基于剂量-反应的代谢组学探究镉对小鼠神经干细胞的影响

Impacts of cadmium on mouse neural stem cells based on dose-response metabomics

[背景]镉是环境中广泛存在的重金属毒物,可在人体内蓄积。研究表明,镉暴露可诱导神经毒性,但具体机制尚不明确。[目的]研究不同浓度镉对小鼠神经干细胞的代谢影响,探讨镉神经毒性的可能机制与潜在生物标志。[方法]分离1日龄新生C57BL/6小鼠的侧脑室下区神经干细胞进行体外培养。细胞培养至第3代时进行镉染毒,分为对照组以及0.5、1.0、1.5μmol·L^(−1)CdCl_(2)染毒组,每组设7皿细胞,其中1皿用于细胞计数。染毒24 h后分别提取细胞内与细胞外代谢物,应用超高效液相色谱-串联质谱(UPLC-MS)技术进行代谢组学检测。采用正交偏最小二乘判别分析(OPLS-DA)研究代谢谱的变化,以变量投影重要度值(VIP)>1与P<0.05为标准筛选差异代谢物。对差异代谢物进行代谢集富集分析(MSEA)与京都基因与基因组百科全书(KEGG)富集分析以确定显著变化的代谢集与代谢途径,并拟合差异代谢物的剂量-反应关系,以代谢物浓度上调或下调10%时的有效剂量(EC)确定镉暴露的潜在生物标志。[结果]细胞内代谢样品中共检测到1201种物质,细胞外代谢样品中共检测到1207种物质。镉染毒组的细胞内外代谢谱均与对照组明显区分,随剂量增加,分离越明显。在0.5、1.0、1.5μmol·L^(−1)染毒浓度下分别筛选到87、83、185个细胞内差异代谢物,161、176、166个细胞外差异代谢物。在4组间有显著差异的代谢物中筛选到176个细胞内差异代谢物,167个细胞外差异代谢物。细胞内外差异代谢物均富集到多种脂类代谢物集。细胞内差异代谢物主要涉及牛磺酸与亚牛磺酸代谢、甘油磷脂代谢、甘油酯类代谢等途径,细胞外差异代谢物主要涉及甘油磷脂代谢、类固醇激素生物合成、半胱氨酸和蛋氨酸代谢等途径。细胞内差异代谢物中拟合到125种代谢物的剂量-反应关系,其中108种代谢物的水平随镉剂量增加呈线性变化,细胞外差异代谢物中拟合到134种,其中86种代谢物呈线性变化。EC值较低的细胞内差异代谢物有亚牛磺酸、乙醇胺、磷脂酰乙醇胺和半乳糖,EC值较低的细胞外差异代谢物有乙酰胆碱和1,5-脱水葡萄糖醇。[结论]镉染毒以剂量依赖性方式显著改变小鼠神经干细胞内外代谢谱,其神经毒性可能与甘油磷脂代谢途径相关,该途径中的乙酰胆碱、乙醇胺和磷脂酰乙醇胺等代谢物是镉神经毒性的潜在生物标志。

[Background]Cadmium(Cd)is a ubiquitous and toxic heavy metal that can accumulate in human body.Previous studies have shown that Cd exposure can induce neurotoxicity,but the underlying mechanism remains unclear.[Objective]To investigate the metabolic impacts of multiple doses of Cd on mouse neural stem cells(NSCs),and to explore the potential mechanism and biomarkers of its neurotoxicity.[Methods]The NSCs were obtained from the subventricular zone(SVZ)of 1-day-old neonatal C57BL/6 mice.The passage 3(P3)NSCs were exposed to CdCl_(2)at designed doses(0,0.5,1.0,and 1.5μmol·L^(−1)).The cells were treated with seven replicates,of which one plate was for cell counting.After 24 h of exposure,the intracellular and extracellular metabolites were extracted respectively and then detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS).The orthogonal partial least-squares discriminant analysis(OPLS-DA)was applied to visualize the alterations of metabolomic profiles and to identify the differential metabolites(DMs)based on their variable importance for the projection(VIP)value>1 and P<0.05.The metabolite set enrichment analysis(MSEA)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were performed to recognize the significantly altered metabolite sets and pathways.The dose-response relationships were established and the potential biomarkers of Cd exposure were identified by 10%up-regulated or 10%down-regulated effective concentration(EC)of target metabolites.[Results]A total of 1201 metabolites were identified in the intracellular metabolomic samples and 1207 for the extracellular metabolomic samples.The intracellular and extracellular metabolome of Cd-treated NSCs were distinct from that of the control group,and the difference grew more distant as the Cd dosage increased.At 0.5,1.0,and 1.5μmol·L^(−1)dosage of Cd,87,83,and 185 intracellular DMs and 161,176,and 166 extracellular DMs were identified,respectively.Within the significantly changed metabolites among the four groups,176 intracellular DMs and 167 extracellular DMs were identified.Both intracellular and extracellular DMs were enriched in multiple lipid metabolite sets.Intracellular DMs were mainly enriched in taurine and hypotaurine metabolism,glycerophospholipid metabolism,and glycerolipid metabolism pathways.Extracellular DMs changed by Cd were mainly enriched in glycerophospholipid metabolism,steroid hormone biosynthesis,and cysteine and methionine metabolism pathways.Among intracellular DMs,125 metabolites were fitted with dose-response relationships,of which 108 metabolites showed linear changes with the increase of Cd dosage.And 134 metabolites were fitted with dose-response relationships among extracellular DMs,of which 86 metabolites showed linear changes.The intracellular DMs with low EC values were hypotaurine,ethanolamine,phosphatidylethanolamine,and galactose,while the extracellular DMs with low EC values were acetylcholine and 1,5-anhydrosorbitol.[Conclusion]Cd treatment can significantly alter the intracellular and extracellular metabolome of mouse NSCs in a dose-dependent manner.The neurotoxicity of Cd may be related to glycerophospholipid metabolism.Acetylcholine,ethanolamine,and phosphatidylethanolamine involved in glycerophospholipid metabolism pathway might be potential biomarkers of Cd-induced neurotoxicity.