Table2_The potential mechanism of Aidi injection against neuroblastoma—an investigation based on network pharmacology analysis.XLSX

Background: Aidi injection, a classic traditional Chinese medicine (TCM) formula, has been used on a broader scale in treating a variety of cancers. In this study, we aimed to explore the potential anti-tumor effects of Aidi injection in the treatment of neuroblastoma (NB) using network pharmacology (NP).
Methods: To elucidate the anti-NB mechanism of Aidi injection, an NP-based approach and molecular docking validation were employed. The compounds and target genes were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN-TCM) database. The protein–protein interaction network was constructed using the STRING database. clusterProfiler (R package) was utilized to annotate the bioinformatics of hub target genes. The gene survival analysis was performed on R2, a web-based genomic analysis application. iGEMDOCK was used for molecular docking validation, and GROMACS was utilized to validate molecular docking results. Furthermore, we investigated the anticancer effects of gomisin B and ginsenoside Rh2 on human NB cells using a cell viability assay. The Western blot assay was used to validate the protein levels of target genes in gomisin B- and ginsenoside Rh2-treated NB cells.
Results: A total of 2 critical compounds with 16 hub target genes were identified for treating NB. All 16 hub genes could potentially influence the survival of NB patients. The top three genes (EGFR, ESR1, and MAPK1) were considered the central hub genes from the drug–compound–hub target gene–pathway network. The endocrine resistance and estrogen signaling pathways were identified as the therapeutic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Gomisin B and ginsenoside Rh2 showed a good binding ability to the target protein in molecular docking. The results of cell experiments showed the anti-NB effect of gomisin B and ginsenoside Rh2. In addition, the administration of gomisin B over-regulated the expression of ESR1 protein in MYCN-amplified NB cells.
Conclusion: In the present study, we investigated the potential pharmacological mechanisms of Aidi against NB and revealed the anti-NB effect of gomisin B, providing clinical evidence of Aidi in treating NB and establishing baselines for further research.

背景：艾迪注射液作为经典中医药（Traditional Chinese Medicine, TCM）方剂，目前已被广泛应用于多种癌症的治疗。本研究旨在借助网络药理学（network pharmacology, NP）方法，探究艾迪注射液治疗神经母细胞瘤（neuroblastoma, NB）的潜在抗肿瘤作用。
方法：为阐明艾迪注射液抗神经母细胞瘤的作用机制，本研究采用基于网络药理学的分析方法与分子对接验证技术。研究中，我们从中药系统药理学数据库与分析平台（Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, TCMSP）以及中药分子机制生物信息学分析工具（Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine, BATMAN-TCM）中获取方剂活性成分与潜在靶基因。通过STRING数据库构建蛋白质相互作用网络，利用R语言包clusterProfiler对核心靶基因（hub target genes）进行生物信息学注释。基于网页端基因组分析应用R2开展基因生存分析，采用iGEMDOCK进行分子对接验证，并借助GROMACS软件对分子对接结果进行二次验证。此外，我们通过细胞活力检测实验，探究了五味子素B（gomisin B）与人参皂苷Rh2（ginsenoside Rh2）对人神经母细胞瘤细胞的抗癌活性；采用蛋白质印迹（Western blot）实验，验证了经五味子素B与人参皂苷Rh2处理的神经母细胞瘤细胞中靶基因的蛋白表达水平。
结果：本研究共筛选出2种关键活性成分与16个核心靶基因，用于神经母细胞瘤的治疗。所有16个核心靶基因均可能对神经母细胞瘤患者的生存预后产生影响。从"药物-成分-核心靶基因-通路"网络中筛选得到的核心枢纽基因为前三者：表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）、雌激素受体1（Estrogen Receptor 1, ESR1）以及丝裂原活化蛋白激酶1（Mitogen-Activated Protein Kinase 1, MAPK1）。通过京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，确定内分泌抵抗与雌激素信号通路为艾迪注射液治疗神经母细胞瘤的潜在通路。分子对接结果显示，五味子素B与人参皂苷Rh2均可与靶蛋白实现良好结合。细胞实验结果证实了五味子素B与人参皂苷Rh2的抗神经母细胞瘤活性。此外，在MYCN扩增的神经母细胞瘤细胞中，五味子素B处理可上调ESR1蛋白的表达水平。
结论：本研究探究了艾迪注射液抗神经母细胞瘤的潜在药理机制，并揭示了五味子素B的抗神经母细胞瘤作用，为艾迪注射液治疗神经母细胞瘤提供了临床依据，同时为后续相关研究奠定了基础。