本研究的目的是探讨布鲁氏菌与GEO的药理作用机制。网络药理学,和体外试验为喉癌的治疗提供理论支持。从TCMSP数据库检索布鲁氏菌的活性成分和匹配目标,虽然与喉癌相关的基因是从GEO获得的,GeneCards,DisGeNet,和DrugBank数据库。此外,组分和目标由PubMed数据库中的文献补充。使用Cytoscape软件创建活性成分-目标网络图。String数据库用于构建PPI网络。在此之后,使用DAVID数据库对核心靶标进行GO富集和KEGG途径分析.最后,AutoDock用于在核心组件和核心靶标之间进行分子对接。探讨β-谷甾醇的生物学效应,使用MTS技术评估了β-谷甾醇治疗后喉癌细胞的活力.在此之后,研究了β-谷甾醇如何影响处理过的细胞培养14天后的集落形成。采用流式细胞术检测细胞凋亡,探讨β-谷甾醇对喉癌细胞凋亡的影响,然后通过RT-qPCR和WesternBlot检测10个关键基因的mRNA和蛋白表达水平。有1258个喉癌相关基因和15个布鲁氏菌成分,β-谷甾醇和木犀草素作为关键成分。布鲁氏菌针对喉癌的主要靶标是IL6,JUN,TNF,IL2,IL4,IFNG,RELA,TP53,CDKN1A,AKT1。GO富集产生41CC,78MF,和383BP。铂类药物耐药性,PI3K-Akt信号通路,p53信号通路,凋亡,HIF-1信号通路,和147个额外的途径已被添加到KEGG。分子对接结果表明,核心成分对核心靶标具有较高的亲和力。细胞实验的结果表明β-谷甾醇以浓度依赖性方式抑制Hep-2细胞活性。此外,β-谷甾醇在Hep-2细胞中具有强大的抗增殖特性。流式细胞仪检测结果显示β-谷甾醇呈浓度依赖性促进喉癌细胞凋亡。RT-qPCR和WesternBlot检测结果显示,TP53、JUN的mRNA和蛋白表达水平,TNF-α,CDKN1A,和IL-2在β-谷甾醇治疗后显著上调,而RELA的mRNA和蛋白表达水平,AKT1,IL-6,IFNG,和IL-4显著下调。这项研究整合了GEO,网络药理学,和体外试验研究了布鲁氏菌抗喉癌活性的可能机制,这可以为未来的其他动物实验提供理论基础。
The goal of this study is to look into the pharmacological mechanism of Bruceae Fructus in conjunction with GEO, network pharmacology, and in vitro assays for the treatment of laryngeal cancer to provide theoretical support for its therapeutic use. The active components and matching targets of Bruceae Fructus were retrieved from the TCMSP database, while genes linked with laryngeal cancer were obtained from the GEO, GeneCards, DisGeNET, and DrugBank databases. Besides, the components and targets were supplemented by literatures in PubMed database. Cytoscape software was used to create the active ingredients-target network diagram. The String database was used to build the PPI network. Following that, the core targets were subjected to GO enrichment and KEGG pathway analysis using the DAVID database. Finally, AutoDock was used to perform molecular docking between the core components and the core targets. To investigate the biological effects of beta-sitosterol, the viability of laryngeal cancer cells was assessed after beta-sitosterol therapy using the MTS technique. Following that, how beta-sitosterol affected colony formation after 14 days of culture of treated cells was researched. Flow cytometry was utilized to detect apoptosis to examine the influence of beta-sitosterol on laryngeal cancer cell apoptosis, and then detected mRNA and protein expression levels of 10 key genes by RT-qPCR and Western Blot assay. There were 1258 laryngeal cancer-related genes and 15 Bruceae Fructus components, with beta-sitosterol and luteolin serving as key components. Bruceae Fructus\' primary targets against laryngeal cancer were IL6, JUN, TNF, IL2, IL4, IFNG, RELA, TP53, CDKN1A, and AKT1. GO enrichment yielded 41 CC, 78 MF, and 383 BP. Platinum drug resistance, the PI3K-Akt signaling pathway, the p53 signaling pathway, apoptosis, the HIF-1 signaling pathway, and 147 additional pathways have been added to KEGG. The results of molecular docking revealed that the core components had a high affinity for the core target. The results of the cell experiment indicate that beta-sitosterol suppressed Hep-2 cell activity in a concentration-dependent manner. Besides, beta-sitosterol has powerful antiproliferative properties in Hep-2 cells. Flow cytometry results showed that beta-sitosterol promoted laryngeal cancer cell apoptosis in a concentration-dependent manner. The results of RT-qPCR and Western Blot assay showed that the mRNA and protein expression levels of TP53, JUN, TNF-α, CDKN1A, and IL-2 were significantly up-regulated after beta-sitosterol treatment, while the mRNA and protein expression levels of RELA, AKT1, IL-6, IFNG, and IL-4 were significantly down-regulated. This study integrating GEO, network pharmacology, and in vitro assays investigated the probable mechanism of Bruceae Fructus\' anti-laryngeal cancer activity, which can give a theoretical foundation for additional future animal experiments.