Department of Anesthesiology, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Bioscience 2, 12705 East Montview Boulevard, Suite 200, Aurora, CO 80045, USA.
Department of Organic Chemistry and Instrumental Analytics, University of Bremen, Leobener Strasse NW2 C0041, 28359 Bremen, Germany.
Sci Rep. 2017 Mar 22;7:45088. doi: 10.1038/srep45088.
The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 μM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 μM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 μM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity.
本研究旨在阐明霉酚酸（MPA）对人肠细胞产生负面影响的分子机制。采用液相色谱-质谱联用技术评估 MPA 暴露和鸟苷补充对 LS180 细胞核苷酸浓度的影响。采用稳定同位素标记氨基酸在细胞培养中的方法进行蛋白质组学分析，结合基于凝胶的液相色谱-质谱和 H 核磁共振波谱进行脂质组学分析。尽管进行了补充，鸟苷核苷酸仍出现耗竭（24 和 72 h 时，p < 0.001；5、100 和 250 μM MPA），尿苷和胞苷核苷酸上调（24 h 时，p < 0.001；5 μM MPA），暴露于 MPA 后。MPA 显著改变了 35 种主要与核苷酸依赖过程和脂质代谢相关的蛋白质。与先前 MPA 相关蛋白变化的研究进行交叉参考广泛证实了这些结果，但显示出可能因模型和/或方法而异的差异。MPA 暴露增加了细胞内脂肪酸、胆固醇和磷脂酰胆碱的浓度（72 h 时，p < 0.01；100 μM MPA），这与脂质代谢蛋白的变化相对应。MPA 影响 LS180 细胞内核苷酸水平、核苷酸依赖过程、结构蛋白表达、脂肪酸和脂质代谢。这些变化可能会损害肠膜完整性，并导致胃肠道毒性。
