目的 通过网络药理学、分子对接和细胞实验探讨4'-去甲基表鬼臼毒素治疗结直肠癌的作用机制。方法 运用Pharmmapper、SEA、ChEMBL数据库预测靶点，运用UniProt数据库将靶点进行标准化处理，以获取相应的药物靶点。利用TTD、DisGeNET、GeneCards、OMIM数据库获得结直肠癌的靶点。将药物及疾病的靶点导入韦恩图绘制软件获得4'-去甲基表鬼臼毒素治疗结直肠癌的潜在靶点。使用Cytoscape 3.7.1软件构建“药物–靶点–疾病”网络。利用仙桃学术数据库对潜在治疗靶点进行基因本体（GO）功能富集分析以及京都基因与基因组百科全书（KEGG）通路分析。同时将潜在治疗靶点导入STRING数据库，以进行蛋白质相互作用（PPI）网络的分析，并使用Cytoscape 3.7.1软件中的CytoNCA插件进行拓扑分析筛选核心靶点。运用AutoDock vina、PyMOL等软件对4'-去甲基表鬼臼毒素和核心靶点进行对接验证。利用结直肠癌模型细胞HCT166进行实验验证，采用CCK-8法检测4'-去甲基表鬼臼毒素对HCT166细胞增殖的影响，流式染色法检验4'-去甲基表鬼臼毒素对HCT166细胞凋亡的影响。通过Western blotting检测磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）通路的关键蛋白表达水平。结果 共获得了270个4'-去甲基表鬼臼毒素的作用靶点基因和3 877个结直肠癌的作用靶点，其中4'-去甲基表鬼臼毒素与结直肠癌共同作用靶点为75个。通过PPI网络分析发现热休克蛋白90α家族A类成员1（HSP90AA1）、酪氨酸激酶受体2（ERBB2）、多聚ADP核糖聚合酶1（PARP1）等为核心靶点。富集结果显示4'-去甲基表鬼臼毒素可能通过PI3K/Akt和低氧诱导因子-1（HIF-1）等信号通路发挥治疗结直肠癌的作用。分子对接的结果表明4'-去甲基表鬼臼毒素和核心靶点具有显著的结合性，对接结合能均低于−7.0 kcal/mol。细胞实验表明，与对照组比较，4'-去甲基表鬼臼毒素抑制HCT116细胞的增殖并促进细胞凋亡（P＜0.001）；Western blotting实验结果提示4'-去甲基表鬼臼毒素抑制HCT116细胞中p-Akt、p-PI3K蛋白的表达（P＜0.001）。结论 通过网络药理学、分子对接以及细胞实验探究发现4'-去甲基表鬼臼毒素可能通过HSP90AA1、ERBB2、PARP1等靶点以及PI3K/Akt信号通路发挥治疗结直肠癌的作用，为4'-去甲基表鬼臼毒素的临床应用和结直肠癌治疗的研究提供依据。

Objective To explore the mechanism of action of 4'-demethylepipodophyllotoxin in treatment of colorectal cancer through network pharmacology, molecular docking, and cellular experiments. Methods The targets were predicted using Pharmmapper, SEA, and ChEMBL databases, and standardized using the UniProt database to obtain the corresponding drug target. The target genes for colorectal cancer were obtained from TTD, DisGeNET, GeneCards, and OMIM databases. The drug and disease target were imported into Venn diagram software to identify potential targets of 4'-demethylepipodophyllotoxin for colorectal cancer treatment. The “drug-target-disease” network was constructed using Cytoscape 3.7.1 software. GO functional enrichment analysis and KEGG pathway analysis were performed on potential therapeutic targets using the Xiantao Academic Database. The potential therapeutic targets were also imported into the STRING database for PPI network analysis, and core targets were screened through topological analysis using CytoNCA in Cytoscape 3.7.1 software. Molecular docking validation of 4'-demethylepipodophyllotoxin and core targets was conducted using AutoDock Vina and PyMOL software. Using the colorectal cancer model cell HCT166 for verification experiment, with the CCK-8 method to detect the effect of 4'-demethylepipodophyllotoxin on HCT166 cell proliferation and flow cytometry to examine the effect of 4'-demethylepipodophyllotoxin on HCT166 cell apoptosis. Western blotting was used to detect the expression levels of key proteins in thePI3K/Akt pathway. Results A total of 270 action target for 4'-demethylepipodophyllotoxin and 3 877 action target for colorectal cancer were obtained, with 75 common action target between 4'-demethylepipodophyllotoxin and colorectal cancer. PPI network analysis identified core target such as HSP90AA1, ERBB2, and PARP1. The enrichment results showed that 4'-demethylepipodophyllotoxin may play a therapeutic role in colorectal cancer through signaling pathways such as PI3K/Akt and HIF-1. Molecular docking results showed that 4'-demethylepipodophyllotoxin has significant binding affinity with core targets, with docking binding energies below -7.0 kcal/mol. Cellular experiments indicated that compared to the control group, 4'-demethylepipodophyllotoxin inhibited HCT116 cell proliferation and promoted apoptosis (P < 0.001), while Western blotting results suggested that 4'-demethylepipodophyllotoxin inhibited the expression of p-Akt and p-PI3K proteins in HCT116 cells (P < 0.001). Conclusion Through network pharmacology, molecular docking, and cellular experiments, it was found that 4'-demethylepipodophyllotoxin may exert therapeutic effects on colorectal cancer through target such as HSP90AA1, ERBB2, and PARP1, as well as the PI3K/Akt signaling pathway, providing a basis for the clinical application of 4'-demethylepipodophyllotoxin and research on colorectal cancer treatment.

R285;R286.5

重庆市长寿区科技计划项目（CSKJ2024028）