HNF-1调节GSTA1表达的机制及与JNK细胞信号通路关系研究

Use drug-induced liver injury in mice with pre-established as experimental animals by the project，and hepatocytes injury model will be established. Compare and analyze the effect on GSTA1 activity and expression with C2- ceramide inhibits the expression of HNF-1 and oltipraz promotes the expression of HNF-1 in vivo and in vitro by using RT-PCR, Western blot and other molecular biology methods. The pGSTA1-LUC contained the GSTA1 promoter will be constructed, and HRE, the potential binding sites for HNF-1 and GSTA1, will be mutated by QuikChange method.Then the recombinant plasmid will be transferred into liver cells by transfecting, detect the expression of GSTA1 and HNF-1 and determine whether HRE is the target on which HNF-1 regulate the expression of GSTA1. In vivo and in vitro, the activation of JNK cell signaling will be measured in hepatocellular injury induced by APAP. With and without JNK inhibitor SP600125 on APAP hepatotoxicity, the relationship between JNK and liver injury will be clear. Compare the influence of GSTA1and HNF-1 on JNK protein expression with C2- ceramide and oltipraz, and explore the relationship among drug-induced liver injury, GSTA1, HNF-1 and JNK. This investigation will lay the theoretical foundation for comprehensive understanding the important role of GSTA1 in stress cell signaling, for the development of new therapeutic targets and new directions for drug research.

本项目以前期建立药物性肝损伤模型小鼠为实验动物，建立肝细胞损伤模型；采用RT-PCR、Western blot等分子生物学手段，在体内、体外比较分析C2-神经酰胺抑制及奥替普拉促进HNF-1表达两种情况下，对GSTA1含量及表达影响。构建包含GSTA1启动子区域的荧光素酶检测报告基因质粒pGSTA1-LUC；采用QuikChange法使HNF-1与GSTA1的可能结合位点HRE突变后转染肝细胞，测定对HNF-1与GSTA1影响，确定HRE是否是HNF-1作用靶位。在体内、体外检测APAP诱导肝细胞损伤中JNK被激活情况；应用JNK抑制剂SP600125前后对APAP肝毒性的影响。比较应用C2和奥替普拉前后HNF-1、GSTA1对JNK蛋白表达影响，探讨药物性肝损伤、GSTA1、HNF-1、JNK之间关系，为全面认识GSTA1在细胞信号中角色，开发新的治疗靶点及药物研发新方向奠定理论基础。

药物性肝损伤已经上升至全球死亡原因的第五位，是药物开发及临床用药安全中的关键问题。醋氨酚（APAP）导致的急性肝损伤可作为最典型的药物性肝损伤模型，因此，本项目采用APAP成功建立了药物性肝损伤体内外模型。在肝损伤中，HNF-1和GSTA1 mRNA及蛋白的表达水平极显著下降，HNF-1对GSTA1的含量和表达具有正向调节作用。构建重组质粒pGSTA1-LUC，采用快速定点突变方法将构建的质粒进行定点突变，命名为pGSTA1-HNF-1-LUC。采用Dual-Luciferase○RReporter Assay System方法检测荧光素酶活性，重组质粒pGSTA1-LUC荧光素酶活性明显降低，重组质粒pGSTA1-HNF1-LUC荧光素酶活性极显著降低，表明转染成功。在转染后的肝细胞中，APAP降低肝细胞存活率，C2-神经酰胺和奥体普拉分别加重和减轻损伤细胞的存活率；APAP导致HNF-1 mRNA及蛋白的表达水平极显著下降，C2-神经酰胺下调HNF-1的表达，奥替普拉上调HNF-1的表达；APAP导致GSTA1 mRNA及蛋白表达极显著降低，HNF-1可正向调节GSTA1的表达，且GSTA1 mRNA及蛋白表达水平的变化与HNF-1的趋势相一致。在转染质粒pGSTA1-LUC的细胞中，APAP导致GSTA1转录活性极显著降低，应用C2-神经酰胺GSTA1转录活性明显下降，应用奥替普拉GSTA1转录活性明显升高，GSTA1转录活性变化与HNF-1表达水平变化趋势相一致；在转染质粒pGSTA1-HNF-1-LUC的肝细胞中，结合位点HRE突变后，HNF-1表达水平的升高与降低对GSTA1的转录活性无明显影响，因此，在APAP诱导肝损伤中，HNF1对GSTA1表达的调节机制是通过与GSTA1启动子中的结合位点HRE作用来实现的。在APAP诱导的肝损伤中，JNK信号通路被激活，应用JNK抑制剂SP600125可以降低JNK信号通路相关蛋白的磷酸化，增加肝脏中GSTA1的表达量，减轻APAP引起的肝损伤和细胞凋亡；HNF-1与JNK信号通路对GSTA1的表达都有调节作用，GSTA1是肝损伤的一个敏感指标。以上研究不仅明确了药物性肝损伤中HNF-1对GSTA1表达的调节作用及调节机制，探明了HNF-1、JNK与GSTA1在药物性肝损伤中的关系，也为保肝药物的研发提供新思路。