Objectives: To investigate the inhibitory effect of Fuzheng Huaji Decoction against non-small cell lung cancer (NSCLC) cells in vitro and explore the underlying mechanism.
Methods: The active ingredients and targets of Fuzheng Huaji Decoction were identified using TCMSP and SwissTargetPrediction databases. NSCLC-related targets from GeneCards and PharmGKB were intersected with the targets of the Decoction, and a protein-protein interaction (PPI) network was constructed to identify the core targets, which were analyzed with GO and KEGG pathway enrichment analysis. Cultured A549 cells were treated with different concentrations of Fuzheng Huaji Decoction-medicated serum, and the changes in cell proliferation, apoptosis, and protein expressions were examined using CCK-8 assay, annexin V-FITC/PI staining and Western blotting.
Results: Fuzheng Huaji Decoction contained 140 active ingredients, and 707 drug-disease intersecting targets were identified. Among these targets, TP53, AKT1, HIF1A, GAPDH, ALB, EGFR, CTNNB1, and TNF were identified as the core targets which were involved in the biological processes related to kinases and receptors and the PI3K-AKT, Ras, calcium, and MAPK pathways. Molecular docking studies indicated strong binding affinity of the active ingredients with TP53, AKT1, and HIF1A. In cultured A549 cells, treatment with 2.5%, 5%, and 10% Fuzheng Huaji Decoction-medicated serum significantly inhibited cell proliferation, promoted cell apoptosis, and downregulated the expression levels of HIF1A, p-AKT (Thr308), and TP53 proteins.
Conclusions: Fuzheng Huaji Decoction inhibits proliferation of NSCLC cells possibly by downregulating the expressions of HIF1A, p-AKT (Thr308), and TP53.
目的: 通过网络药理学、分子对接和体外实验探究扶正化积汤治疗非小细胞肺癌（NSCLC）的作用机制。方法: 通过TCMSP、SwissTargetPrediction数据库筛选扶正化积汤活性成分及其作用靶点。通过在GeneCards、PharmGKB数据库获取NSCLC相关靶点，与扶正化积汤活性成分靶点取交集。利用STRING数据库建立蛋白相互作用（PPI）网络，使用Cytoscape3.8.2软件CytoNCA插件筛选PPI网络核心靶点，利用DAVID6.8数据库进行GO和KEGG通路富集分析。使用A549细胞开展体外实验进行靶点验证，CCK-8法检测扶正化积汤含药血清对A549细胞增殖的影响。Annexin V-FITC/PI染色检测含药血清对A549细胞凋亡的影响。Western blotting检测含药血清对关键通路中相关蛋白表达的影响。结果: 扶正化积汤活性成分共140个，药物和疾病交集靶点共707个，PPI网络核心靶点有TP53，AKT1，HIF1A，GAPDH，ALB，EGFR，CTNNB1，TNF等，GO分析显示主要生物学过程为转移酶、激酶、受体、水解酶、丝氨酸/苏氨酸蛋白激酶、氧化还原酶等，KEGG通路富集显示主要信号通路为PI3K-AKT信号通路、Ras信号通路、钙离子信号通路、MAPK信号通路等。分子对接结果显示，药物有效成分与TP53、AKT1、HIF1A均有效对接。体外实验结果：CCK-8实验确定以2.5%、5%、10%为扶正化积汤含药血清的低、中、高浓度（P<0.05，P<0.01，P<0.0001）。细胞凋亡实验显示扶正化积汤含药血清不同程度促进A549细胞凋亡（P<0.01，P<0.001）。Western blotting结果显示，扶正化积汤下调A549细胞中HIF1A、p-AKT 308和TP53蛋白的表达水平（P<0.05，P<0.01，P<0.001，P<0.0001）。结论: 扶正化积汤可能通过调控HIF1A，p-AKT 308，TP53蛋白的表达，抑制A549细胞增殖，发挥治疗NSCLC的潜在作用。.
Keywords: Fuzheng Huaji Decoction; in vitro experiments; molecular docking; network pharmacology; non-small cell lung cancer.