蚕豆胁迫下小鼠肝脏和血浆的代谢组学特征及其与肝细胞基因表达谱的关系

This objective of the project is to analyze the metabolomic characteristics of liver and plasma and to look for the hemolysis-associated metabolites produced in the process of metabolism of fava bean in model mouse under fava bean stress by using GC/MS, NMR and UPLC/MS/MS methods and multivariate data analyses PCA, PLS, PLS-DA, OPLS-DA. To look for the related target genes associated with favism, the project will also analyze the interactive links between the characteristic metabolites in the liver and plasma, the liver cell gene expression profile and oxidative stress in mice under fava bean stress and to analyze the metabolic pathways related to fava bean metabolism by integrative analysis of the liver cell gene expression profile and the oxidative stress index and database analysis. The results will be helpful for elucidating the specific metabolites and the related metabolic pathways associated with favism due to G6PD deficiency and provide valuable clues for further identifying the related genes in human being. It is of significant for elucidating the molecular mechanism of favism as well as for preventing and treating favism. The results will also provide preliminary scientific evidence for understanding the interaction between the food and the genome and for understanding individual differences of food metabolism, which is of significance for evaluating food safety and guiding the reasonable personalized dietary intake.

本课题以小鼠为模型，应用GC/MS、NMR和UPLC/MS/MS分析技术和PCA、PLS、PLS-DA、OPLS-DA等多维统计学方法分析蚕豆胁迫下小鼠肝脏和血浆代谢组学的变化特征，寻找蚕豆在体内代谢过程中产生的与溶血相关的代谢标志物，同时结合肝细胞基因表达谱和体内氧化应激状态的情况，通过数据库分析了解与蚕豆代谢相关的代谢通路，阐明蚕豆胁迫下小鼠肝内和血浆中代谢物的变化特征及其与肝细胞内基因表达和氧化应激的关系，寻找与蚕豆病个体差异相关的基因靶点。研究结果将有助于阐明蚕豆引起G6PD缺乏症患者溶血的物质基础及相关的代谢通路，为进一步在人类中寻找与蚕豆病的相关基因提供有价值的线索，对于阐明蚕豆病溶血的分子机制以及蚕豆病的防治具有非常重要的意义。同时，本研究也可为了解食物与基因组的相互作用以及食物代谢的个体差异提供初步的科学依据，对于食品的安全性评估以及指导合理的个性化膳食也具有重要的意义。

本项目以G6PD缺乏鼠为模型，在蚕豆胁迫下分别从基因表达谱、代谢组及氧化应激等方面进行研究，阐明蚕豆病发生的物质基础及分子机制。.（1）获得G6PD缺乏（G6PDx）小鼠；检测蚕豆胁迫下G6PD正常(C3H)小鼠和G6PDx小鼠体内氧化应激应答的数据，结果表明进食蚕豆后，在G6PD正常和G6PD缺乏状态下对小鼠体内均发生氧化应激。.（2）获得蚕豆胁迫下G6PD正常小鼠和G6PD缺乏小鼠肝细胞的基因表达谱，揭示蚕豆对肝细胞基因表达谱的影响。在C3H小鼠中，蚕豆进食诱导95个基因显著上调，54个基因显著下调。在G6PDx小鼠中，蚕豆进食诱导273个基因显著上调，165个基因显著下调。与C3H对照组小鼠相比， G6PDx对照组显示174个基因显著上调，146个基因显著下调。与C3H蚕豆组小鼠相比， G6PDx蚕豆组显示205个基因显著上调，147个基因显著下调。.（3）获得蚕豆胁迫下G6PD正常小鼠和G6PD缺乏小鼠血浆和肝脏代谢组的特征。GC-MS检测小鼠血浆，共检测到403个代谢产物并进行主成分分析。通过OPLS-DA模型分析表明，C3H和G6PDx小鼠的蚕豆组和对照组间具有显著性的差异。进食蚕豆后，在C3H中发现有7个差异性代谢物；在G6PDx小鼠中发现有21个差异性代谢物。进食蚕豆后，C3H小鼠血浆差异性代谢物组分包括β-丙氨酸，天冬酰胺，肌酐，DHA，油酸，亚油酸，和肌醇。这些组分主要与脂类和氨基酸代谢相关。在进食蚕豆后，在G6PDx小鼠血浆中发现21个差异性代谢物组分，与氨基酸代谢，糖代谢和脂类代谢相关。GC-MS检测小鼠肝脏，共检测到316个代谢产物并进行主成分分析。通过OPLS-DA模型的进一步分析消除干扰以获得更加准确的数据，分析表明C3H和G6PDx小鼠的蚕豆组和对照组间具有显著性的差异。在进食蚕豆后，C3H小鼠肝脏差异性代谢物组分与碳代谢，核苷酸代谢，能量代谢和氨基酸代谢相关。在进食蚕豆后，在G6PDx小鼠肝脏中发现25个差异性代谢物组分，与碳代谢，氨基酸代谢，核苷酸代谢和脂类代谢相关。..（4）将基因表达谱、代谢组及氧化应激整合分析，发现脂类代谢，氨基酸代谢在转录组及代谢组中都发生显著差异，而而生物转化相关基因P450基因家族中9个基因发生显著变化，这可能预示蚕豆诱导的P450基因变化与蚕豆诱发小鼠发生氧化应激相关。