硝唑尼特对骨肉瘤的影响及机制研究

目的 以网络药理学为基础,探讨硝唑尼特(nitazoxanide,NTZ)对骨肉瘤的影响及作用机制。方法 利用化合物靶点预测数据库,筛选NTZ在骨肉瘤治疗中的潜在靶点,构建蛋白互作网络,并在Cytoscape软件中对蛋白互作网络进行可视化分析,通过计算度值,将排名前20的基因视为关键靶基因。借助David数据库对潜在靶点进行富集分析,预测药物潜在作用途径。通过体外实验验证NTZ的抗骨肉瘤的作用,设置NTZ处理组0,10,20,30,40 μmol·L^-1和0.05%DMSO对照组分别作用于骨肉瘤细胞143B细胞,结晶紫、MTT实验检测NTZ对骨肉瘤143B细胞增殖的影响;划痕和Transwell侵袭实验观察迁移侵袭能力;流式细胞术和Hoechst 33258检测细胞凋亡;蛋白印迹法(Western blot)检测细胞蛋白水平的变化。结果 NTZ作用于骨肉瘤的潜在靶点共有78个;Gene Ontology(GO)富集分析涉及306个条目,京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)分析得到80个条目,涉及肿瘤通路、细胞周期信号通路、受体酪氨酸蛋白激酶(ErbB)信号通路等。实验结果表明,NTZ抑制骨肉瘤细胞增殖和迁移侵袭,诱导凋亡,细胞外调节蛋白激酶(ERK1/2)信号通路关键分子ERK1/2的磷酸化显著抑制。结论 NTZ对骨肉瘤的作用涉及多靶点、多过程,能调节ERK1/2信号通路,有效抑制骨肉瘤。

Effect of Nitazoxanide on Human Osteosarcoma Cells and Its Underlying Mechanism

OBJECTIVE To investigate the effect of nitazoxanide(NTZ) on osteosarcoma cells 143B and explore its underlying mechanism based on network pharmacology. METHODS The Swiss Target Prediction database and CTD database were used to obtain the drug target of NTZ, and the target genes of osteosarcoma were collected from Gene Cards database, then the drug-disease intersection genes were obtained by the intersection. The interaction network was constructed using STRING database and visualized with Cytoscape to obtain the drug-osteosarcoma-target protein regulatory network. Through calculating the degree value of each node, the first 20 genes were selected as key genes. Through GO and KEGG signaling pathway enrichment analysis in David database, the potential action pathway of the drug was predicted. In vitro experiments were validated. Human osteosarcoma cells were treated with 0,10,20,30,40 μmol·L^-1 NTZ and 0. 05% DMSO,respectively. The effect of NTZ on the proliferation of osteosarcoma 143B cells was detected by crystal violet staining and MTT assay. Wound-healing assay and Transwell invasion assay were used to observe the migration and invasion ability of 143B cells after NTZ treatment. Apoptosis was detected by flow cytometry and Hoechst 33258 staining. The Protein level was detected by Western blot. RESULTS There are 78 potential targets of NTZ in osteosarcoma. GO enrichment analysis involved 306 items,and KEGG enrichment analysis obtained 80 items,involving tumor pathways,cell cycle signaling pathways,ErbB signaling pathways,etc. The results showed that NTZ could inhibit the proliferation,migration and invasion of osteosarcoma cells. Meanwhile,apoptosis was induced by NTZ,and significantly inhibit the phosphorylation of ERK1/2,a key molecule of ERK1/2 signaling pathway. CONCLUSION The effect of NTZ on osteosarcoma involves multi-target and multi-process,which has an inhibitory effect on osteosarcoma,and can inhibit the activation of ERK1/2 signaling pathway.