目的 采用网络药理学与体外细胞实验相结合的策略，系统探讨钩吻素子发挥抗胃癌效应的潜在分子机制。方法 通过GeneCards数据库获取胃癌相关疾病靶点；利用Pharmmapper、SwissTargetPrediction及TargetNet平台预测钩吻素子的潜在作用靶点；借助STRING数据库构建蛋白质-蛋白质相互作用（PPI）网络，并进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析；使用Cytoscape构建“成分-疾病-靶点-通路”可视化网络；通过分子对接与分子动力学模拟对核心靶点进行初步验证。同时采用CCK-8法、Annexin V FITC/PI双染流式细胞术及Western blotting实验对预测结果开展体外验证。结果 共筛选得到钩吻素子相关靶点176个、胃癌疾病靶点5 535个，二者交集靶点106个；核心靶点包括：丝裂原活化蛋白激酶1（MAPK1）-细胞外调节蛋白激酶2（ERK2）、生长因子受体结合蛋白2（GRB2）、丝裂原活化蛋白激酶激酶1（MAP2K1）-丝裂原/细胞外信号调节激酶激酶1（MEK1）、糖原合成激酶3β（GSK3B）、丝裂原活化蛋白激酶14（MAPK14）-p38丝裂原活化蛋白激酶（p38）、肝细胞生长因子受体（MET）、酪氨酸蛋白激酶JAK2-JAK2等。分子对接结果显示钩吻素子与MAPK1、MAP2K1、MAPK14等靶点具有较强结合亲和力，分子动力学模拟证实钩吻素子可与MAPK1形成稳定复合物。细胞实验表明，不同浓度钩吻素子作用MGC-803细胞24 h后，与对照组相比，细胞活力呈浓度相关性下降，差异具有统计学意义；细胞凋亡率随给药浓度升高而显著上升；钩吻素子可明显下调p-ERK1/2表达并上调p-p38表达水平。结论 钩吻素子通过多靶点、多通路协同发挥抗胃癌作用。体外实验证实其能够抑制胃癌MGC-803细胞增殖，并促进细胞凋亡，调控MAPK信号通路是其发挥抗肿瘤作用的重要潜在机制之一。

Objective To explore the potential mechanism of koumine against gastric cancer combined network pharmacology and in vitro cell experiments. Methods Gastric cancer-related targets were retrieved from the GeneCards database. The potential targets of koumine were predicted using Pharmmapper, SwissTargetPrediction and TargetNet. The protein-protein interaction (PPI) network was constructed via STRING, followed by GO and KEGG enrichment analyses. The “component-disease-target-pathway” network was visualized by Cytoscape, and key targets were preliminarily verified by molecular docking and molecular dynamics simulation. In addition, CCK-8 assay, Annexin V-FITC/PI flow cytometry and Western blotting were performed to validate the bioinformatic predictions. Results A total of 176 koumine-related targets and 5 535 gastric cancer-related targets were screened, among which 106 overlapping targets were identified. The core targets included MAPK1 (ERK2), GRB2, MAP2K1 (MEK1), GSK3B, MAPK14 (p38), MET, JAK2, etc. Molecular docking showed that koumine had strong binding affinity with MAPK1, MAP2K1 and MAPK14, and molecular dynamics simulation confirmed that koumine could form a stable complex with MAPK1. Cell experiments demonstrated that after treatment with different concentrations of koumine for 24 h, the viability of MGC-803 cells was decreased in a concentrationdependent manner, and the apoptosis rate was significantly increased. Meanwhile, koumine obviously down-regulated the expression of p-ERK1/2 and up-regulated the expression of p-p38. Conclusion Koumine exerts anti-gastric cancer effects through multiple targets and signaling pathways. In vitro experiments confirmed that koumine inhibits the proliferation and promotes the apoptosis of gastric cancer MGC-803 cells, and the regulation of the MAPK signaling pathway serves as one of its key underlying mechanisms.

R285.5

福建省科技创新联合基金项目（2021Y9038，2024Y9255）