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Tandem Mass Tag-based quantitative proteomics analysis of metabolic associated fatty liver disease induced by high fat diet in mice
Nutrition & Metabolism ( IF 4.5 ) Pub Date : 2020-11-18 , DOI: 10.1186/s12986-020-00522-3
Hu Li , Wei Huang , Mingjie Wang , Peizhan Chen , Li Chen , Xinxin Zhang

Although metabolic associated fatty liver disease (MAFLD) is the most common chronic liver disease worldwide, the exact molecular mechanism of MAFLD progression remains unknown. In the present study, Tandem Mass Tag-labeled quantitative proteomic technology was used to elucidate the protein expression patterns of liver tissues in the progression of MAFLD, providing new potential therapeutic targets of it. Five 6-week-old male C57BL/6 mice were fed with high fat diet (HFD) for 22 weeks to establish the MAFLD mouse models. Five C57BL/6 mice of the same age were fed with normal diet (ND) and taken as controls. Mice serum were sampled for biochemical tests, and livers were isolated for histopathological examinations. Six mouse liver samples (three from each group) were performed for proteomic analysis. Differentially expressed proteins were defined using fold change of > 1.5 or < 0.67 and p value < 0.05 as thresholds. Bioinformatic analysis was used to identify the hub proteins. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), Gene Expression Omnibus dataset, western blotting and immunohistochemistry were used to validate the expression of identified hub proteins. After 22 weeks on HFD diet, all mice developed MAFLD demonstrated by histopathological examination. Mouse body weights, liver weights, serum alanine transaminase and aspartate transaminase levels were significantly higher in the HFD group than ND group. Proteomics technology identified 4915 proteins in the mouse livers, among which 71 proteins were differentially expressed. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that majority of the differentially expressed proteins were involved in the peroxisome and peroxisome proliferator-activated receptor signaling pathway, as well as biosynthesis of unsaturated fatty acids. Protein–protein interaction analysis showed that these differentially expressed proteins interacted with each other and formed a complex network. Ten hub proteins were identified and validated using RT-qPCR. Five of these proteins were validated in the Gene Expression Omnibus dataset. Finally, Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase protein was validated in mouse liver tissue samples using western blotting and immunohistochemistry. Our data showed that lipid metabolism-related pathways are closely associated with the development of MAFLD. The identified hub proteins might be novel targets for treating MAFLD.

中文翻译:

基于串联质量标签的高脂饮食诱发小鼠代谢相关性脂肪肝疾病的定量蛋白质组学分析

尽管代谢相关性脂肪肝病(MAFLD)是全世界最常见的慢性肝病,但MAFLD进展的确切分子机制仍然未知。在本研究中,串联质谱标签标记的定量蛋白质组学技术用于阐明MAFLD进展过程中肝组织的蛋白质表达模式,为其提供了新的潜在治疗靶标。给五只6周大的雄性C57BL / 6小鼠喂食高脂饮食(HFD)22周,以建立MAFLD小鼠模型。给五只相同年龄的C57BL / 6小鼠喂食正常饮食(ND),并作为对照。抽取小鼠血清进行生化测试,并分离肝脏以进行组织病理学检查。进行了六只小鼠肝脏样品(每组三只)进行蛋白质组学分析。使用> 1.5或<0.67的倍数变化和p值<0.05为阈值来定义差异表达的蛋白质。生物信息学分析用于鉴定毂蛋白。实时定量聚合酶链反应(RT-qPCR),基因表达Omnibus数据集,蛋白质印迹和免疫组织化学被用来验证已鉴定的毂蛋白的表达。在接受HFD饮食22周后,所有小鼠均发生了由组织病理学检查证实的MAFLD。HFD组的小鼠体重,肝脏重量,血清丙氨酸转氨酶和天冬氨酸转氨酶水平显着高于ND组。蛋白质组学技术在小鼠肝脏中鉴定出4915种蛋白质,其中71种蛋白质被差异表达。京都基因与基因组百科全书通路分析表明,大多数差异表达的蛋白质都参与了过氧化物酶体和过氧化物酶体增殖物激活的受体信号传导途径,以及不饱和脂肪酸的生物合成。蛋白质-蛋白质相互作用分析表明,这些差异表达的蛋白质彼此相互作用,形成了复杂的网络。使用RT-qPCR鉴定并验证了十种毂蛋白。这些蛋白中的五个在Gene Expression Omnibus数据集中得到了验证。最后,使用免疫印迹和免疫组织化学方法在小鼠肝脏组织样品中验证了Enoyl-CoA水合酶和3-羟酰基CoA脱氢酶蛋白。我们的数据表明,脂质代谢相关的途径与MAFLD的发展密切相关。
更新日期:2020-11-18
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