Short Bowel Syndrome (SBS) has Widespread Effects Beyond Altering Nutrient Absorption: RNA Sequencing a Zebrafish SBS Model. Danio rerio

Purpose: Most of the morbidity associated with short bowel syndrome (SBS) are attributed to effects of decreased enteral nutrition and administration of total parenteral nutrition (TPN). We hypothesized that acute SBS alone has significant systemic effects, and tested this in a zebrafish SBS model. Methods: With IACUC approval, adult male wild-type zebrafish underwent SBS (laparotomy, proximal stoma, distal ligation,n=3) or sham (laparotomy alone,n=3) surgery. After 2 weeks, the proximal intestine was harvested, RNA isolated and external RNA controls consortium (ERCC) controls spiked into each sample, sequenced and aligned to reference genome with gene ontology (GO) enrichment analysis performed. CyclinD1, CyclinB1, SAA1, IFN-gamma, and CYP7A1 gene expression were confirmed by qPCR. Results: RNA-seq analysis identified 1346 up-regulated genes and 678 down-regulated genes in SBS zebrafish compared to sham. The up-regulated genes were involved in acute phase response signaling, complement system, coagulation, cell proliferation, cellular barrier, production of nitric oxide & reactive oxygen species and bile acid biosynthesis. The down-regulated genes were involved in folate synthesis, gluconeogenesis, glycogenolysis, fatty-acid oxidation & activation, and drug & steroid metabolism. CyclinD1 gene expression was 2-fold higher, CyclinB1 2.8-fold higher, SAA1 4.5-fold higher, IFN-gamma 2.1-fold higher, and CYP7A1 25-fold higher in SBS than sham by qPCR. Conclusion: The gene expression of SBS demonstrates complex and extensive alteration of multiple pathways, some previously implicated as effects of TPN. The systemic complications of SBS alone are significant and extend beyond the complications of therapy. Overall design: Profiles of paired fed and unfed zebrafish intestine were generated by deep sequencing in triplicate.

研究目的：短肠综合征（short bowel syndrome, SBS）相关的绝大多数发病症状，均由肠内营养摄入不足与全肠外营养（total parenteral nutrition, TPN）的治疗效应所导致。本研究推测，单纯急性短肠综合征即可引发显著的全身效应，并以此在斑马鱼短肠综合征模型中对该假说进行验证。
研究方法：本研究经动物实验伦理审查委员会（Institutional Animal Care and Use Committee, IACUC）批准，选取成年雄性野生型斑马鱼，分为短肠综合征造模组（实施剖腹手术、近端造口、远端结扎，n=3）与假手术组（仅实施剖腹手术，n=3）。术后2周，采集近端肠组织，提取总RNA，并向每份样本中加入外源RNA质控联合体（External RNA Controls Consortium, ERCC）内参，随后进行RNA测序，将测序序列比对至参考基因组，并开展基因本体（Gene Ontology, GO）富集分析。通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qPCR）验证CyclinD1、CyclinB1、SAA1、IFN-γ及CYP7A1的基因表达水平。
研究结果：RNA测序分析显示，与假手术组相比，短肠综合征斑马鱼体内存在1346个上调基因与678个下调基因。上调基因主要富集于急性期反应信号通路、补体系统、凝血过程、细胞增殖、细胞屏障功能、一氧化氮与活性氧生成以及胆汁酸生物合成等通路；下调基因则主要参与叶酸合成、糖异生、糖原分解、脂肪酸氧化与活化，以及药物及类固醇代谢等生物学过程。经qPCR验证，短肠综合征组的CyclinD1基因表达量较假手术组升高2倍，CyclinB1升高2.8倍，SAA1升高4.5倍，IFN-γ升高2.1倍，CYP7A1升高25倍。
研究结论：短肠综合征的基因表达谱呈现多条通路复杂且广泛的改变，其中部分通路此前被认为与全肠外营养的治疗效应相关。单纯短肠综合征即可引发显著的全身并发症，且其影响范围超出了治疗相关并发症范畴。
整体实验设计：通过深度测序技术，对三组重复的配对喂食与未喂食斑马鱼肠组织的转录表达谱进行构建。