基于网络药理学与实验验证探...岩宁方治疗非小细胞肺癌机制_桑舒柳.pdf

安 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Aug，27（8）基于网络药理学与实验验证探讨肺岩宁方治疗非小细胞肺癌机制桑舒柳，丁蓉珍，姜靖洁，龚亚斌作者单位：上海中医药大学附属岳阳中西医结合医院肿瘤科，上海200437通信作者：龚亚斌，男，主任医师，博士生导师，研究方向为中医药防治恶性肿瘤，Email：基金项目：国家自然科学基金资助项目（82074339）摘要：目的 通过网络药理学筛选肺岩宁方治疗非小细胞肺癌（NSCLC）的潜在靶点和相关通路并通过实验验证其可能的作用机制。方法 检索TCMSP数据库和相关文献，获取肺岩宁方的有效化学成分和潜在作用靶点，查询Genecards、DisGeNET疾病数据库预测非小细胞肺癌疾病相关靶点。以非小细胞肺癌疾病靶点与肺岩宁方潜在作用靶点的交集靶点作为研究对象，采用Cytoscape 3.7.2软件构建活性成分-靶点网络图。利用STRING数据库构建蛋白质-蛋白质相互作用网络图，通过设置拓扑参数筛选出肺岩宁方治疗非小细胞肺癌的重要靶点，进行GO和KEGG分析，通过GEPIA数据库得到与肺癌病人生存相关的核心靶点。制备不同浓度的肺岩宁方干预HCC827细胞，采用CCK8法检测细胞增殖情况并计算IC50；平板克隆形成实验检测肺岩宁方对HCC827细胞克隆形成能力的影响；Transwell 侵袭实验检测肺岩宁方对HCC827细胞侵袭能力的抑制作用。采用qPCR array技术检测网络药理学预测的核心靶点mRNA的表达水平。结果 肺岩宁方共有133个活性成分、521个相关靶点作用于非小细胞肺癌，GO和KEGG分析表明肺岩宁方通过11种成分影响71个重要靶点而发挥抗非小细胞肺癌作用，其中有14个靶点与肺癌病人生存相关，可能与PI3K-AKT等多种信号通路相关。肺岩宁方能抑制HCC827细胞的增殖能力，克隆形成能力以及侵袭能力，并下调PI3K-AKT信号通路的关键基因KIT的mRNA水平。结论 肺岩宁方治疗NSCLC的作用机制可能是通过下调KIT调控PI3K-AKT信号通路来实现的。关键词：癌，非小细胞肺；网络药理学；肺岩宁方；信号通路Exploring the molecular mechanism of Feiyanning in the treatment of non-small cell lung cancer based on network pharmacology and experimental verificationSANG Shuliu,DING Rongzhen,JIANG Jingjie,GONG YabinAuthor Affiliation:Department of Oncology,Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200437,ChinaAbstract：Objective To screen the potential targets and related pathways of Feiyanning in the treatment of non-small cell lung cancer(NSCLC)through network pharmacology,and to verify its possible mechanism of action by experiments.Methods The bioactive compounds and potential targets of Feiyanning were obtained by the TCMSP database and related literatures.The targets related to NSCLC were predicted by the Genecards database and DisGeNET database.The intersection target of non-small cell lung cancer disease target and the potential action target of Feiyanning was selected as the study object.The active ingredient-target network map was constructed by Cytoscape 3.7.2 software.A protein-protein interaction(PPI)network map was developed by using the STRING database,and the core targets of Feiyanning in the treatment of NSCLC were screened by setting topology parameters to perform GO and KEGG analysis.Core targets associated with the survival of lung cancer patients were identified through the GEPIA database.HCC827 cells were treated with different concentrations of Feiyanning.The cell proliferation was detected by CCK8,and IC50 was calculated.The colony formation assay was performed to