目的 通过网络药理学、分子对接及实验验证探讨雷公藤红素通过调控铁死亡抑制胃癌的机制。方法 通过TCMSP、SwissTargetPrediction数据库收集药物作用靶点，Genecards、Drukbank、OMIM、TTD及Pharmgkb数据库收集胃癌疾病作用靶点，通过jvenn在线网站获取二者交集靶点，通过STRING数据库及Cytoscape 3.7.2软件进行网络可视化，利用CytoNCA插件及CytoHubba扩展程序计算节点得分，获得Hub基因，然后使用R软件进行基因本体（gene ontology，GO）功能、基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析；通过FerrDb V2数据库检索铁死亡相关靶点，与药物-疾病靶点取交集获得核心靶点，并根据度值大小初步确定雷公藤红素通过调控铁死亡治疗胃癌的作用靶点；采用SYBYL-X 2.0、RCSB、AutoDock Vina、Discovery Studio等软件进行分子对接验证。体外培养人胃腺癌AGS细胞，通过细胞计数试剂盒-8（cell counting kit-8,CCK-8）检测细胞活力；通过克隆形成实验和EdU实验探讨雷公藤红素对胃癌细胞增殖能力的影响；划痕实验和Transwell实验探讨雷公藤红素对细胞迁移能力的影响；流式细胞术检测细胞内活性氧（reactive oxygen species，ROS）积累水平；荧光显微镜观察线粒体膜电位水平的变化；试剂盒检测细胞内亚铁离子（Fe²⁺）、丙二醛（malondialdehyde，MDA）及还原型谷胱甘肽/氧化型谷胱甘肽（glutathione/oxidized glutathione，GSH/GSSG）水平；Western blotting检测核心靶点及铁死亡相关蛋白表达情况。结果 共获得124个雷公藤红素靶点、2 343个胃癌相关靶点，取交集得到89个共有靶点。蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络筛选得到10个Hub基因。GO功能富集分析显示，雷公藤红素主要通过影响细胞增殖、转录、凋亡和氧化应激等来发挥功能，KEGG通路富集分析显示，雷公藤红素参与调控多条肿瘤相关信号通路。获得雷公藤红素-胃癌-铁死亡交集靶点25个，结合度值大小及PPI得分确定信号转导和转录激活因子3（signal transducer and activator of transcription 3，STAT3）为雷公藤红素通过调控铁死亡治疗胃癌的核心作用靶点。分子对接结果提示，雷公藤红素与STAT3有较好的结合活性。与对照组比较，雷公藤红素组AGS细胞活力、克隆数量、EdU阳性率、细胞迁移率显著降低（P＜0.05、0.01），ROS水平明显升高（P＜0.01），线粒体膜电位明显下降（P＜0.01），MDA及Fe²⁺水平显著升高（P＜0.01），GSH/GSSG值显著降低（P＜0.01），且呈剂量相关性。给予铁死亡抑制剂（ferrostatin-1，Fer-1）干预后显著逆转雷公藤红素对以上指标的作用（P＜0.05、0.01）。结论 雷公藤红素通过调控STAT3诱导胃癌细胞铁死亡，抑制胃癌细胞的恶性增殖及迁移。

Objective To investigate the mechanism of celastrol on inhibiting gastric cancer through regulating ferroptosis using network pharmacology, molecular docking and experimental validation. Methods Drug targets were collected from TCMSP and SwissTargetPrediction databases, while gastric cancer disease targets were obtained from Genecards, DrugBank, OMIM, TTD and PharmGKB databases. The intersection of these targets was identified using jvenn, followed by network visualization using STRING and Cytoscape 3.7.2 software. CytoNCA and CytoHubba plugins were used to calculate node scores and identify Hub genes. Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed using R software. Ferroptosis-related targets were retrieved from FerrDb V2, and the intersection with drug-disease targets was used to identify core targets. Molecular docking was carried out using SYBYL-X 2.0, RCSB, AutoDock Vina and Discovery Studio. AGS cells were cultured in vitro, and cell viability was measured using cell counting kit-8 (CCK-8). Cloning formation experiments and EdU experiments were used to investigate the effect of celastrol on cell proliferation. Scratch assays and Transwell assays were used to assess the effects of celastrol on cell migration. Flow cytometry was used to detect intracellular reactive oxygen species (ROS) levels. Fluorescence microscopy was used to observe mitochondrial membrane potential (MMP) changes. Kits were used to measure the levels of Fe²⁺, malondialdehyde (MDA) and glutathione/oxidized glutathione (GSH/GSSG). Western blotting was performed to determine the expression levels of key target and ferroptosis-related proteins. Results A total of 124 potential targets for celastrol and 2 343 gastric cancer-related targets were identified. The intersection of these targets revealed 89 common targets. Protein-protein interaction (PPI) network analysis identified 10 Hub genes. GO function enrichment analysis showed that celastrol mainly exerted its effects by influencing cell proliferation, transcription, apoptosis and oxidative stress. KEGG pathway enrichment analysis showed that celastrol was involved in regulating multiple tumor-related signaling pathways. A total of 25 common targets between celastrol, gastric cancer and ferroptosis were identified, signal transducer and activator of transcription 3 (STAT3) was determined as the core target through which celastrol regulateing ferroptosis in gastric cancer treatment based on docking score and PPI score. Molecular docking results indicated a strong binding affinity between celastrol and STAT3. Compared with control group, the viability, colony number, EdU positivity rate and cell migration rate of AGS cells in celastrol group were significantly decreased (P < 0.05, 0.01), ROS level was significantly increased (P < 0.01), MMP was significantly decreased (P < 0.01), levels of MDA and Fe²⁺ were significantly increased (P < 0.01), GSH/GSSG value was significantly decreased (P < 0.01), and showed a dose-dependent relationship. After intervention with ferrostatin-1 (Fer-1), the effects of celastrol on the above indicators were significantly reversed (P < 0.05, 0.01). Conclusion Celastrol induces ferroptosis in gastric cancer cells by regulating STAT3, inhibiting malignant proliferation and migration of gastric cancer cells.

R285.5

国家自然科学基金资助项目（82460561）；甘肃省自然科学基金资助项目（24JRRA586）；国家卫健委重点实验室硕博基金项目（NHCDP2022005）；甘肃省人民医院院内科研基金项目（22GSSYD-38）