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基于网络药理学及体外实验探究温下方正丁醇部位联合吉非替尼治疗非小细胞肺癌的作用机制 被引量:2

Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment
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摘要 运用网络药理学、分子对接和体外实验探究温下方正丁醇部位(n-butanol fraction of Wenxia Formula,NWXF)联合吉非替尼(gefitinib,GEF)治疗非小细胞肺癌(non-small cell lung cancer,NSCLC)的潜在作用机制。超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(UPLC-Q-Orbitrap MS)检测NWXF的主要化学成分,利用SwissADME数据库筛选NWXF的活性成分,SwissTargetPrediction数据库获取NWXF活性成分的潜在作用靶点,OMIM和GeneCards数据库搜集NSCLC相关疾病靶点。运用Cytoscape 3.9.0软件和STRING数据库对共同靶点进行蛋白-蛋白互作(protein-protein interaction,PPI)网络构建和关键靶点筛选,利用DAVID数据库进行基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,预测其可能的作用机制。运用SYBYL-X 2.0软件对核心成分与关键靶点进行分子对接验证。MTT法检测NWXF和GEF单用及联用对人非小细胞肺癌细胞(A549、PC-9)增殖的抑制作用以及NWXF对人胚肺成纤维细胞MRC-5的作用,运用Q值评价两药联用的效果,TUNEL法检测NWXF和GEF单用及联用对A549、PC-9细胞凋亡的影响,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)法检测NWXF和GEF单用及联用对A549、PC-9细胞EGFR、JNK、Bax mRNA表达的影响,蛋白印迹(Western blot,WB)法检测NWXF和GEF单用及联用对A549和PC-9细胞EGFR、p-EGFR、JNK、p-JNK、Bax蛋白表达的影响。结果共获取NWXF治疗NSCLC的77种活性成分、488个潜在靶点及49个关键靶点。GO富集分析结果显示,NWXF干预NSCLC可能与调控细胞增殖、凋亡过程及蛋白质磷酸化等生物学过程相关,KEGG富集分析结果显示,NWXF干预NSCLC可能与调控MAPK信号通路、PI3K-AKT信号通路、HIF-1信号通路及肿瘤中的microRNA等信号通路相关。NWXF中核心成分与关键靶点分子对接结果显示,91.9%的对接分值>5,表明核心成分与关键靶点有较好的结合能力。� This study combined network pharmacology,molecular docking,and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC).Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF.The active components of NWXF were retrieved from SwissADME,and the candidate targets of these active components were retrieved from SwissTargetPrediction.Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC.Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC.Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms.Finally,molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0.The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9).Additionally,the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed.The effectiveness of the drug combination was evaluated based on the Q value.The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF.Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR),c-Jun N-terminal kinase(JNK),and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF.Western blot was employed to determine the protein levels of EGFR,p-EGFR,JNK,p-JNK,and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF.A total of 77 active components,488 potential targets
作者 陈瑞杰 毕倩宇 施杰民 钟婧 韩晋涛 季旭明 CHEN Rui-jie;BI Qian-yu;SHI Jie-min;ZHONG Jing;HAN Jin-tao;JI Xu-ming(Huzhou Central Hospital,Huzhou 313000,China;School of Basic Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,China;Laboratory of Neuropharmacology and Translational Medicine of Zhejiang Province,Hangzhou 310053,China;Academy of Chinese Medical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China)
出处 《中国中药杂志》 CAS CSCD 北大核心 2024年第2期471-486,共16页 China Journal of Chinese Materia Medica
基金 国家自然科学基金项目(81774198,82274406)。
关键词 温下方正丁醇部位 吉非替尼 非小细胞肺癌 凋亡 网络药理学 分子对接 机制 n-butanol fraction of Wenxia Formula gefitinib non-small cell lung cancer apoptosis network pharmacology mole-cular docking mechanism
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