目的 通过网络药理学和分子对接方法研究表没食子儿茶素没食子酸酯（EGCG）抗小细胞肺癌的潜在作用机制，并通过体外实验进一步验证。方法 通过检索PubChem、Swiss Target Prediction和Stitch数据库预测EGCG的潜在作用靶点，检索DrugBank、GeneCards、TTD、Omin和Pharmgkb数据库获得小细胞肺癌的相关靶点；使用R4.3.1软件获取交集靶点。使用STRING数据库构建EGCG治疗小细胞肺癌潜在靶点的蛋白质–蛋白质相互作用（PPI）网络；使用Cytoscape软件对STRING数据库筛选得到的靶点进行3次核心靶点筛选；使用R4.3.1软件中的“ClusterProfiler”对核心靶点进行基因本体（GO）和京都基因与基因组百科全书（KEGG）信号通路富集分析，进一步通过分子对接和分子动力学模拟研究EGCG治疗SCLC的潜在作用机制。使用梯度浓度EGCG处理人小细胞肺癌H82和H562细胞，通过Western blotting检测核心靶点蛋白的表达水平，并通过流式细胞术分析EGCG对小细胞肺癌细胞凋亡和细胞周期的影响。结果 预测到779个EGCG潜在作用靶点，29 599个小细胞肺癌相关靶点，最终得到交集靶点678个，进一步筛选得到E1A结合蛋白p300（EP300）、信号转导及转录激活因子3（STAT3）、Jun和肿瘤蛋白p53（TP53）4个核心靶点。GO和KEGG富集分析结果显示，EGCG可能通过磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（Akt）信号通路、丝裂原激活蛋白激酶（MAPK）信号通路及蛋白多糖调节等机制发挥对小细胞肺癌的治疗作用。分子对接结果显示EGCG与4个核心靶点的对接结合能均小于−8.7 kcal/mol；分子动力学模拟显示EGCG与4个核心靶点的结合稳定性均较强。体外实验中，EGCG可显著降低EP300、STAT3、Jun蛋白的表达水平，而不降低TP53蛋白的表达水平；40 μmol/L EGCG引起细胞周期阻滞于G₂/M期，同时促进了细胞凋亡。结论 EGCG对小细胞肺癌的治疗作用可能通过多种靶点和多个通路途径实现。

Objective To investigate the potential mechanism of epigallocatechin gallate (EGCG) against small cell lung cancer (SCLC) using network pharmacology and molecular docking methods, and to further validate the findings through in vitro experiments. Methods Potential targets of EGCG were predicted by searching the PubChem, Swiss Target Prediction, and Stitch databases. SCLC-related targets were obtained from the DrugBank, GeneCards, TTD, Omim, and PharmGkb databases. Intersection targets were identified using R4.3.1 software. A protein-protein interaction (PPI) network of potential targets of EGCG against SCLC was constructed using the STRING database. Three rounds of core target screening were performed using Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the core targets were carried out using the “ClusterProfiler” package in R4.3.1 software. Molecular docking and molecular dynamics simulations were further employed to explore the potential mechanism of EGCG against SCLC. Human SCLC H82 and H562 cells were treated with gradient concentrations of EGCG. The expression levels of core target proteins were detected by Western blotting, and the effects of EGCG on apoptosis and cell cycle of SCLC cells were analyzed by flow cytometry. Results A total of 779 potential targets of EGCG and 29 599 SCLC-related targets were predicted, yielding 678 intersection targets. After further screening, four core targets were identified: E1A binding protein p300 (EP300), signal transducer and activator of transcription 3 (STAT3), Jun, and tumor protein p53 (TP53). GO and KEGG enrichment analyses indicated that EGCG may exert its therapeutic effects on SCLC through mechanisms such as the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and proteoglycan regulation. Molecular docking results showed that the binding energies of EGCG with all four core targets were less than −8.7 kcal/mol. Molecular dynamics simulations revealed that EGCG stably binds to all four core targets. In in vitro experiments, EGCG significantly reduced the expression levels of EP300, STAT3, and Jun proteins, but did not reduce the expression level of TP53 protein. Treatment with 40 μmol/L EGCG induced cell cycle arrest at the G₂/M phase and promoted apoptosis. Conclusion The therapeutic effect of EGCG on SCLC may be achieved through multiple targets and multiple pathways.

R285;R286.4

国家自然科学基金资助项目(82073388)