detect the effect of Feiyanning on the clone formation ability of HCC827 cells.Transwell assay was performed to detect the inhibition effect of Feiyanning on the invasion ability of HCC827 cells.qPCR array technique was used to explore the expression levels of mRNA of core targets predicted by network pharmacology.Results There were 133 bioactive compounds and 521 relevant targets acting on NSCLC in Feiyanning.GO and KEGG analysis showed that Feiyanning exerted anti-NSCLC effects by influencing 71 important targets with 11 components,among which 14 targets were associated with the survival of lung cancer patients,possibly related to various signaling pathways such as PI3K-AKT.Feiyanning inhibited the proliferation,clonality,and invasion ability of HCC827 cells,and suppressed the mRNA of KIT which was the key gene in the PI3K-AKT pathway.Conclusion The action mechanism of Feiyanning in the treatment of NSCLC may be realized by down-regulating KIT to regulate the PI3K-AKT signaling pathway.Key words：Carcinoma,non-small-cell lung;Network pharmacology;Feiyanning;Signaling pathway药学研究引用本文：桑舒柳，丁蓉珍，姜靖洁，等.基于网络药理学与实验验证探讨肺岩宁方治疗非小细胞肺癌机制 J.安徽医药，2023，27（8）：1516-1520.DOI：10.3969/j.issn.1009-6469.2023.08.008.1516网络首发时间：2023-07-06 16:17:02网络首发地址：https:/ 徽 医 药 Anhui Medical and Pharmaceutical Journal 2023 Aug，27（8）据统计，目前肺癌的死亡率仍居首位，是癌症死亡的主要原因1。非小细胞肺癌（non-small cell lung cancer，NSCLC）约占肺癌总发病率的 85%2。肺癌治疗方法包括手术、放化疗、靶向、免疫等3-4，但病人预后欠佳。据调查显示约40%的NSCLC病人存在表皮生长因子受体（epidermal growth factor receptor，EGFR）突变5，靶向治疗在临床应用越来越广泛，但病人仍出现明显的不良反应。多项研究已证实中医药在恶性肿瘤的治疗中应用广泛，可增效减毒，提高病人生存质量、延长生存期等6。肺岩宁方是上海市名中医徐振晔教授总结多年临床实践而研发出来的抗癌经验方，由生黄芪、黄精、石见穿、七叶一枝花等组成，具有益气养精，解毒散结之功7。临床研究表明肺岩宁方能有效抑制晚期肺癌的侵袭和转移8，并能有效提高NSCLC病人的生存质量9，但肺岩宁方治疗EGFR突变型NSCLC的具体分子机制仍未阐明。本研究于 2020 年 11 月至 2021年 2月以网络药理学为切入点，预测肺岩宁方治疗EGFR突变型NSCLC的主要活性成分、作用靶点及作用通路，并通过细胞实验进行生物学验证，探讨肺岩宁方治疗EGFR突变型NSCLC的潜在作用机制，为其临床上更广泛应用提供依据。1材料与方法1.1化合物有效成分和靶点的收集及疾病靶点的预测以“白术”“蜂房”“干蟾皮”“黄精”“黄芪”“灵芝”“山慈菇”“石见穿”“淫羊藿”“重楼”“山茱萸”为检索词，在 TCMSP 数据库（https：/old.tcmsp- smile号，并通过 Swiss Target Prediction收集对应的靶点。将关键词设置为“non-small cell lung cancer”，检索 Genecard（www.genecards.org）和 DisGeNET（www.disgenet.org）数 据 库，选 择“Homo sapiens”，检 索NSCLC的疾病靶点。然后将药物靶点与疾病相关靶点取并集，得到韦恩图，并集得到的靶点则为肺岩宁方治疗NSCLC的潜在靶点。1.2肺岩宁方活性成分-靶点网络和蛋白质相互作用网络（PPI）构建将上述获得的活性成分及靶点对应关系导入到软件Cytoscape 3.7.2中进行相关的网络构建和分析，画出肺岩宁方“活性成分-靶点网络”图，采用“Network Analyze”插件进行网络特征分析来筛选重要的活性成分。将肺岩宁方与NSCLC的并集靶点导入STRING（https：/string-db.org）平台，选择“Homo sapiens”，筛选条件为置信度0.7，下载蛋白互作文件后将其导入到 Cytoscape 3.7.2 中，生成 PPI 网络图，以“Degree”“Be