在APAP、TAA诱导的肝细胞药物性损伤中C/EBPβ对FXR的调控机制

Regulation of FXR by C/EBPβin drug-induced injury triggered by APAP or TAA in hepatocytes

目的:通过生物信息学分析寻找对乙酰氨基酚(APAP)诱导药物性肝损伤(DILI)过程中的关键基因,并研究在APAP、硫代乙酰胺(TAA)介导的小鼠肝细胞药物性损伤中,CCAAT/增强子结合蛋白β(C/EBPβ)对法尼醇X受体(FXR)的调控机制。方法:采用生信分析筛选APAP诱导的DILI模型中的关键基因;通过RT-qPCR、Western blot法检测不同浓度的TAA(0、13、33 mmol/L)、APAP(0、2、5μmol/L)对小鼠肝细胞AML12中C/EBPβ及FXR的mRNA及蛋白水平表达的影响。针对C/EBPβ基因设计两条不同的shRNA,并分别克隆至pLKO载体,用于构建C/EBPβ稳定敲低的AML12细胞系;通过流式细胞术分析TAA、APAP对AML12细胞凋亡的影响。结果:(1)FXR是APAP所致DILI模型中的关键性基因;(2)TAA和APAP导致AML12小鼠肝细胞损伤中FXR的mRNA及蛋白表达水平均显著降低;(3)TAA和APAP导致AML12小鼠肝细胞损伤中C/EBPβ蛋白表达水平显著降低;(4)C/EBPβ稳定敲低的AML12细胞系中FXR的mRNA水平显著下调。(5)奥贝胆酸(OCA)可以抑制凋亡基因聚ADP-核糖聚合酶(PARP)的表达。结论:在TAA及APAP诱导的肝细胞损伤中,C/EBPβ可能是调控FXR的关键基因。

Objective:To analyze the key genes in acetaminophen(APAP)-induced drug-induced liver injury(DILI)by bioinformatics and investigate the regulation of farnesoid X receptor(FXR)by CCAAT/enhancer binding proteinβ(C/EBPβ)in drug-induced injury triggered by APAP or thioacetamide(TAA)in hepatocytes of mice.Methods:Key genes in APAP-induced DILI model were screened by bioinformatic analysis.The effect of different concentrations of TAA(0,13 and 33 mmol/L)and APAP(0,2 and 5μmol/L)on the mRNA and proteins expression of C/EBPβand FXR in mice liver AML12 cells were detected by Real-time qPCR and Western blot.Two shRNAs were designed for C/EBPβgene and cloned into pLKO vector,respectively,to construct C/EBPβ-stable knockdown AML12 cell lines.The effect of TAA and APAP on apoptosis of AML12 cells was analyzed by flow cytometry.Results:FXR is a key gene in drug-induced liver injury model triggered by APAP.TAA and APAP significantly reduced the mRNA and protein expression levels of FXR and protein expression levels of C/EBPβin injury of mice liver AML12 cells.The mRNA levels of FXR in C/EBPβknockdown AML12 cell lines were significantly down regulated.OCA can inhibit the expression of apoptosis gene PARP.Conclusion:C/EBPβmay be a key gene regulating FXR in hepatocyte injury induced by TAA or APAP in AML12 cells.