Ileum transcriptional response to quercetin in WT and POR-null mice. Mus musculus

Using mice deficient in hepatic cytochrome-P450 oxidoreductase (POR), which disables the liver cytochrome P450 system, the metabolism and biological response of the anti-carcinogenic flavonoid, quercetin, was examined. Profiling circulating metabolites revealed similar profiles over 72 h in wild type (WT) and POR-null (KO) mice, showing that hepatic P450 and reduced biliary secretion do not affect quercetin metabolism. Transcriptional profiling at 24 h revealed that 2-3 fold more genes responded significantly to quercetin in WT compared to KO in the jejunum, ileum, colon, and liver, suggesting that hepatic P450s mediate many of the biological effects of quercetin, such as immune function, estrogen receptor signaling and lipid, glutathione, purine, and amino acid metabolism, even though quercetin metabolism is not modified. The functional interpretation of expression data in response to quercetin (single dose of 7 mg/animal) revealed a molecular relationship between the liver and jejunum. In WT animals, amino acid and sterol metabolism were predominantly modulated in the liver, fatty acid metabolism response was shared between the liver and jejunum, and glutathione metabolism was modulated in the small intestine. In contrast, KO animals do not regulate amino acid metabolism in the liver or small intestine, they share the control of fatty acid metabolism between the liver and jejunum, and regulation of sterol metabolism is shifted from the liver to the jejunum and that of glutathione metabolism from the jejunum to the liver. This demonstrates that the quercetin-mediated regulation of these biological functions in extrahepatic tissues is dependent on the functionality of the liver POR. In conclusion, using a systems biology approach to explore the contribution of hepatic phase I detoxification on quercetin metabolism demonstrated the resiliency and adaptive capacity of a biological organism in dealing with a bioactive nutrient when faced with a tissue-specific molecular dysfunction. Keywords: nutritional intervention, comparative genomic response, genotype variation Overall design: All animals were adapted to the RM3 (E) 801710 Soya-free powdered diet (B. S & S. [Scotland] Ltd, UK) over a period of 14 days. Quercetin was added separately to the semi-purified diets at a concentration of 6200 ppm (0.62 %; 6.2 g per kg). 65 male cytochrome P450 reductase null (KO) mice and 65 wild type (WT) C57BL/6 mice were reared, all aged between 6-8 weeks. Animals were housed 3 per cage, where both temperature and relative humidity were maintained within a range of 19-23oC and 40-70%, respectively. Twelve-hour periods of light were cycled with twelve-hour periods of darkness. For each strain of mouse, the following experimental design was used: a control group (25 mice) receiving powdered RM3 diet ad libitum and a group (25 mice) receiving a ‘high dose’ of quercetin (7 mg / mouse). The experimental diet was administered on day 15, following a 14-day adaptation period to the RM3 diet. Animals were sacrificed after 24 h. RNA samples destined for microarray analysis were only accepted and pooled into three groups if no aberrant signs of degradation (e.g. multiple peaks) were observed. The comprehensive gene expression profiles of the liver, jejunum, ileum, and colon were analyzed. In all cases, except those listed, RNA from 3 mice was pooled to form sample 1, another 3 mice to form sample 2, etc. Thus, 9 mice were used for the four experimental groups: wild-type, wild-type+quercetin, POR-null, POR-null+quercetin.

本研究利用肝组织细胞色素P450氧化还原酶（cytochrome-P450 oxidoreductase, POR）缺陷型小鼠——该模型可使肝脏细胞色素P450系统失活——对抗癌类黄酮化合物槲皮素（quercetin）的代谢过程与生物学效应进行了考察。对循环代谢物的谱学分析显示，野生型（wild type, WT）与POR敲除（POR-null, KO）小鼠在72小时内的代谢谱无显著差异，表明肝脏P450系统与胆汁分泌受损均不会对槲皮素的代谢过程产生影响。24小时时的转录组谱学分析结果显示，在空肠、回肠、结肠与肝脏组织中，野生型小鼠内受槲皮素显著调控的基因数量较POR敲除小鼠高出2-3倍，这表明尽管肝脏P450系统并未改变槲皮素的代谢过程，但它可介导槲皮素的多数生物学效应，包括免疫功能、雌激素受体信号通路以及脂质、谷胱甘肽、嘌呤与氨基酸代谢等。对单次给药剂量为7 mg/只的槲皮素处理后的基因表达数据进行功能注释分析，揭示了肝脏与空肠之间的分子调控关联。在野生型小鼠中，氨基酸与固醇代谢主要在肝脏中被调控，脂肪酸代谢的响应同时存在于肝脏与空肠中，而谷胱甘肽代谢则在小肠内发生调控。与之相反，POR敲除小鼠既无法在肝脏与小肠中调控氨基酸代谢，仅在肝脏与空肠之间共享脂肪酸代谢的调控通路，且固醇代谢的调控位点从肝脏转移至空肠，谷胱甘肽代谢的调控位点则从空肠转移至肝脏。上述结果表明，槲皮素在肝外组织中对上述生物学功能的调控作用依赖于肝脏POR的正常功能。综上，本研究通过系统生物学方法探究肝脏I相解毒通路对槲皮素代谢的影响，揭示了生物体在遭遇组织特异性分子功能缺陷时，应对生物活性营养素的弹性与适应能力。
关键词：营养干预、比较基因组响应、基因型变异

实验设计：所有实验动物均在14天的适应期内饲喂RM3(E) 801710无大豆粉末饲料（英国苏格兰B. S & S.有限公司生产）。将槲皮素以6200 ppm（0.62%，即每千克饲料含6.2 g槲皮素）的浓度单独添加至半纯化饲料中。共饲养65只雄性POR敲除（KO）小鼠与65只雄性野生型（WT）C57BL/6小鼠，所有小鼠的周龄均为6-8周。每笼饲养3只动物，饲养环境的温度与相对湿度分别控制在19-23℃与40%-70%范围内。光照与黑暗周期均为12小时交替循环。针对两种小鼠品系，均采用如下实验设计：对照组（25只）自由采食RM3粉末饲料，槲皮素处理组（25只）饲喂添加了高剂量槲皮素（7 mg/只）的饲料。在14天的饲料适应期结束后，于第15天开始饲喂实验饲料。于给药24小时后处死动物。用于芯片分析的RNA样品仅在未出现降解异常（如多峰现象）的情况下才可被接收，并合并为三组。对肝脏、空肠、回肠与结肠组织的全基因表达谱进行了分析。除另有说明外，所有样品均由3只小鼠的RNA混合制备：即样本1取自3只小鼠，样本2取自另外3只小鼠，以此类推。因此，四个实验组（野生型、野生型+槲皮素处理、POR敲除、POR敲除+槲皮素处理）共使用了9只小鼠。