Table_5_Network Pharmacology Study and Experimental Validation of Yiqi Huayu Decoction Inducing Ferroptosis in Gastric Cancer.xlsx

ObjectiveThis study aimed to identify the mechanism of Yiqi Huayu Decoction (YQHY) induced ferroptosis in gastric cancer (GC) by using network pharmacology and experimental validation.
MethodsThe targets of YQHY, ferroptosis-related targets, and targets related to GC were derived from databases. Following the protein–protein interaction (PPI) network, the hub targets for YQHY induced ferroptosis in GC were identified. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the hub targets from a macro perspective. We verified the hub targets by molecular docking, GEPIA, HPA, and the cBioPortal database. Finally, we performed cell viability assays, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, lipid peroxidation, and GSH assays to explore the mechanism of YQHY induced ferroptosis in GC.
ResultsWe identified the main active compounds and hub targets: Quercetin, DIBP, DBP, Mipax, Phaseol and TP53, ATM, SMAD4, PTGS2, and ACSL4. KEGG enrichment analyses indicated that the JAK2-STAT3 signaling pathway may be a significant pathway. Molecular docking results showed that the main active compounds had a good binding activity with the hub targets. The experimental results proved that YQHY could induce ferroptosis in AGS by increasing the MDA content and reducing the GSH content. qRT–PCR and Western blot results showed that YQHY can induce ferroptosis in GC by affecting the JAK2-STAT3 pathway and the expression of ACSL4.
ConclusionsThis study indicated that YQHY can induce ferroptosis in GC by affecting the JAK2–STAT3 pathway and the expression of ACSL4, and induction of ferroptosis may be one of the possible mechanisms of YQHY’s anti-recurrence and metastasis of GC.

本研究旨在通过网络药理学与实验验证，明确益气化瘀汤（Yiqi Huayu Decoction，YQHY）诱导胃癌（gastric cancer，GC）细胞铁死亡（ferroptosis）的作用机制。
方法 本研究从公共数据库中获取益气化瘀汤（YQHY）的作用靶点、铁死亡（ferroptosis）相关靶点以及胃癌（GC）相关靶点。基于蛋白质相互作用（protein–protein interaction，PPI）网络，筛选出YQHY诱导胃癌细胞铁死亡的核心靶点。进一步采用基因本体（gene ontology，GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析，从宏观层面解析核心靶点的功能富集特征。随后通过分子对接、GEPIA数据库、人类蛋白质图谱（Human Protein Atlas，HPA）以及cBioPortal数据库对核心靶点进行验证。最后开展细胞活力检测、实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）、蛋白质印迹法（western blotting）、脂质过氧化检测以及谷胱甘肽（glutathione，GSH）含量测定，以探究YQHY诱导胃癌细胞铁死亡的具体机制。
结果 本研究筛选得到YQHY的主要活性成分与核心靶点：主要活性成分为槲皮素、邻苯二甲酸二异丁酯（DIBP）、邻苯二甲酸二丁酯（DBP）、Mipax、菜豆素（Phaseol）；核心靶点包括TP53、ATM、SMAD4、PTGS2以及ACSL4。KEGG富集分析结果显示，JAK2-STAT3信号通路可能为关键调控通路。分子对接结果表明，YQHY的主要活性成分与核心靶点具有良好的结合活性。体外实验结果证实，YQHY可通过升高AGS胃癌细胞中丙二醛（malondialdehyde，MDA）含量、降低GSH含量，诱导胃癌细胞铁死亡。qRT-PCR与蛋白质印迹实验结果显示，YQHY可通过调控JAK2-STAT3通路以及ACSL4的表达，诱导胃癌细胞铁死亡。
结论 本研究表明，YQHY可通过调控JAK2-STAT3通路与ACSL4的表达诱导胃癌细胞铁死亡，而铁死亡的诱导可能是YQHY抗胃癌复发与转移的潜在作用机制之一。