Transcriptomic response in colon of rat with increased dietary protein content

Increase of fecal hazardous compounds and decrease of butyrate-producing bacteria has been implicated to be detrimental to colonic health during weight loss by high-protein diet in human. The question remains that whether the change of luminal microenvironment were paralleled with the alteration of colonic function in vivo.To reveal the change of gene expression involved in metabolic or immmunological process, we have employed the whole genome microarray profiling to identify differentially expressed genes in rat dietary normal protein (20% protein) or high protein (45% protein) diet. Colonic epithelium were sampled in adult male wistar rats (weighing 180-200g) fed each diet for six weeks.Each group contained six replicates.A total of 181 transcripts were significantly changed: 88 genes were up-regulated and 93 genes were down-regulated in high protein compared with normal protein rats. Transcriptome analysis identified the upregulation of chemotaxis, TNF-a signal process, antigen presentation, apoptosis, glutathione S-transferase activity and downregulation of innate immune, O-linked glycosylation of mucin, oxidative phosphorylation.Expression of ten genes, including calmodulin1,acetyl-CoA acyltransferase 2,isovaleryl-CoA dehydrogenase and occludin, were quantified in the same RNA samples by real-time PCR, validating the microarray analysis.人体高蛋白饮食减重过程中，粪便有害化合物增多、产丁酸菌减少，已被证实会对结肠健康产生不利影响。目前仍有待解答的问题是，体内管腔微环境的变化是否与结肠功能的改变相一致。为揭示代谢或免疫过程相关基因表达的变化，本研究采用全基因组微阵列（whole genome microarray）分析技术，对喂食正常蛋白（20%蛋白）或高蛋白（45%蛋白）饲料的大鼠体内差异表达基因进行筛选。本研究选取体重180~200g的成年雄性Wistar大鼠，分别喂食两种饲料六周后，采集其结肠上皮组织；每组设置6个生物学重复。最终共筛选得到181个显著差异表达的转录本：与正常蛋白饮食组大鼠相比，高蛋白饮食组大鼠体内有88个基因上调表达，93个基因下调表达。转录组（transcriptome）分析结果显示，高蛋白饮食组大鼠体内趋化作用、肿瘤坏死因子-α（TNF-α）信号通路、抗原呈递、细胞凋亡及谷胱甘肽S-转移酶活性相关通路显著上调，而固有免疫、黏蛋白O-连接糖基化及氧化磷酸化相关通路显著下调。本研究采用实时荧光定量PCR（real-time PCR）技术，对同一份RNA样本中的10个基因（包括钙调蛋白1、乙酰辅酶A酰基转移酶2、异戊酰辅酶A脱氢酶及闭合蛋白）的表达量进行定量验证，以确认微阵列分析结果的可靠性。