乌头碱对大鼠粪样代谢谱影响的代谢组学研究

Metabonomics study of feces from rats treated with aconitine

目的研究乌头碱对Wistar大鼠急性毒性作用下粪样的代谢谱的变化。方法 15只雄性Wistar大鼠适应性饲养1周后,随机分为给药组(10只)、对照组(5只),给药组按照2.54 mg/kg的剂量灌胃给予乌头碱,对照组给予同体积饮用水,采集给药6 h后的粪样,处死大鼠后采集心脏、肝脏、肾脏,并计算脏器系数。使用核磁共振技术检测粪便水提物的代谢物谱,应用模式识别技术对积分数据进行正交偏最小方差判别分析(OPLS-DA)及皮尔森相关分析,并进一步对差异代谢物使用SPSS 17.0软件进行Student t检验,使用MATLAB软件做相关系数图。结果给药组大鼠的脏器系数相对于对照组出现不同程度的降低,其中心脏、肝脏差异有统计学意义(P<0.05)。在粪便水提物中,核磁检测到的代谢物包括短链脂肪酸、氨基酸、单糖、三羧酸循环中间体及嘧啶和嘌呤类等40余种代谢产物。统计学分析表明,与对照组大鼠相比,给药组大鼠的粪便水提物中α-氨基戊酸、谷氨酸、苯丙氨酸的含量出现显著性降低,差异有统计学意义(P<0.05)。由此可见。乌头碱对大鼠肠道菌群产生了毒性作用,使得这些肠道菌群代谢及分解胃肠道食物的能力下降,致使粪样中代谢产物含量发生了变化。结合代谢通路的分析发现,乌头碱通过影响肠道菌群造成了大鼠体内氨基酸及能量代谢的异常,并且也一定程度上造成了主要脏器的损伤。结论本文客观地反映了大鼠粪样中代谢物谱图与乌头碱毒性的关系,指明了肠道菌群对整个机体内环境的平衡起到的重要作用。潜在差异代谢物及代谢通路分析表明,建立粪便代谢组学方法对于进一步研究评价药物毒性具有重要意义。

Objective To investigate the metabolic profiles of feces from rats treated with aconitine using NMR-based metabolomics approach. Methods After a 7-day acclimatization period, 15 male Wistar rats were randomly assigned to two treatment groups （control and aconitine）, each group were administrated with aconitine at a dose of 2.54 mg/kg or the equal volume of drinking water, respectively. Feces samples were collected at 6 h after the administration, then ac- quired the main organs （heart, liver, kidney） after anesthesia and calculated their organic indexes. 1H-NMR spectra were performed on Varian INOVA 600 NMR spectrometer. OPLS-DA and Pearson＇s correlation analysis were applied to the aligned integral data. The plots of correlation coefficient were carried out in MATLAB. Additionally, a two- tailed Student＇s t-test was performed for metabolites by using SPSS 17.0. Results Compared to the control group, or- ganic indexes were reduced in aconitine-induced group, in which heart and liver were caused significant differences （P 〈 0.05）. These metabolites had been confirmed including the short-chain fatty acid （SFCAs）, monosaccharides, amino acids, tricarboxylic acid （TCA） cycle intermediates, pyrimidine and purine metabolites in fecal water extracts. Multivariate statistical analysis showed that when compared to control group, the levels of α-ketoisovalerate, glutamate, phenylalanine in aconitine-induced group decreased significantly （P 〈 0.05）. Therefore, intestinal flora of rats pro- duced significantly toxic effects with aconitine, and reduced gut flora metabolism and ability to decompose the gastrointestinal food, resulting in changes in the fecal metabolite content. These results combined with pathway analysis indicated the disorder of amino acid metabolism and energy metabolism in dosed group. Conclusion This work objectively reflects the relationship of metabolite profiling in rats feces and aconitine toxicity and points out the important role played of the intestinal flora balance for the whole environment of the body. And the analysis of potential biomarkers and metabolic pathway also that fecal metabolomics is of great significance for further evaluating drug toxicity.