果糖诱导斑马鱼肝脏脂肪变性的转录组学分析

Transcriptomic analysis of fructose induced liver steatosis in larval zebrafish

目的非酒精性脂肪肝(NAFLD)的发病呈低龄化趋势,本研究旨在建立一种模拟低龄人群的高糖诱导的NAFLD模型,揭示其基因表达谱特征,为疾病的机制研究和药物筛选提供基础。方法采用高果糖饮食诱导斑马鱼产生肝脏脂肪变性,油红O染色分析肝脏脂肪变性率,组织病理学方法观察肝脏脂质堆积,并分析肝脏生化指标变化。利用转录组测序观察基因表达谱变化,通过基因与基因组百科全书(KEGG)和基因本体(GO)数据库进行差异基因功能富集,分析其可能影响的信号通路与生物过程。结果斑马鱼幼鱼高果糖喂养10d后发生肝脏脂肪变性,肝脏切片HE染色和油红O染色结果发现,果糖喂饲的幼鱼肝脏出现微小脂质空泡,呈大面积红色着色。肝脏甘油三酯、葡萄糖含量显著高于正常对照组。经转录组测序发现果糖喂饲的幼鱼肝脏表达谱发生明显改变,共发现485个差异表达基因(变化倍数>3,P<0.05)。差异基因KEGG分析表明,PPAR信号通路为显著富集通路,real-time PCR结果确认PPAR通路中fads2、fabp7b、plin2、adipoqa基因水平显著上调。下调基因的GO分析结果显示,病毒防御为显著富集的生物学过程。结论建立了一种果糖诱导的斑马鱼NAFLD模型,可应用于斑马鱼幼体肝脏脂肪变性的机制研究和药物筛选。能量代谢相关基因表达的变化可为果糖致NAFLD的作用机制研究提供依据。

Objective The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in young people.The purpose of this study was to establish a sugar-induced NAFLD model and reveal the characteristics of its gene expression profile for the mechanism studies of NAFLD and drug screening in youth.Methods Zebrafish larvae were fed with a high fructose diet or normal diet respectively.The liver steatosis was analyzed by hepatic lipid accumulation and biological indexes.Transcriptome sequencing was employed to observe the changes of gene expression profile,and the functional enrichment of differentially expressed genes was conducted by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database.Results Zebrafish larvae were fed with a high fructose diet for 10 days developed liver steatosis.The results of pathological showed that lipid vacuoles appeared in the liver of the fructose fed larvae with red staining.The triglyceride and glucose contents of liver were significantly increased in fructose group compared with the control group.Transcriptome sequencing showed significant changes of gene expression profile in fructose-fed larvae.A total of 485 differentially expressed genes were identified (change fold > 3,P < 0.05).Analysis of the differentially expressed genes by KEGG showed that PPAR signaling pathway was significantly enriched.Four genes involved in PPAR pathway were validated by real-time PCR.The GO analysis from down-regulated gene showed that GO term:defense response to virus was significantly enriched.Conclusion This study has established a fructose-induced zebrafish NAFLD model.Particularly,variant expression of energy metabolism related genes in fatty livers supports a role of fructose diet in NAFLD pathology and provides a foundation for mechanism studies and drug screening of NAFLD.