本研究旨在利用网络药理学预测山柰子（OS）在三阴性乳腺癌（TNBC）中的治疗机制，并通过体外实验证实。从中药系统药理数据库检索了OS的活性成分和靶蛋白，并从GeneCards数据库获取了与TNBC相关的靶基因。利用重叠的基因构建蛋白质相互作用（PPI）网络，通过String数据库。此外，我们采用在线生物信息学分析平台（https://www.bioinformatics.com.cn/）进行基因本体论和基因组百科全书信号通路富集分析，评估生物过程、分子功能和细胞组分，并生成模拟信号通路。此外，采用分子对接方法评估小分子药物与信号通路靶点的结合能力。通过CCK8实验检测小分子药物对TNBC细胞存活性的影响，并利用Western Blot验证AKT、VEGF和低氧诱导因子1α（HIF-1α）蛋白的表达。从中药系统药理数据库获取了OS的15个活性成分和166个治疗靶点。Venn图显示，其中163个靶点与TNBC相关。蛋白质相互作用网络分析确定了AKT1，IL-6，JUN，血管内皮生长因子A（VEGFA），CASP3和HIF-1α作为OS可能治疗TNBC的潜在核心靶点。此外，分子对接结果表明，OS中的活性成分金莲花苷与VEGFA和HIF-1α具有良好的结合能力。CCK8实验结果表明，金莲花苷抑制了MDA-MB-231和BT-20细胞的存活性。Western Blot显示，与对照组相比，金莲花苷干预导致VEGFA和HIF-1α蛋白表达下降（P <.05），PARP的裂解增加。OS可能通过多个细胞信号通路发挥其对TNBC的治疗作用。OS的活性成分金莲花苷可以通过靶向VEGFA/HIF-1α通路增强TNBC细胞的凋亡。本研究为TNBC中OS的潜在治疗机制提供了新的见解，并可能有助于发展新的TNBC治疗方法。Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.

This study aimed to use network pharmacology to predict the therapeutic mechanism of oroxyli semen (OS) on triple-negative breast cancer (TNBC) and validate it through in vitro experiments.The active ingredients and target proteins of OS were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, and the TNBC-related target genes were obtained from the GeneCards database. The overlapping genes were used to construct a protein-protein interaction (PPI) network via the String database. Furthermore, we employed an online bioinformatics analysis platform (https://www.bioinformatics.com.cn/) to perform gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses to evaluate biological processes, molecular functions, and cellular components and generate simulated signal pathways. Additionally, molecular docking was used to evaluate the binding ability of small molecule drugs and signaling pathway targets. CCK8 assay was conducted to detect the effect of small molecule drugs on TNBC cell viability, and Western Blot was utilized to verify the expression of AKT, VEGF, and hypoxia-inducible factor 1-alpha (HIF-1α) proteins.Fifteen active ingredients and 166 therapeutic targets of OS were obtained from the Traditional Chinese Medicine Systems Pharmacology database. The Venn diagram revealed that 163 targets were related to TNBC. The protein-protein interaction network analysis identified AKT1, IL-6, JUN, vascular endothelial growth factor A (VEGFA), CASP3, and HIF-1α as potential core targets through which OS may treat TNBC. Furthermore, the molecular docking results indicated that the active ingredient chryseriol in OS had good binding ability with VEGFA, and HIF-1α. CCK8 assay results indicated that chryseriol inhibited the viability of MDA-MB-231 and BT-20 cells. Western Blot demonstrated that chryseriol intervention led to a decrease in VEGFA, and HIF-1α protein expression compared with the control group (P < .05), increased the cleaved PARP.OS may exert its therapeutic effects on TNBC through multiple cellular signaling pathways. Chryseriol, the active component of OS, can enhance the apoptosis of TNBC cells by targeting VEGFA/HIF-1α pathway. This study provided new insights into the potential therapeutic mechanism of OS for TNBC and may aid in the development of novel therapeutic approaches for TNBC.Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.