目的 通过网络药理学、分子对接和体外实验探讨汉防己甲素（TET）治疗胰腺癌的潜在作用靶点，并阐明其相关分子机制。方法 利用中药系统药理学数据库与分析平台（TCMSP）、SwissTargetPrediction和PharmMapper数据库构建TET的潜在作用靶点数据集，与在GeneCards和OMIM数据库中获得的PAAD相关靶点取交集，得到TET治疗PAAD的潜在作用靶点。利用STRING数据库获取交集靶点蛋白的蛋白质–蛋白质相互作用（PPI）网络信息并导入Cytoscape 3.8.0筛选核心靶点；运用Metascape数据库对交集靶点进行基因本体论（GO）功能富集分析和京都基因与基因组百科全书（KEGG）信号通路富集分析。使用AutoDock和PyMol软件进行分子对接验证。采用CCK-8法检测不同梯度浓度TET对胰腺癌细胞增殖活性的影响；胰腺癌细胞分为对照组和汉防己甲素6.25、12.5、25 μmol/L组，采用细胞划痕愈合实验、克隆形成实验和流式细胞术检测各组细胞迁移率、克隆形成情况和细胞周期变化。实时荧光定量PCR（qRT-PCR）法检测各组细胞中磷脂酰肌醇3激酶（PI3K）–蛋白激酶B（Akt）通路相关基因表达水平。结果 获得TET影响胰腺癌的潜在靶点140个，分析构建胰腺癌与TET之间的PPI网络，获得核心靶点Akt1、表皮生长因子受体（EGFR）和PIK3CA等。分子对接显示这些核心靶点与TET都具有良好的结合活性。GO和KEGG富集分析表明TET治疗胰腺癌的关键靶点主要涉及磷酸化等生物学过程和PI3K-Akt等信号通路。胰腺癌细胞的死亡率随着TET浓度的增加而升高。与对照组比较，TET组细胞迁移率降低（P＜0.01），TET组PANC-1细胞克隆形成数减少（P＜0.001）；与对照组比较，TET组G₀/G₁期比例升高（P＜0.001）。与对照组相比，TET组血小板源性生长因子受体α（PDGFRA）基因表达下调（P＜0.05），而T细胞白血病/淋巴瘤1A（TCL1A）、Jun、ILK、哺乳动物雷帕霉素靶蛋白（mTOR）、核转录因子-κB抑制剂α（NF-κBIA）、PIK3CA、丝裂原活化蛋白激酶8（MAPK8）基因表达水平显著升高（P＜0.05）。结论 TET可抑制人胰腺癌细胞增殖、迁移和克隆，造成细胞周期阻滞，其作用机制可能与PI3K-Akt信号通路有关。

Objective To analyze the potential therapeutic targets of tetrandrine (TET) in treatment of pancreatic adenocarcinoma (PAAD) by network pharmacology, molecular docking, and in vitro cellular test, and to clarify the related molecular mechanism. Methods Potential target datasets for TET were compiled utilizing the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), Swiss Target Prediction and PharmMapper. To obtain target information pertinent to pancreatic cancer, the term “pancreatic ductal adenocarcinoma” was employed in searches within the GeneCards and OMIM databases. The intersection of TET targets and pancreatic cancer targets was identified to determine the potential therapeutic targets for pancreatic cancer. Subsequently, a protein-protein interaction (PPI) network was constructed using the STRING database and imported into Cytoscape 3.8.0 software to identify core targets. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were conducted using the Metascape database. Molecular docking validation was performed utilizing AutoDock and PyMOL software. Cell proliferation activity was detected by CCK-8 assay. Additionally, pancreatic cancer cells were categorized into a control group and TET treatment group (6.25, 12.5, 25 μmol/L) to evaluate cell migration via the scratch assay, clone formation ability through colony formation assay, and cell cycle distribution using flow cytometry. The mRNA expression on level of PI3K-Akt pathway-related genes in the cells were detected using the real-time fluorescence quantitative PCR (qRT-PCR) method. Results A total of 140 potential targets of TET affecting pancreatic cancer were obtained, the PPI network between TET and pancreatic cancer was analyzed and constructed, and core targets such as Akt1, epidermal growth factor receptor (EGFR) and PIK3CA were obtained. Molecular docking showed that these core targets had good binding activity with TET. GO and KEGG enrichment analysis revealed that the key targets of TET in the treatment of pancreatic cancer were mainly involved in biological processes such as phosphorylation and PI3K-Akt, MAPK and other signaling pathways. It was observed that the mortality rate of pancreatic cancer cells treated with TET increased proportionally with the concentration of the drug, compared to the control group. Additionally, the migration rate of PANC-1 cells in the TET treatment group was significantly reduced (P < 0.01) relative to the control group. Furthermore, compared with the blank control group, the number of colony formation of PANC-1 cells in the TET treatment group was significantly decreased (P < 0.001), indicating a dose-dependent effect. Flow cytometry analysis revealed a significant increase in the proportion of cells in the G₀/G₁ phase in the TET treatment group compared with control group (P < 0.001). Compared with the control group, the expression levels of PDGFRA mRNA in the TET group were significantly decreased (P < 0.05). The mRNA expression levels of TCL1A, Jun, ILK, mTOR, NF-κBIA, GRB10, FOS, CASP9, PIK3CA, CD40, and MAPK8 were significantly increased (P < 0.05). Conclusion TET can inhibit PAAC cell proliferation, migration, cloning, and causing cell cycle arrest, and the underlying mechanism of which may involve the PI3K-Akt signaling pathways.

R285

福建省自然科学基金资助项目（2022J01531，2023J01250）