基于1H-NMR代谢组学技术的D-半乳糖致衰老大鼠尿液代谢谱的动态变化

摘要

OBJECTIVE To investigate the dynamic changes in urine metabolic profiles in rats induced by D-galactose (D-Gal),and to study the correlations between the differential metabolites and behavior indicators using the proton nuclear magnetic resonance (1H-NMR)-based metabonomics.METHODS Subcutaneous injection of D-Gal 100 mg· kg-1 for 10 weeks was adopted in the model group.The sample of urine was collected at day 0 (dO),d14,d28,d42,d56 and d70.NMR metabonomics technique was used for acquisition of data,which was analyzed by multivariate statistical analysis.The ability of learning and memory were measured by Morris water maze test from d70.After the behavioral test,the rats were sacrificed and the hippocampus was observed by hematoxylin-eosin staining.RESULTS Principal component analysis (PCA) results revealed that there was considerable difference between the model group and the normal control group at d70.According to the varible importance plot (VIP) calculation and S-plot scores,a total of 12 metabolites were screened and identified as potential biomarkers at d70.The differences of metabolites and Morris water maze test were subjected to correlation analysis,and the results showed that the levels of choline,lactate and dimethylglycine in the model group were significantly increased and negatively correlated with the times of crossing the platform (r =-0.90,-0.50 and-0.52;n=10).Formate was significantly negatively correlated with the time spent in the target area (r =-0.51,n=10),but choline and formate were significantly positively correlated with the escape latency (r =0.72 and 0.53;n=10).However,the levels of creatine and taurine decreased in the model group,which was significantly positively correlated to the times of acrossing platforms (r =0.89 and 0.71;n=10),while alanine was significantly positively correlated to the time spent in the target area(r =0.74,n=10).Taurine,alanine and creatine were significantly negatively correlated with the escape latency (r =-0.66,-0.50 and-0.85;n=10).The correlations between the differential metabolites and the behavioral indicators were further proved.CONCLUSION The metabolic profile changes in urine from D-Gal induced aging model rats are significantly correlated with impairement of ability in learning and memory.1H-NMR metabonomics in urinary metabolic profile changes may be used as an evaluation index in the D-Gal induced aging rats model.%目的 通过代谢组学技术研究D-半乳糖(D-Gal)致衰老大鼠尿液代谢谱的动态变化,并探究差异代谢物和行为学指标的相关性.方法 大鼠连续SC给予D-Gal 100 mg·kg-110周,并分别在第0,14,28,42,56和70天收集每只实验鼠的尿液,采用代谢组学技术对实验大鼠6次尿液样本进行核磁共振(NMR)数据采集并结合多元统计进行分析.第70天开始采用Morris水迷宫检测实验大鼠的学习记忆能力.行为学实验结束后,处死并制备脑切片,HE染色观察海马病理改变.结果 对第0,14,28,42,56和70天模型组和正常对照组大鼠的尿液进行主成分分析发现,造模2～4周时,两组的代谢谱无差异;造模6～8周,两组大鼠逐渐不同;造模10周时,两组完全不同.采用正交校正的偏最小二乘判别分析寻找两组之间的差异物,在第10周发现,乳酸、丙氨酸、α-酮戊二酸和胆碱等12个峰面积具有显著性差异的潜在生物标志物.将差异代谢物和穿越平台次数、潜伏期以及目标象限停留时间进行关联分析.结果表明,模型组中含量显著性升高的差异代谢物胆碱、乳酸和二甲基甘氨酸与穿越平台次数具有显著性负相关(r =-0.90,-0.50和-0.52;n=10),甲酸与目标象限停留时间呈显著性负相关(r=-0.51,n=10);胆碱和甲酸与潜伏期呈显著性正相关(r=0.72和0.53;n=10);而模型组含量显著性降低的差异代谢物肌酸和牛磺酸与穿越平台次数具有显著性正相关(r=0.89和0.71;n=10),而丙氨酸与目标象限停留时间呈显著性正相关(r=0.74;n=10);牛磺酸、丙氨酸和肌酸与潜伏期呈显著性负相关(r=-0.66,-0.50和-0.85;n=10);进一步验证了差异代谢物与行为学指标的相关性.结论 D-Gal诱导的衰老大鼠尿液代谢谱变化和其学习记忆能力损伤有一定的相关性,基于1H-NMR代谢组学的尿液代谢谱变化规律,可作为D-Gal致衰老大鼠模型成功与否的评价指标之一.