目的 通过网络药理学和分子对接技术分析钩吻素甲（GEL）抗舌鳞状细胞癌（TSCC）的靶点及作用机制，并通过实验验证GEL对人舌鳞癌CAL27细胞增殖、凋亡的影响及关键靶点的作用。方法 运用网络药理学预测GEL和TSCC的共有靶点，绘制蛋白质-蛋白质相互作用（PPI）网络图，并进行基因本体（GO）功能富集分析和京都基因与基因组百科全书（KEGG）通路富集分析，使用Autodock vina软件进行分子对接。通过Incucyte S3活细胞动态分析系统观察GEL作用下CAL27细胞形态，并拟合半数抑制浓度（IC₅₀）值，采用细胞增殖与活性检测-8（CCK-8）实验、平板克隆实验、细胞周期实验检测细胞增殖能力；通过Hoechst 33258染色、Rhodamine 123染色检测细胞凋亡情况，Western blotting检测B淋巴细胞瘤2基因相关X基因（Bax）、B淋巴细胞瘤2基因（Bcl-2）、含半胱氨酸的蛋白水解酶3基因（Caspase-3）蛋白表达。结果 筛选出药物-疾病共有靶点49个，核心靶点为雌激素受体1（ESR1）、含半胱氨酸的蛋白水解酶3基因（CASP3）、基质金属蛋白酶9（MMP-9）、B淋巴细胞瘤2基因样1（BCL2L1）、非受体酪氨酸激酶（SRC），KEGG通路富集较高的为癌症途径、PI3K/Akt通路等。分子对接结合良好，其中GEL与CASP3和BCL2L1有强结合性。体外实验显示，GEL可以抑制CAL27细胞增殖并阻滞细胞周期于G₂/M期，GEL干预下CAL27细胞染色质固缩，线粒体膜电位降低，Bax、Caspase-3蛋白表达增多，Bcl-2蛋白表达降低。结论 GEL通过多靶点、多途径抑制人舌鳞癌CAL27细胞增殖并促进其凋亡，可能与激活Bax/Bcl-2/Caspase-3通路有关。

Objective To investigate the targets and mechanism of action of GEL against tongue squamous cell carcinoma (TSCC) by network pharmacology and molecular docking techniques, and to verify the effects of GEL on the proliferation, apoptosis of human tongue squamous carcinoma CAL27 cells and the effects of key targets by experimental methods. Methods The shared targets of GEL and TSCC were predicted by network pharmacology, and the PPI network diagram was drawn. GO function and KEGG pathway enrichment analysis were performed, and molecular docking was performed using Autodock vina software. The cell morphology of CAL27 cells under GEL action was observed by Incucyte S3 live cell dynamic analysis system and the IC₅₀ value was fitted. The cell proliferation ability was detected by CCK-8 experiment, plate colony experiment and cell cycle experiment. The cell apoptosis was detected by Hoechst 33258 staining and Rhodamine 123 staining, and the expression of Bax, Bcl-2 and Caspase-3 proteins was detected by Western blotting. Results A total of 49 common targets were screened, including ESR1, CASP3, MMP-9, BCL2L1 and SRC, with the KEGG pathway enrichment being higher in cancer pathway, PI3K/Akt pathway, etc. The molecular docking was combined well, with GEL having a strong binding to CASP3 and BCL2L1. The in vitro experiment showed that GEL could inhibit the proliferation of CAL27 cells and block the cell cycle at the G₂/M phase. Under GEL intervention, the chromatin condensation of CAL27 cells was observed, the mitochondrial membrane potential was lowered, the expression of Bax, Caspase-3 proteins increased, and the expression of Bcl-2 protein decreased. Conclusion GEL can inhibit the proliferation of CAL27 cells through multiple targets and pathways, promote their apoptosis, and may be related to activating the Bax/Bcl-2/Caspase-3 pathway.

R285.5

广西自然科学基金资助项目（2018GXNSFDA050009）；广西中医药大学博士启动基金项目（2020BS034）；广西国际壮医医院引进人才科研启动基金项目（GZ2021RC016）