基于代谢组学方法研究乌头碱和苯甲酰乌头原碱对BeWo细胞的毒性机制

Metabolomics Study of Aconitine and Benzoylaconine Induced Reproductive Toxicity in Be Wo Cell

以BeWo细胞建立体外胎盘模型,采用基于气相色谱与质谱联用技术(GC-MS/MS)的代谢组学方法,研究乌头碱及其代谢产物苯甲酰乌头原碱的生殖毒性机制。收集不同时间点(0,6,12,24和36 h)药物干预后的BeWo细胞裂解液,通过GC-MS/MS分析获得细胞代谢轮廓;正交偏最小二乘法-判别分析(OPLSDA)显示,BeWo细胞内代谢物在药物干预后呈动态变化。进一步通过变量重要性投影(VIP)值和单因素方差分析筛选差异性代谢物,最终鉴定出脯氨酸、甘氨酸、苏氨酸、谷氨酸、谷氨酰胺、乙酰谷氨酰胺、赖氨酸、组氨酸、葡萄糖、半乳糖、琥珀酸共11个差异性代谢物。经Metaboanalyst分析,主要涉及谷氨酰胺和谷氨酸代谢,甘氨酸、丝氨酸和苏氨酸代谢,丙氨酸、天冬氨酸和谷氨酸代谢,赖氨酸降解,精氨酸和脯氨酸代谢,组氨酸代谢等6条代谢通路。实验结果表明,乌头碱和苯甲酰乌头原碱主要通过影响氨基酸代谢对BeWo细胞产生生殖毒性作用。

An intracellular metabonomics method based on gas chromatography coupled with mass spectrometer（ GC-MS / MS） was established to explore the toxicity mechanism of aconitine and benzoylaconine. Be Wo,the continuous cell lines originated from human placenta were selected to establish the in vitro placenta model. The intracellular metabolites were extracted after exposed with 5 μg / m L aconitine and benzoylaconine for 0,6,12,24 and 36 h,respectively. The intracellular metabolic profiling was acquired by GC-MS / MS. Orthogonal partial least squares discriminant analysis（ OPLS-DA） score plot showed the timedependent change of the metabolomics profiles between the control and intervention groups. After screened by variable influence on projection（ VIP） value,one-way analysis of variance and searched with NIST 2014 database, we identified 11 metabolites, including proline, glycine, threonine, glutamic acid,N-acetylglutamine,glutamine,lysine,histidine,succinic acid,glucose and galactose,which mainly involved these pathways：（ 1） glutamine and glutamate metabolism,（ 2） glycine,serine and threonine metabolism,（ 3） alanine,aspartate and glutamate metabolism,（ 4） lysine degradation,（ 5） arginine and proline metabolism and（ 6） histidine metabolism. The results demonstrated that amino acid metabolism was the main metabolic pathway and responsible for the placental and fetal toxicity of aconitine and benzoylaconine.