论文部分内容阅读
红系衍生的核因子相关因子2(Nrf2)是调节抗氧化基因表达的关键转录因子.γ-谷氨酰半胱氨酸合成酶(γ-GCS)是肺内主要的抗氧化基因,但Nrf2如何调节γ-GCS表达目前仍不清楚.该文采用香烟烟雾提取物(CSE)诱导大鼠气道上皮细胞为研究对象,探索Nrf2调节γ-GCS基因表达机制.细胞免疫荧光显示,Nrf2蛋白质在CSE处理1、3和6h组,主要在胞核中表达.细胞免疫化学与Western印迹显示,γ-GCS蛋白质在CSE1、3、6h组表达明显增强,其中Western印迹结果高于对照组(P<0.05);Nrf2胞核蛋白质表达增强.p-aPKCι/ζ蛋白质在CSE1、3h组表达增强,与对照组相比差异显著(P<0.05).RT-PCR结果表明,γ-GCSmRNA在CSE1、3、6h组表达明显高于对照组(P<0.05).γ-GCS活性在CSE1、3、6h组增高并高于对照组(P<0.05).GSH含量在CSE3、6h组明显高于对照组(P<0.05).预先加入aPKCι/ζ抑制剂RO813220,Nrf2胞浆蛋白质表达增强,GSH含量、p-aPKCι/ζ蛋白质、γ-GCS蛋白质与其mRNA和活性均明显低于CSE3h组(P<0.05).相关性分析显示Nrf2与γ-GCS、γ-GCS活性、p-aPKCι/ζ呈正相关,p-aPKCι/ζ与Nrf2、γ-GCS、γ-GCS活性呈正相关(P<0.05).以上结果表明,CSE可能通过aPKCι/ζ-Nrf2信号通路调节γ-GCS的表达水平和活性.
The erythroid-derived nuclear factor-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes.γ-glutamylcysteine synthetase (γ-GCS) is the major antioxidant gene in lung, but Nrf2 How to regulate the expression of γ-GCS remains unclear.In this paper, cigarette smoke extract (CSE) -induced airway epithelial cells of rats as the research object to explore Nrf2 regulate γ-GCS gene expression mechanism.Cell immunofluorescence showed that Nrf2 protein in The expression of γ-GCS protein in CSE-1, 3, 6h groups was significantly enhanced by CSE treatment for 1, 3, and 6h, and mainly expressed in the nucleus.Cell immunochemistry and Western blotting showed that the expression of γ-GCS protein in CSE1,3,6h group was higher than that in control group 0.05). The expression of nuclear factor of Nrf2 was enhanced.The expression of p-aPKCι / ζprotein in CSE1,3h group was significantly higher than that in control group (P <0.05) .RT-PCR results showed that the expression of γ-GCSmRNA in CSE1,3 (P <0.05) .Gamma-GCS activity increased in CSE1,3,6h group and higher than that in control group (P <0.05) .GSH content in CSE3,6h group was significantly higher than that in control group (P <0.05) .Compared with RO813220, the protein expression of Nrf2 in cytoplasm increased, GSH content, p-aPKCι / ζ protein , Γ-GCS protein and its mRNA and activity were significantly lower than CSE3h group (P <0.05) .Correlation analysis showed that Nrf2 was positively correlated with γ-GCS, γ-GCS activity, p-aPKCι / ζ, p-aPKCι / Γ-GCS, γ-GCS activity (P <0.05) .This result indicated that CSE may regulate the expression level and activity of γ-GCS through aPKCι / ζ-Nrf2 signaling pathway.