基于1H-NMR技术的SD大鼠弥漫性轴索损伤血浆代谢组学研究

Application of 1H-NMR-based pattern recognition in plasma metabolomics of rats with diffuse axonal injury

摘要:

目的：采用核磁共振氢谱（1H-NMR）结合模式识别的方法研究SD大鼠弥漫性轴索损伤（diffuse axonal injury，DAI）后血浆代谢的变化，探讨1H-NMR在DAI中应用的可行性，并筛选出DAI的早期差异代谢物。方法：采用改良的Marmarou A法建立SD大鼠DAI模型，分为打击致伤组（n=6），假手术组（n=6，给予打击致伤组相同的打击前准备，但不予以打击），于致伤后1 d抽取心血，分离出血浆。应用1H-NMR结合模式识别技术对大鼠血浆进行代谢组学分析，筛选差异代谢物。结果：通过对打击致伤组与假手术组血浆代谢组差异代谢物的相关性分析及独立样本t检验，筛选出相关系数丨r丨＞0.6且P＜0.05的差异代谢物。结果显示，与假手术组相比，打击致伤组血浆中丙酮酸（Pyruvate）（r=-0.831，P=0.019）、赖氨酸（Lysine）（r=-0.773，P=0.030）、谷氨酸（Glutamate）（r=-0.772，P=0.042）、谷氨酰胺（Glutamine）（r=-0.772，P=0.042）、异丁酸（Isobutyrate）（r=-0.631，P=0.045）、甘油（Glycerol）（r=-0.844，P=0.032）明显升高，丙二酸二乙酯（Malonate）（r=0.699，P=0.038）、L2（r=0.786，P=0.047）、L3（r=0.666，P=0.031）、L4（r=0.715，P=0.022）、L10（r=0.663，P=0.025）、β-葡萄糖（β-Glucose）（r=0.800，P=0.030）、α-葡萄糖（α-Glucose）（r=0.808，P=0.015）明显降低。结论：基于1H-NMR结合模式的方法对DAI后血浆的代谢组学分析是可行的，所筛选出的13种差异代谢物及其表达变化，不仅为进一步探索DAI的病理生理机制提供了新的研究基础；同时，对于临床DAI的早期诊断、治疗及法医学鉴定提供了重要的方法和依据。

Abstract:

Objective：To investigate the feasibility of applying 1H-NMR-based pattern recognition in the studies of plasma metabo－nomics of Sprague-Dawley（SD） rats with diffuse axonal injury（DAI） and identify potential metabolic markers of DAI at early stage. Methods：Modified Marmarou A method was used to establish the model of DAI in SD rats. All animals were sacrificed at 1 d post in－sult. Plasma was prepared with heart blood. 1H-NMR technique was applied for detection of plasma samples from sham-injured group（n=6） and injured group（n=6）. Pattern recognition of partial least squares discriminant analysis（PLS-DA） and orthogonal signal correction-partial least squares discriminant analysis（OPLS-DA） were used to analyze and determine the differences of plasma metabolites between the two groups. Results：Different metabolites between sham-injured group and injured group were found by cor－relation analysis and student’s t test，metabolites were selected on condition that correlation coefficient丨r丨＞0.6 and P＜0.05. Finally，Pyruvate（r=-0.831，P=0.019），Lysine（r=-0.773，P=0.030），Glutamate（r=-0.772，P=0.042），Glutamine（r=-0.772，P=0.042），Isobutyrate（r=-0.631，P=0.045） and Glycerol（r=-0.844，P=0.032） significantly increased in injured group compared with those of sham-injured group. Malonate（r=0.699，P=0.038），L2（r=0.786，P=0.047），L3（r=0.666，P=0.031），L4（r=0.715，P=0.022），L10（r=0.663，P=0.025），β-Glucose（r=0.800，P=0.030） and α-Glucose（r=0.808，P=0.015） significantly decreased in in－jured group compared with those of sham-injured group. Conclusion：1H-NMR technique is effective in the study of plasma metabo－nomics of SD rats with DAI. The different metabolites may suggest the possible pathophysiological mechanism of DAI and lay the foundation for the research of early diagnosis，treatment and forensic identification of DAI.