LC-MS分析清热活血方共筛选出中68个活性成分,对应靶标223个;通过检索疾病数据库,去除重复后共得到与AS相关疾病靶点3291个;将清热活血方对应靶标与AS靶点取交集得到136个核心基因。GO富集分析结果显示包含炎症反应的调控、膜微域和配体-激活转录因子活性等的参与;KEGG通路富集分析结果显示涉及PI3K/Akt等47条清热活血方抗AS信号通路。WB和RT-qPCR结果提示,清热活血方可以下调p-PI3K/PI3K和p-Akt/Akt水平,且可下调PI3K/Akt通路的13个基因表达水平。LC-MS定量分析得出黄芩苷和丹酚酸B是含量最多的两个成分,与PI3K/Akt通路上的13个基因进行分子对接,结果提示RXRA基因与黄芩苷和丹酚酸B自由结合能最低。结论清热活血方可能通过介导RXRA基因调控PI3K/Akt信号通路实现抗AS作用,本研究为清热活血方干预AS提供新思路和新靶点。
Objective To explore the potential target of Qingre Huoxue recipe for prevention and treatment of atherosclerosis(AS),combined with liquid-mass spectrometry(LC-MS)analysis and network pharmacological methods.Methods Firstly,LC-MS qualitative analysis was used to identify the chemical components of Qingre Huoxue Formula,and the obtained components were input into Swiss target prediction database for target prediction.Cytoscape3.7.1 was used to construct the active ingredient-core target network of Qingre Huoxue Formula.Secondly,AS-related targets were collected from disease databases.The AS-related targets were intersect with the target of Qingre Huoxue Formula to obtain the anti-AS target of Qingre Huoxue Formula,and the STRING database was used to make the target protein interaction network,then we do GO and KEGG enrichment analysis.Next,50 male ApoE^(-/-) mice were divided into normal group,model group,Qingre Huoxue Formula low,middle and high dose groups,and ApoE^(-/-) mice were fed by high fat diet to establish AS model.Qingre Huoxue Formula groups were given the low,middle and high dose of drug intervention;the aorta was isolated at the end of the 16^(th) week,the real-time fluorescent quantitative PCR(RT-qPCR)and immune protein imprinting(WB)to filter out pathways of gene and protein level of validation.Finally,LC-MS quantitative analysis was performed to screen out the two components with the highest content,and molecular docking was conducted with the screened pathway genes to screen the target genes for the second time.Results A total of 68active ingredients were screened out by LC-MS analysis of the Qingre Huoxue Formula,corresponding to 223 targets.By searching the disease database,a total of 3291 AS-related disease targets were obtained after removing duplicates.The corresponding targets of Qingre Huoxue Formula were intersected with AS targets to obtain 136 core genes.The results of GO enrichment analysis showed that it included the regulation of inflammatory response,membrane microdomain and ligand-activated transcription factor activity.KEGG pathway enrichment analysis showed that 47 anti-AS signaling pathways including PI3K/Akt were involved.WB and RT-qPCR results indicated that Qingre Huoxue Formula could down-regulate the levels of p-PI3K/PI3K and p-Akt/Akt,and down-regulate the expression levels of 13 genes in the PI3K/Akt pathway.LC-MS quantitative analysis showed that baicalin and salvianolic acid B were the two components with the highest content,and the RXRA gene had the lowest free binding energy with baicalin and salvianolic acid B by molecular docking with 13 genes in the PI3K/Akt pathway.Conclusion Qingre Huoxue Formula may achieve anti-AS effect by mediating RXRA gene regulation of PI3K/Akt signal pathway.This study provides a new idea and new target for Qingre Huoxue Formula to interfere with AS.
作者
金政
罗元
崔嘉妍
李梓源
李俊龙
皮建彬
卿立金
吴伟
Jin Zheng;Luo Yuan;Cui Jiayan;Li Ziyuan;Li Junlong;Pi Jianbin;Qing Lijin;Wu Wei(Zhujiang Hospital of Southern Medical University,Guangzhou 510280,China;Guangzhou University of Chinese Medicine,Guangzhou 510405,China;The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2022年第7期2619-2636,共18页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会面上项目(82074356):基于P2RY12受体介导PI3K/Akt/mTOR自噬信号通路探讨栀子苷干预晚期AS的机制研究,负责人:吴伟
广东省基础与应用基础研究基金委员会广东省区域联合基金-青年基金项目(2021A1515110287):清热活血法介导外泌体miR-26a-5p/PTGS2/NF-κB通路调控巨噬细胞极化改善AS的机制研究,负责人:金政。
关键词
清热活血方
动脉粥样硬化
液相-质谱
分子对接
Qingre Huoxue formula
Atherosclerosis
Liquid chromatography-mass spectrometry
Molecular docking
