目的: 采用基于气相色谱-质谱联用 (GC-MS) 的代谢组学技术探究甲氨蝶呤 (MTX) 的毒性机制。方法: 喂养第7天时小鼠腹腔注射MTX (20 mg·kg-1),对照组小鼠腹腔注射等量的生理盐水。摘除眼球后采集血样,并收集心脏、肝脏、肾脏、肺、肠、胃和海马进行代谢组学分析。通过正交偏最小二乘判别分析 (OPLS-DA) 进行差异代谢物筛选,随后采用MetaboAnalyst 5.0 (http://www.metaboanalyst.ca) 及京都基因和基因组百科全书数据库 (KEGG;http://www.kegg.jp) 进行通路分析。最后以肠组织为例,进行病理学分析及代谢物验证。结果: 非靶向代谢组学分析显示,MTX暴露后,小鼠整体代谢轮廓发生改变,其中肝脏与肠道中代谢轮廓改变最为显著;通路分析结果表明MTX主要影响小鼠体内多种氨基酸代谢/合成、能量代谢、泛酸盐和辅酶A生物合成、嘧啶代谢以及谷胱甘肽代谢等代谢通路。MTX处理能够明显破坏肠道上皮结构,同时炎性细胞增加。通过全自动氨基酸分析仪分析发现,L-谷氨酸,L-天冬氨酸和甘氨酸的含量在MTX处理后明显上升,这与代谢组学分析的结果一致。结论: 本研究阐明了甲氨蝶呤暴露后对小鼠各组织代谢轮廓的影响,识别MTX毒性相关的代谢生物标记物和代谢通路,为MTX毒理机制的研究提供新见解。
Objective: To investigate the metabolic changes after methotrexate (MTX) exposure in mice using a gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics approach. Methods: MTX (20 mg·kg-1) was intraperitoneally injected into experimental mice (n=9) at the 7th day, and mice in control group (n=9) were treated with the same amount of saline. A gas chromatography-mass spectrometry (GC-MS) approach was employed to identify discriminant metabolites in serum and various organs including the heart, liver, kidney, lung, intestine, stomach, and hippocampus. The potential metabolites were identified using orthogonal partial least squares discrimination analysis (OPLS-DA). Subsequently, the MetaboAnalyst 5.0 (http://www.metaboanalyst.ca) and Kyoto Encyclopedia of genes and genomes database (KEGG, http://www.kegg.jp) were employed to depict the metabolic pathways. Pathological analysis and metabolite verification were carried our using intestinal tissue as an example. Results: Untargeted metabolomics analysis showed that MTX exposure affected the comprehensive metabolic profiling of mice, especially in the liver and intestine. Pathway analysis identified that MTX affected several metabolic pathways in mice, such as amino acid metabolism/ synthesis, energy metabolism, pantothenate and coenzyme A biosynthesis, pyrimidine metabolism, and glutathione metabolism. MTX treatment can obviously damage the intestinal epithelial structure and increase the numbers of inflammatory cells. Meanwhile, it was found that the contents of L-glutamic acid, L-aspartic acid and glycine were significantly increased after MTX treatment, which was consistent with the results of metabonomic analysis. Conclusion: This study clarified the metabolic profiling of methotrexate exposure on various tissues of mouse, identified metabolic biomarkers and metabolic pathways related to MTX toxicity, and provided new insights for the MTX toxicological mechanism.
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