目的 通過網絡藥理學、分子對接和體外實驗探討漢防己甲素（TET）治療胰腺癌的潛在作用靶點，并闡明其相關分子機制。方法 利用中藥系統(tǒng)藥理學數據庫與分析平臺（TCMSP）、SwissTargetPrediction和PharmMapper數據庫構建TET的潛在作用靶點數據集，與在GeneCards和OMIM數據庫中獲得的PAAD相關靶點取交集，得到TET治療PAAD的潛在作用靶點。利用STRING數據庫獲取交集靶點蛋白的蛋白質–蛋白質相互作用（PPI）網絡信息并導入Cytoscape 3.8.0篩選核心靶點；運用Metascape數據庫對交集靶點進行基因本體論（GO）功能富集分析和京都基因與基因組百科全書（KEGG）信號通路富集分析。使用AutoDock和PyMol軟件進行分子對接驗證。采用CCK-8法檢測不同梯度濃度TET對胰腺癌細胞增殖活性的影響；胰腺癌細胞分為對照組和漢防己甲素6.25、12.5、25 μmol/L組，采用細胞劃痕愈合實驗、克隆形成實驗和流式細胞術檢測各組細胞遷移率、克隆形成情況和細胞周期變化。實時熒光定量PCR（qRT-PCR）法檢測各組細胞中磷脂酰肌醇3激酶（PI3K）–蛋白激酶B（Akt）通路相關基因表達水平。結果 獲得TET影響胰腺癌的潛在靶點140個，分析構建胰腺癌與TET之間的PPI網絡，獲得核心靶點Akt1、表皮生長因子受體（EGFR）和PIK3CA等。分子對接顯示這些核心靶點與TET都具有良好的結合活性。GO和KEGG富集分析表明TET治療胰腺癌的關鍵靶點主要涉及磷酸化等生物學過程和PI3K-Akt等信號通路。胰腺癌細胞的死亡率隨著TET濃度的增加而升高。與對照組比較，TET組細胞遷移率降低（P＜0.01），TET組PANC-1細胞克隆形成數減少（P＜0.001）；與對照組比較，TET組G₀/G₁期比例升高（P＜0.001）。與對照組相比，TET組血小板源性生長因子受體α（PDGFRA）基因表達下調（P＜0.05），而T細胞白血病/淋巴瘤1A（TCL1A）、Jun、ILK、哺乳動物雷帕霉素靶蛋白（mTOR）、核轉錄因子-κB抑制劑α（NF-κBIA）、PIK3CA、絲裂原活化蛋白激酶8（MAPK8）基因表達水平顯著升高（P＜0.05）。結論 TET可抑制人胰腺癌細胞增殖、遷移和克隆，造成細胞周期阻滯，其作用機制可能與PI3K-Akt信號通路有關。

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

福建省自然科學基金資助項目（2022J01531，2023J01250）