Effects of dietary curcumin on the colon proteome and transcriptome in the Mdr1a-/- mouse model of inflammatory bowel disease

This study assessed gene expression changes associated with inflammation in the colon tissue of multi-drug resistance targeted mutation (Mdr1a-/-) mice, a model of human IBD. It also tested the anti-inflammatory potential of curcumin in the colon of these mice with a particular focus on the proteome. A colonic histological injury score was determined for each mouse to assess the level of colon inflammation in Mdr1a-/- mice, and to establish the effect of curcumin on inflammation. Insights into mechanisms of colonic inflammation in the Mdr1a-/- mice were gained using transcriptome (microarray) and proteome (2-D gel electrophoresis and LCMS protein identification) analyses. These data were compared to changes in the colon proteome in response to curcumin, and to previously described transcriptomic analyses [Nones et al, PMID: 18761777; GSE10684] in response to curcumin, in this model. A total of 24 male Mdr1a-/- mice, and 24 male FVB/N mice, purchased from Taconic (Hudson, NY, USA) at 4-5 weeks of age, were used for this study. 12 mice were randomly assigned to each of two different dietary groups: control (AIN-76A powdered diet), or curcumin (AIN-76A + 0.2% curcumin). At 21 and 24 weeks of age, mice were euthanized and colon samples taken for histological, microarray, and proteomics analyses. The total histology score (HIS) in the colon was determined according to previously described criteria. Total RNA was isolated using TRIzol® reagent. Colon RNA from four Mdr1a-/- mice (with high HIS) on the control diet (i.e. AIN-76A powdered diet) was compared with colon RNA from four FVB/N mice with low HIS also on the control diet (i.e. AIN-76A powdered diet). All individual RNA samples were hybridized against a common reference RNA on separate slides. The reference RNA was prepared using equimolar RNA extracts from small intestine, colon, kidney and liver of normal healthy growing Swiss mice plus RNA extracts from Swiss mouse fetuses. These data were compared with previously reported transcriptomics analysis of colon RNA from the same four Mdr1a-/- mice on the AIN-76A diet (with high HIS) compared with colon RNA from four Mdr1a-/- mice on the curcumin diet (with low HIS; Nones et al 2009, PMID: 18761777)本研究评估了多药耐药靶向突变（multi-drug resistance targeted mutation, Mdr1a-/-）小鼠结肠组织中与炎症相关的基因表达变化，该动物模型可用于模拟人类炎症性肠病（Inflammatory Bowel Disease, IBD）。同时，本研究还检测了姜黄素对该小鼠结肠的抗炎潜力，重点聚焦于蛋白质组（proteome）层面。 研究人员为每只小鼠计算结肠组织学损伤评分，以此评估Mdr1a-/-小鼠的结肠炎症程度，并明确姜黄素对炎症的干预效果。通过转录组（transcriptome，微阵列（microarray））与蛋白质组（二维凝胶电泳（2-D gel electrophoresis）及液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LCMS）蛋白质鉴定）分析，本研究揭示了Mdr1a-/-小鼠结肠炎症的潜在机制。将上述实验数据与姜黄素处理后小鼠结肠蛋白质组的变化进行比对，并与该模型中已发表的姜黄素响应转录组分析[Nones等人，PMID: 18761777；GSE10684]进行对照。 本研究共纳入48只雄性小鼠，其中24只为Mdr1a-/-小鼠，24只为FVB/N小鼠，所有小鼠均于4~5周龄时从美国纽约哈德逊的Taconic公司购得。将小鼠随机分为两组，每组12只：对照组（饲喂AIN-76A粉状饲料）与姜黄素组（饲喂添加0.2%姜黄素的AIN-76A粉状饲料）。 分别在21周龄和24周龄时对小鼠实施安乐死，采集结肠样本用于组织学、微阵列及蛋白质组学分析。参照已发表的标准，计算结肠组织总组织学评分（HIS）。 采用TRIzol®试剂（TRIzol® reagent）提取总RNA。将对照组饲喂AIN-76A粉状饲料且组织学评分较高的4只Mdr1a-/-小鼠的结肠RNA，与同样饲喂AIN-76A粉状饲料且组织学评分较低的4只FVB/N小鼠的结肠RNA进行杂交比对。所有单个RNA样本均与通用参考RNA在独立玻片上完成杂交。通用参考RNA由正常健康生长的瑞士小鼠小肠、结肠、肾脏和肝脏的等摩尔RNA提取物，以及瑞士小鼠胎儿的RNA提取物混合制备而成。 将上述转录组数据与已发表的同类分析结果进行对比：该分析针对同一批4只饲喂AIN-76A粉状饲料且组织学评分较高的Mdr1a-/-小鼠，与4只饲喂姜黄素饲料且组织学评分较低的Mdr1a-/-小鼠的结肠RNA展开[Nones等人，2009，PMID: 18761777]。