论文部分内容阅读
目的:探讨1型多聚二磷酸腺苷核糖合成酶(PARP-1)对去甲肾上腺素(NE)诱导培养的大鼠心脏成纤维细胞基质金属蛋白酶(MMP)-1,MMP-9及金属蛋白酶组织抑制剂(TIMP)-1表达的调节作用及其机制。方法:①培养乳鼠心脏成纤维细胞,10μmol/LNE刺激细胞24h,使用实时定量PCR法检测MMP-1、MMP-9及TIMP-1的基因表达水平;使用PARP抑制剂3-氨基苯甲酰胺(3-aminobenzamide,3AB)后,观察PARP-1对上述基因表达的影响。②检测心脏成纤维细胞内活性氧(ROS)水平、PARP酶活性的变化。③采用凝胶阻滞实验检测心脏成纤维细胞内转录因子AP-1的DNA结合能力,研究PARP-1对AP-1DNA结合能力的影响。结果:NE诱导心脏成纤维细胞内MMP-1,MMP-9及TIMP-1的基因表达水平明显增加。细胞内ROS产生增加,PARP酶被激活。核内转录因子AP-1的DNA结合能力明显增强。PARP抑制剂3AB可明显减少NE诱导的MMP-1、MMP-9及TIMP-1的基因表达水平,同时显著抑制AP-1的DNA结合能力。使用抗氧化剂vitC减少ROS产生,抑制了NE诱导的PARP-1活性增加及AP-1的DNA结合,进而显著降低了NE诱导的MMP-1、MMP-9及TIMP-1的基因表达水平。结论:NE刺激心脏成纤维细胞内ROS产生明显增多,大量的ROS激活了PARP使其酶活性显著增高,PARP通过调节转录因子AP-1的DNA结合调控了MMP-1,MMP-9及TIMP-1的基因表达。PARP可能是心脏纤维化过程中的重要调节机制之一。
Objective: To investigate the effect of PARP-1 on the expression of matrix metalloproteinase (MMP-1) and MMP-9 in rat cardiac fibroblasts induced by norepinephrine (NE) Modulation of proteasome inhibitor (TIMP) -1 expression and its mechanism. Methods: ① The cultured rat cardiac fibroblasts were cultured in vitro and the cells were stimulated with 10μmol / LNE for 24 hours. The mRNA expression of MMP-1, MMP-9 and TIMP-1 were detected by real-time quantitative PCR. PARP inhibitor 3-aminobenzamide (3-aminobenzamide, 3AB), observed PARP-1 on the expression of these genes. ② The levels of reactive oxygen species (ROS) and PARP activity in cardiac fibroblasts were detected. (3) The DNA binding capacity of AP-1 in cardiac fibroblasts was detected by gel blocking assay, and the effect of PARP-1 on AP-1 DNA binding ability was investigated. Results: The gene expression of MMP-1, MMP-9 and TIMP-1 in NE-induced cardiac fibroblasts increased significantly. Intracellular ROS production increases and PARP enzyme is activated. DNA binding capacity of nuclear transcription factor AP-1 is significantly enhanced. 3AB, a PARP inhibitor, significantly reduced the gene expression of MMP-1, MMP-9 and TIMP-1 induced by NE and significantly inhibited the DNA binding ability of AP-1. Antioxidant vitC reduced the production of ROS, inhibited NE-induced increase of PARP-1 activity and DNA binding of AP-1, and then significantly reduced the gene expression of MMP-1, MMP-9 and TIMP-1 induced by NE. Conclusion: NE stimulated ROS production in cardiac fibroblasts significantly increased, a large number of ROS activated PARP to significantly increase its activity, PARP regulation of transcription factor AP-1 DNA regulation of MMP-1, MMP-9 and TIMP- 1 gene expression. PARP may be one of the important regulatory mechanisms in cardiac fibrosis.