Transcription profiling of mouse colon response to quercetin in WT and POR-null animals

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. Experiment 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. Experiment Overall Design: 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. Experiment Overall Design: 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.本研究利用肝细胞色素P450氧化还原酶（hepatic 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，探究了抗癌黄酮类化合物槲皮素（quercetin）的代谢过程与生物学效应。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小时内的代谢谱特征相似，表明肝脏P450系统与胆汁分泌减少均不会影响槲皮素的代谢。 在24小时时开展的转录组谱分析（transcriptional profiling）显示，空肠（jejunum）、回肠（ileum）、结肠（colon）与肝脏（liver）中，野生型小鼠对槲皮素产生显著响应的基因数量较敲除型小鼠多出2-3倍，这提示尽管槲皮素的代谢未发生改变，但肝脏P450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。 对单次给药（每只动物7mg）槲皮素后的表达数据进行功能注释后发现，肝脏与空肠之间存在分子层面的关联。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被调控。与之相反，敲除型小鼠既不会在肝脏也不会在小肠内调控氨基酸代谢，其脂肪酸代谢的调控由肝脏与空肠共同承担，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。这表明槲皮素对肝外组织中这些生物学功能的调控，依赖于肝脏POR的功能完整性。 综上，本研究通过系统生物学（systems biology）方法探究肝脏I相解毒作用（phase I detoxification）对槲皮素代谢的贡献，揭示了当生物体遭遇组织特异性分子功能障碍时，其应对生物活性营养素的韧性与适应能力。 ## 实验总体设计 1. 所有实验动物均在14天内适应RM3（E）801710无大豆粉末饲料（英国B. S & S. [苏格兰]有限公司生产）。将槲皮素以6200 ppm（0.62%；每千克6.2g）的浓度添加至半纯化饲料中。 2. 饲养65只雄性POR敲除小鼠与65只雄性野生型C57BL/6小鼠，所有小鼠均为6-8周龄。每笼饲养3只动物，环境温度维持在19-23℃，相对湿度维持在40%-70%，光照与黑暗周期各为12小时。 3. 针对每种小鼠品系，实验设计如下：对照组（25只小鼠）自由摄食RM3粉末饲料，实验组（25只小鼠）摄食含"高剂量"槲皮素（每只小鼠7mg）的饲料。实验饲料于第15天开始投喂，此前动物已完成14天的RM3饲料适应期。所有动物在给药后24小时被处死。 4. 仅当RNA样本未出现降解异常信号（如多峰）时，方可用于微阵列分析（microarray analysis）并合并为三组。对肝脏、空肠、回肠与结肠的全面基因表达谱进行分析。除特殊说明外，每组样本均由3只小鼠的RNA混合而成（如样本1由3只小鼠的RNA混合，样本2由另外3只小鼠的RNA混合，依此类推）。因此，四组实验（野生型、野生型+槲皮素、POR敲除、POR敲除+槲皮素）共使用了9只小鼠。