黄酮类化合物分析及Launaea cornuta（Hochst. ex Oliv. & Hiern）C. Jeffrey乙酸乙酯分馏组对人宫颈癌细胞系抗增殖活性的体外与体计算研究

引言：宫颈癌是全球妇女死亡的主要原因之一，原因在于现有治疗方法的局限性及其相关的不良副作用。Launaea cornuta作为传统药物，用于治疗包括癌症在内的多种疾病。然而，目前尚无科学验证其对宫颈癌的抗增殖活性。目的：本研究旨在评估L. cornuta的选择性抗增殖、细胞毒性及抗迁移作用，并通过网络分析方法探索其在人体宫颈癌细胞系（HeLa-229）中的潜在治疗机制。材料与方法：采用MTT法评估L. cornuta乙酸乙酯分馏组对宫颈癌细胞增殖的细胞毒性作用，伤口愈合试验评估其抗迁移能力。利用定性颜色法和气相色谱-质谱联用技术（GC-MS）分析化合物。随后，进行蛋白-蛋白相互作用（PPI）网络分析、基因本体（GO）和京都基因与基因组百科全书（KEGG）分析，以筛选L. cornuta潜在抗宫颈癌治疗靶点基因。采用分子对接（MD）预测和理解配体与宫颈癌相关靶点的分子相互作用。通过RT-qPCR验证网络分析结果。结果：L. cornuta乙酸乙酯分馏组对HeLa-229细胞具有显著的细胞毒性作用（IC50为20.56 ± 2.83 μg/mL），选择性指数（SI）为2.36，对非癌细胞（Vero-CCL 81，IC50为48.83 ± 23.02）影响较小。初步筛选显示存在苷类、酚类、皂苷、萜类、醌类和单宁。GC-MS分析识别了13种化合物。获得124个与L. cornuta乙酸乙酯分馏组作用相关的潜在靶基因，包括AKT1、MDM2、CDK2、MCL1和MTOR，且PI3K/Akt1、Ras/MAPK、FoxO和EGFR信号通路被认为是显著富集的通路。分子对接表明，stigmasteryl甲醚与CDK2、AKT1、BCL2、MDM2和Casp9具有良好的结合亲和力，结合能范围为-7.0至-12.6 kcal/mol。Tremulone与TP53和P21的结合亲和力也较好，分别为-7.0和-8.0 kcal/mol，提示乙酸乙酯分馏组化合物与宫颈癌靶点基因的分子作用具有稳定性。此外，RT-qPCR分析显示，相较于阴性对照（DMSO 0.2%），HeLa-229细胞中CDK2、MDM2和BCL2表达下调，而Casp9和P21表达上调。结论：结果表明，L. cornuta乙酸乙酯分馏组中的植物化学物质通过调控多种分子靶点和信号通路，表现出对HeLa-229细胞的选择性抗增殖和细胞毒性作用。本研究为开发创新性抗宫颈癌药物奠定了基础。

Introduction: Cervical cancer is one of the leading causes of death among women globally due to the limitation of current treatment methods and their associated adverse side effects. Launaea cornuta is used as traditional medicine for the treatment of a variety of diseases including cancer. However, there is no scientific validation on the antiproliferative activity of L. cornuta against cervical cancer. Objective: This study aimed to evaluate the selective antiproliferative, cytotoxic and antimigratory effects of L. cornuta and to explore its therapeutical mechanisms in human cervical cancer cell lines (HeLa-229) through a network analysis approach. Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate fraction on the proliferation of cervical cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay and the antimigratory effect was assessed by wound healing assays. Compounds were analysed using the qualitative colour method and gas chromatography-mass spectroscopy (GC-MS). Subsequently, bioinformatic analyses, including the protein-protein interaction (PPI) network analysis, Gene Ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, were performed to screen for potential anticervical cancer therapeutic target genes of L. cornuta. Molecular docking (MD) was performed to predict and understand the molecular interactions of the ligands against cervical cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the network analysis results. Results: L. cornuta ethyl acetate fraction exhibited a remarkable cytotoxic effect on HeLa-229 proliferation (IC50 of 20.56 ± 2.83 μg/mL) with a selectivity index (SI) of 2.36 with minimal cytotoxicity on non-cancerous cells (Vero-CCL 81 (IC50 of 48.83 ± 23.02). The preliminary screening revealed the presence of glycosides, phenols, saponins, terpenoids, quinones, and tannins. Thirteen compounds were also identified by GC-MS analysis. 124 potential target genes associated with the effect of L. cornuta ethyl acetate fraction on human cervical cancer were obtained, including AKT1, MDM2, CDK2, MCL1 and MTOR were identified among the top hub genes and PI3K/Akt1, Ras/MAPK, FoxO and EGFR signalling pathways were identified as the significantly enriched pathways. Molecular docking results showed that stigmasteryl methyl ether had a good binding affinity against CDK2, ATK1, BCL2, MDM2, and Casp9, with binding energy ranging from -7.0 to -12.6 kcal/mol. Tremulone showed a good binding affinity against TP53 and P21 with -7.0 and -8.0 kcal/mol, respectively. This suggests a stable molecular interaction of the ethyl acetate fraction of L. cornuta compounds with the selected target genes for cervical cancer. Furthermore, RT-qPCR analysis revealed that CDK2, MDM2 and BCL2 were downregulated, and Casp9 and P21 were upregulated in HeLa-229 cells treated with L. cornuta compared to the negative control (DMSO 0.2%). Conclusion: The findings indicate that L. cornuta ethyl acetate fraction phytochemicals modulates various molecular targets and pathways to exhibit selective antiproliferative and cytotoxic effects against HeLa-229 cells. This study lays a foundation for further research to develop innovative clinical anticervical cancer agents.