基于UPLC-Q-TOF-MS/MS与网络药理学研究新“浙八味”衢枳壳黄酮抗非小细胞肺癌有效成分及作用机制

Study on the Active Components and Mechanism of Action of New “Zhe Eight Flavors” Quzhou Fructus Aurantii against Non-Small Cell Lung Cancer Based on UPLC-Q-TOF-MS/MS and Network Pharmacology

关键词:  衢枳壳黄酮  网络药理学  非小细胞肺癌  SMAD3

Key words:  Pure Total Flavonoids From Fruit of Citrus aurantium 'Changshan-huyou  network pharmacology  non-small cell lung cancer  SMAD3.

摘要:

目的 基于UPLC-Q-TOF-MS/MS与网络药理学预测并通过实验验证新“浙八味”衢枳壳黄酮活性成分抗非小细胞肺癌的有效成分及作用机制。方法 采用UPLC-Q-TOF-MS/MS技术检测衢枳壳黄酮的主要化学物质，并通过TCMSP、ETCM、BATMAN-TCM及Swiss Target Prediction数据库收集衢枳壳黄酮类化合物的潜在活性成分及作用靶点；应用GeneCards、CTD、Disgenet和OMIM数据库构建非小细胞肺癌疾病靶点集；将衢枳壳黄酮潜在靶点与非小细胞肺癌靶点取交集以获取关键靶点蛋白，建立PPI蛋白互作网络；对核心靶点进行GO 功能和KEGG 通路富集分析，并构建成分-靶点-通路-疾病网络；通过分子对接验证衢枳壳黄酮活性成分与SMAD3核心靶点的结合活性；采用增殖、克隆、愈合与迁移实验分析衢枳壳黄酮对H1299细胞活力的影响；JC-1染色法观察H1299细胞的线粒体膜电位变化；运用RT-qPCR技术和Western blot验证衢枳壳黄酮对网络药理学预测的关键靶点SMAD3的调控作用。结果 从衢枳壳黄酮中共鉴定出148个化合物，其中12个作为潜在活性成分，主要作用于SRC、ESR1、AKT1与SMAD3等核心靶点。GO富集分析结果显示PTFC可能作用细胞凋亡过程、蛋白质磷酸化、表皮生长因子受体信号通路，KEGG 富集分析发现衢枳壳黄酮可能通过PI3K-Akt信号通路、FoxO信号通路发挥抗NSCLC作用。分子对接结果显示，其中10种活性成分与SMAD3蛋白靶点均有较好的结合能力。实验结果表明衢枳壳黄酮能够影响非小细胞肺癌的增殖、克隆、愈合与迁移能力，降低线粒体膜电位。RT-qPCR结果表明其可显著下调SMAD3 mRNA的表达，Western blot结果表明其可降低p-Smad2/3蛋白表达，证明了网络药理学分析的可靠性。 结论 本研究揭示了衢枳壳黄酮通过SMAD3靶点治疗非小细胞肺癌的潜在分子作用机制，为其临床防治非小细胞肺癌的应用奠定基础。

Abstract:

OBJECTIVE To explore the active components and mechanisms of the flavonoids from new “Zhe Eight Flavors” Quzhou Fructus Aurantii against non-small cell lung cancer (NSCLC) through UPLC-Q-TOF-MS/MS and network pharmacology, and experiment verification. METHODS The main chemical substances of Pure Total Flavonoids From Citrus aurantium 'Changshan-huyou' (PTFC) were detected using UPLC-Q-TOF-MS/MS. The potential active components and target proteins of PTFC were collected through TCMSP, ETCM, BATMAN-TCM, and Swiss Target Prediction databases. NSCLC disease target sets were constructed using GeneCards, CTD, Disgenet, and OMIM databases. The intersection of potential targets of PTFC and NSCLC targets was obtained to identify key target proteins, and a protein-protein interaction network was constructed. The molecular docking was conducted to verify the binding activity between the active ingredients of PTFC and the core target SMAD3. GO function and KEGG pathway enrichment analysis were performed on core targets, and Components - Targets - Pathways – Diseases network was established. The effects of PTFC on the vitality of H1299 NSCLC cells were analyzed through proliferation, cloning, wound healing, and migration assays. The changes in mitochondrial membrane potential in H1299 Cells were observed by JC-1 staining. RT-qPCR and Western blot were used to verify the regulatory effect of PTFC on the key target SMAD3 predicted by network pharmacology. RESULTS A total of 148 compounds were identified from PTFC, with 12 compounds considered potential active ingredients, primarily acting on core targets such as SRC, ESR1, AKT1, and SMAD3. GO enrichment analysis results suggested that PTFC might be involved in biological processes including apoptosis, protein phosphorylation, and epidermal growth factor receptor signaling pathway. KEGG enrichment analysis revealed that PTFC might exert anti-NSCLC effects through the PI3K-Akt signaling pathway and FoxO signaling pathway. The molecular docking experiments showed that ten active ingredients exhibited good binding binding affinity with the SMAD3 protein target. Experimental results demonstrated that PTFC could inhibit proliferation, clonogenicity, wound healing, and migration capabilities of NSCLC, leading to a decrease in mitochondrial membrane potential. RT-qPCR results showed significant downregulation of SMAD3 mRNA expression, and Western blot analysis confirmed reduced p-Smad2/3 protein expression, thereby validating the reliability of network pharmacology predictions. CONCLUSION This study elucidates the potential molecular mechanism by which PTFC target SMAD3 to treat NSCLC, laying a foundation for its clinical application in NSCLC.