本研究致力于通过综合策略，整合网络药理学、代谢组学和分子生物学验证，评估蟾毒素在肺腺癌（LUAD）治疗中的疗效和治疗机制。通过PharmMapper和Swiss Target Prediction数据库确定蟾毒素的潜在目标，通过GeneCard数据库的目标过滤和GEO数据库的数据挖掘获得与LUAD相关的目标。构建PPI网络筛选核心目标，并通过几个公共数据库评估其临床意义。进行GO和KEGG通路分析，以确定具有特定生物主题的基因可能的富集。采用分子对接和分子动力学模拟确定蟾毒素与核心目标之间的相关性和结合模式。通过体外和体内模型实验验证网络药理分析预测的蟾毒素作用于LUAD的潜在机制。最后，通过非靶向代谢组学研究，探讨了蟾毒素干预LUAD裸鼠代谢物和代谢通路的效果。收集了209个蟾毒素靶点和1082个与LUAD相关的靶点，识别出51个交叉靶点。通过网络拓扑分析，选出了包括Akt1、STAT3、EGFR、CASP3和SRC在内的10个核心靶点，并通过分子对接和分子动力学模拟表明它们与蟾毒素具有潜在的结合活性。PPI网络的中心模块与细胞增殖和凋亡有密切关系。GO和KEGG富集分析表明，蟾毒素通过PI3K/Akt、FoxO1和MAPK/ERK信号通路可能通过抑制增殖和促进凋亡发挥LUAD的治疗作用，并且这些发现已经通过一系列体外研究和肿瘤组织的HE、TUNEL和Ki-67染色得到了证实。进一步的代谢组学分析显示，蟾毒素主要调节ABC转运蛋白和重新构建AA代谢通路，从而有助于治疗LUAD。从分子和代谢角度来看，本研究成功地过滤出相关的关键靶基因、差异内源性代谢物和信号通路，提供了一种独特的认识蟾毒素对抗LUAD可能机制的视角，并提出了一种新的有望的LUAD治疗策略。版权所有©2023 Elsevier Ltd.，由作者发布。保留所有权利。

This study aims to evaluate the efficacy and therapeutic mechanism of bufalin on lung adenocarcinoma (LUAD) through a comprehensive strategy integrating network pharmacology, metabolomics and molecular biology verification.The putative targets of bufalin were discerned from PharmMapper and Swiss Target Prediction database. LUAD-related targets were obtained by target filtering of GeneCard database and data mining of GEO database. PPI network was constructed to screen the core targets, and their clinical significance was assessed through several public databases. GO and KEGG pathway analyses were performed to identify possible enrichment of genes with specific biological themes. Molecular docking and molecular dynamics (MD) simulation were employed to determine the correlation and binding pattern between bufalin and core targets. The potential mechanisms of bufalin acting on LUAD, as predicted by network pharmacology analyses, were experimentally validated using in-vitro and in-vivo models. Finally, the effects of bufalin intervention on metabolite profile and metabolic pathway in LUAD nude mice were investigated by non-targeted metabolomics.209 bufalin targets and 1082 LUAD-associated targets were harvested, of which 51 intersection targets were identified. 10 core targets including Akt1, STAT3, EGFR, CASP3 and SRC were picked out through network topology analysis, and they had a potent binding activity with bufalin as indicated by molecular docking and MD simulation. Hub module of PPI network was closely related to cell proliferation and apoptosis. GO and KEGG enrichment analyses suggested that bufalin exerted therapeutic effects on LUAD possibly by inhibiting proliferation and promoting apoptosis via PI3K/Akt, FoxO1 and MAPK/ERK pathways, which were confirmed by a series of in-vitro studies as well as HE, TUNEL and Ki-67 staining of tumor tissues. Further metabolomics analysis revealed that bufalin mainly regulated ABC transporter and remodeled AA metabolism, thereby contributing to the treatment of LUAD.From molecular and metabolic perspective, the present study not only provided a unique insight into the possible mechanisms of bufalin against LUAD after successfully filtering out associated key target genes, differential endogenous metabolites, and signaling pathways, but also proposed a novel promising therapeutic strategy for LUAD.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.