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[摘要] 运用代谢组学方法研究黄连-黄芩药对对脑缺血模型大鼠代谢的作用机制。采用线栓法致大脑中动脉阻塞复制缺血再灌脑损伤大鼠模型（MCAO）。通过超高效液相色谱-串联四级杆飞行时间质谱（UPLC-Q-TOF/MS）的方法、Markerlynx软件、以及主成分分析法和偏最小二乘判别法分析正常大鼠、脑缺血模型大鼠以及分别给予黄连（HL）、黄芩（HQ）和黄连-黄芩药对（LQ）干预后大鼠尿液样本中的内源性代谢物的差异，结合内源性代谢物的一级及二级碎片离子的精确信息，检索并鉴定可能的生物标志物，进而在MetPA数据库中构建代谢通路。结果在脑缺血模型大鼠的尿液中发现并鉴定出了20种生物标志物，并对其相对含量及代谢途径进行分析。主要涉及神经递质调节、氨基酸代谢、能量代谢、脂质代谢等。这些代谢通路在脑缺血模型大鼠的体内发生紊乱，主成分分析表明，正常与脑缺血模型得到明显区分，而给予HL，HQ，LQ后可以使之向着正常状态转变。此研究对脑缺血大鼠代谢组及黄连-黄芩药对干预的作用机制进行阐释，体现代谢组学可反映生物体的生理及代谢状态，能较好地体现药对是中医遣方用药的特色优势，具有内在的组合变化规律以及中医药的整体观、系统观以及多成分协同或拮抗作用的特点。
[关键词] 脑缺血；黄连-黄芩药对；代谢组学；UPLC-Q-TOF/MS
[Abstract] The metabolic effect of Huanglian-Huangqin herb pairs on cerebral ischemia rats was studied by using metabolomic method. The rat model of ischemia reperfusion injury induced by introduction of transient middle cerebral artery occlusion （MCAO） followed by reperfusion. Ultra high performance liquid chromatography-series four pole time of flight mass spectrometry method（UPLC-Q-TOF/MS），Markerlynx software，and principal component analysis and partial least-squares discriminant analysis were used to analyze the different endogenous metabolites among the urine samples of sham rats，cerebral ischemia model rats，Huanglian groups （HL），Huangqin groups （HQ） and Huanglian-Huangqin herb pairs groups （LQ） was achieved，combined with accurate information about the endogenous metabolites level and secondary fragment ions，retrieval and identification of possible biological markers，metabolic pathway which build in MetPA database. The 20 potential biomarkers were found in the urine of rats with cerebral ischemia，which mainly involved in the neurotransmitter regulation，amino acid metabolism，energy metabolism，lipid metabolism and so on. Those metabolic pathways were disturbed in cerebral ischemia model rats，the principal component analysis showed that the normal and cerebral ischemia model is clearly distinguished，and the compound can be given to the normal state of change after HL，HQ，LQ administration. This study index the interpretation of cerebral ischemia rat metabolism group and mechanism，the embodiment of metabonomics can reflect the physiological and metabolic state，which can better reflect the traditional Chinese medicine as a whole view，system view and the features of multi ingredient synergistic or antagonistic effects.
[Key words] cerebral ischemia；Huanglian-Huangqin herb pairs；metabolomics；UPLC-Q-TOF/MS
药对是单味中药与若干方剂之间的桥梁，是许多方剂隐含的规律性特征与辨证施治内涵体现[1]。黄连和黄芩作为黄连解毒汤中的君药和臣药是其重要的组成部分，方中黄连与黄芩配伍比例为3∶2。黄连-黄芩属清热燥湿配伍的经典药对，两药配伍主治上、中二焦，火邪去而热毒清，均能清热燥湿、泻火解毒[2-3]。代谢组学是对一个生物系统的细胞在给定时间和条件下所有小分子代谢物质的定性定量分析，从而描述生物内源性代谢物质的整体规律，并且将在内外因素作用下产生的代谢物质的动态变化与病理生理相关联。代谢组学处于生命信息传递的终端，它是能表现出生物体系整体功能或状态的最终结果而抛开体内复杂的生理过程的一种系统生物学科学[4-6]。本实验室前期研究黄连解毒汤在脑缺血模型大鼠体内药代动力学和药效学，探究其在体内的分布及代谢特点，寻找中药复方中蕴含的潜在规律和方证对应的合理性，结果黄连解毒汤中有效成分对脑缺血病症大鼠具有一定的调节作用。在此基础上本实验以大脑中动脉栓塞（middle cerebral artery occusion，MCAO）模型大鼠为研究对象，采用代谢组学技术、模式识别方法并结合代谢组网络数据库对大鼠尿液中代谢物进行研究，探讨正常与模型大鼠其尿液中的内源性代谢物的整体变化，分析鉴定潜在生物标志物及代谢通路，寻找黄连解毒汤干预后代谢产物动态变化规律及抗脑缺血的生物學作用机制。