目的:分析葛花解酒地治汤(,GJDD)通过蛋白质组学方法对酒精性脂肪活病(AFLD)进行研究。
方法:将雄性C57BL/6J小鼠随机分为4组:对照组,模型组,GJDD组和白藜芦醇组。在Lieber-DeCarli经典方法的基础上,通过一次灌胃酒精成功制备AFLD模型后,GJDD组和白藜芦醇组分别灌胃GJDD(4900mg/kg)和白藜芦醇(400mg/kg),每天一次,连续9d。观察肝脏组织的脂肪沉积,并通过油红O(ORO)染色进行评估。采用4DLabel-free定量蛋白质组方法测定和定量各实验组肝组织中的蛋白表达。根据蛋白质表达差异倍数筛选差异表达的蛋白质,然后通过基因本体分类和京都百科全书对基因和基因组途径进行富集分析。最后,来自对照组的差异共表达蛋白的表达验证,通过靶向蛋白质组学定量技术对模型组和GJDD组进行验证。
结果:在ORO的半定量分析中,各种脂肪变性(ToS,MaS,与GJDD或白藜芦醇处理的小鼠相比,AFLD小鼠中的MiS)被评估为更高。4DLabel-free蛋白质组学分析结果显示,共鉴定出4513种蛋白质,其中对3763种蛋白质进行了定量,筛选了946种差异表达蛋白质。与对照组相比,模型组肝组织中145个蛋白表达上调,148个蛋白表达下调。此外,与模型组相比,GJDD组肝组织中92个蛋白上调,135个蛋白下调。在每两组之间发现15种差异共表达的蛋白质(模型组与对照组,GJDD组与模型组比较,GJDD组与对照组),参与了许多生物过程。其中,11个差异共表达的关键蛋白(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2和Slco1a1)通过靶向蛋白质组学定量技术进一步鉴定,其表达模式与4D无标记蛋白质组学分析结果一致。
结论:我们的研究提供了基于蛋白质组学的证据,证明GJDD通过调节肝脏蛋白表达缓解AFLD,可能是通过调节脂质代谢,胆汁酸代谢和抗氧化应激的发挥。
OBJECTIVE: To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction (, GJDD) on alcoholic fatty live disease (AFLD) by using proteomic methods.
METHODS: The male C57BL/6J mouse were randomly divided into four groups: control group, model group, GJDD group and resveratrol group. After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method, the GJDD group and resveratrol group were intragastrically administered with GJDD (4900 mg/kg) and resveratrol (400 mg/kg) respectively, once a day for 9 d. The fat deposition of liver tissue was observed and evaluated by oil red O (ORO) staining. 4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group. The differentially expressed proteins were screened according to protein expression differential multiples, and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Finally, expression validation of the differentially co-expressed proteins from control group, model group and GJDD group were verified by targeted proteomics quantification techniques.
RESULTS: In semiquantitative analyses of ORO, all kinds of steatosis (ToS, MaS, and MiS) were evaluated higher in AFLD mice compared to those in GJDD or resveratrol-treated mice. 4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified, of which 3763 proteins were quantified and 946 differentially expressed proteins were screened. Compared with the control group, 145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group. In addition, compared with the model group, 92 proteins were up-regulated and 135 proteins were down-regulated in the liver tissue of the GJDD group. 15 differentially co-expressed proteins were found between every two groups (model group vs control group, GJDD group vs model group and GJDD group vs control group), which were involved in many biological processes. Among them, 11 differentially co-expressed key proteins (Aox3, H1-5, Fabp5, Ces3a, Nudt7, Serpinb1a, Fkbp11, Rpl22l1, Keg1, Acss2 and Slco1a1) were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.
CONCLUSIONS: Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression, likely through the modulation of lipid metabolism, bile acid metabolism and with exertion of antioxidant stress.