基于NMR技术对中药干预Wilson病模型大鼠的代谢组学研究

目的：以基于核磁共振（nuclear magnetic resonance,NMR）的代谢组学方法对Wilson病（Wilson＇sdisease,WD）模型大鼠及中药干预后的WD模型大鼠血清进行代谢组学研究,分析中药干预后大鼠血清中小分子代谢物的变化,从小分子代谢物层面上探讨中药治疗Wilson病的内在机制。方法：33只雄性Wistar大鼠,体重180±20g,随机被分为健康对照组（n=11）,WD模型组（n=11）和中药干预组（n=11）。健康对照组自由摄水饮食,WD模型组和中药干预组采用铜负荷法制作Wilson病大鼠模型,以肝豆灵治疗中药干预组大鼠。采用NMR技术对大鼠血清进行检测,以MestRe-C 2.3软件及自编软件对谱图进行手动调相、基线校正和谱峰对齐。对样品进行分段积分,将积分数据归一化后构成数据矩阵,并利用PCA（Principle ComponentAnalysis）方法对数据矩阵进行统计分析。将健康对照组和WD模型组相对比,WD模型组和中药给药组相对比,以分析中药给药之后大鼠血清内小分子代谢物的变化。结果：相对于健康对照组,WD模型组大鼠血清甜菜碱（betaine）,氧化三甲胺（TAMO）、低密度脂蛋白（LDL）、极低密度脂蛋白（VLDL）、葡萄糖（glucose）含量有显著降低,胆碱（choline）、胆碱磷酸（phospho-rylcholine）有所降低,乳酸（lactate）、谷氨酰胺（glutamine）、糖蛋白（glycoprotein）有显著升高,肌酸酐（creatinine）,精氨酸（arginine）有所升高。相对于WD模型组,中药干预组大鼠血清糖蛋白（glycopro-tein）、3-羟基丁酸（3-hydroxybutyrate）、脂质（Lipid,mainly VLDL/LDL）、葡萄糖（glucose）、胆碱（cho-line）、磷酸胆碱（phosphorylcholine）有显著升高,亮氨酸（leucine）、异亮氨酸（isoleucine）有所升高,乳酸（lactate）显著下降,谷氨酰胺（glutamine）、肌氨酸＋肌氨酸酐（Creatine＋creatinine）、精氨酸（arginine）有所下降。结论：这些发生改变的代谢物可以作为中药干预WD的小分子代谢标志物参考,为进一步研究中药治疗WD的内在代谢机制提供思路。

1H-NMR Spectroscopy-based Metabonomic Research on Serum of Traditional Chinese Medicine Treated Model Rats of Wilson's Disease

Objective： Applying 1H nuclear magnetic resonance（1H-NMR） spectroscopy-based metabonomic approach to investigate the changes of small molecular metabolites in the serum from traditional Chinese drug treated model rats of Wilson＇s disease（WD）contrasted with those of the control group.To explore the mechanism of traditional Chinese drug treating Wilson＇s disease from small molecular aspect.Methods： 33 male Wistar rats（weight=180±20g） were divided into three groups randomly,the model group（n=11）,the control group（n=11）,and the traditional Chinese drug treating group,Gandouling group（n=11）.The models were established with excessive copper method,and the traditional Chinese drug group was treated with Gandouling formula.The serum was tested with 1H-NMR technology.The spectra were edited with MestRe-C2.3 and self-programmed software and then principal component analysis（PCA） was applied to make differentiation between the model and control groups,and the model and Gandouling groups respectively.Then,small modular changes in the serum after Gandouling treatment were analyzed to explore the mechanism of traditional Chinese drug treating WD rats.Results： Contrasting the small molecular changes between the control and model groups,we found choline and phosphorylcholine concentrations were lower and TAMO＋betaine,LDL,VLDL and glucose were significantly lower,while creatine＋ creatinine and arginine concentrations were higher and lactate,glutamine and glycoprotein were significantly higher in the serum of the model group.Contrasting the small molecular changes between the Gandouling and model groups,we found glycoprotein,3-hydroxybutyrate,Lipid,glucose,choline,phosphorylcholine concentrations were significantly higher,and leucine,isoleucine concentrations were higher,while lactate concentration was significantly lower,and glutamine Creatine＋ creatinine,arginine concentrations were lower in the serum of Gandouling group.The small molecular metabolites above may contribute to the discrimination,and serve as references for further research on the mechanism of traditional Chinese drug treating WD.