目的:探讨桑皮对骨肉瘤(OS)的治疗机制。我们进行了生物信息学预测,然后进行了体外实验验证。
方法:从GEO数据库获得来自正常组织和OS组织的基因表达数据,并进行差异分析。从中药系统药理学数据库中提取了具有活性的桑皮成分和靶基因。通过将这些靶标与OS中差异表达的基因相交,我们确定了潜在的药物作用靶点.使用STRING数据库,构建了蛋白质-蛋白质相互作用网络。随后对这些交叉基因的分析,包括基因本体论富集和京都百科全书的基因和基因组途径富集,使用R软件来阐明生物过程,分子功能,和细胞成分,导致信号通路的模拟。分子对接评估了小分子与信号通路靶标的结合能力。在U-2OS细胞上进行体外验证。CCK8测定用于确定药物在OS细胞中诱导的细胞毒性,蛋白质印迹法用于验证AKT的表达,细胞外信号调节激酶(ERK),幸存者,和细胞周期蛋白D1蛋白。
结果:通过正常组织和OS组织之间的差异基因表达分析,我们确定了12,364个差异表达基因。从TCSMP数据库,得出与OS相关的39个活性成分和185个治疗靶标。蛋白质相互作用网络表明AKT1、IL-6、JUN、VEGFA,和CASS3可能是OS的MoriCortex的中心目标。分子对接显示Mori皮质中的活性化合物Morusin对AKT和ERK表现出强的结合亲和力。CCK8实验表明,莫鲁素显著抑制U-2OS细胞的活力。WesternBlot显示p-AKT/AKT比率降低,p-ERK/ERK比值,幸存者,和细胞周期蛋白D1。
结论:MoriCortex可能通过多种细胞信号通路对OS发挥治疗作用。莫鲁辛,MoriCortex的活性成分,可以通过靶向AKT/ERK通路抑制OS细胞的细胞周期调控和促进细胞死亡。
OBJECTIVE: To explore the therapeutic mechanism of Mori Cortex against osteosarcoma (OS), we conducted bioinformatics prediction followed by in vitro experimental validation.
METHODS: Gene expression data from normal and OS tissues were obtained from the GEO database and underwent differential analysis. Active Mori Cortex components and target genes were extracted from the Traditional Chinese Medicine System Pharmacology database. By intersecting these targets with differentially expressed genes in OS, we identified potential drug action targets. Using the STRING database, a protein-protein interaction network was constructed. Subsequent analyses of these intersected genes, including Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment, were performed using R software to elucidate biological processes, molecular functions, and cellular components, resulting in the simulation of signaling pathways. Molecular docking assessed the binding capacity of small molecules to signaling pathway targets. In vitro validations were conducted on U-2 OS cells. The CCK8 assay was used to determine drug-induced cytotoxicity in OS cells, and Western Blotting was employed to validate the expression of AKT, extracellular signal-regulated kinases (ERK), Survivin, and Cyclin D1 proteins.
RESULTS: Through differential gene expression analysis between normal and OS tissues, we identified 12,364 differentially expressed genes. From the TCSMP database, 39 active components and 185 therapeutic targets related to OS were derived. The protein-protein interaction network indicated that AKT1, IL-6, JUN, VEGFA, and CASP3 might be central targets of Mori Cortex for OS. Molecular docking revealed that the active compound Morusin in Mori Cortex exhibits strong binding affinity to AKT and ERK. The CCK8 assay showed that Morusin significantly inhibits the viability of U-2 OS cells. Western Blot demonstrated a reduction in the p-AKT/AKT ratio, the p-ERK/ERK ratio, Survivin, and Cyclin D1.
CONCLUSIONS: Mori Cortex may exert its therapeutic effects on OS through multiple cellular signaling pathways. Morusin, the active component of Mori Cortex, can inhibit cell cycle regulation and promote cell death in OS cells by targeting AKT/ERK pathway.