高铜对于肝细胞Wnt/β-catenin信号通路的影响

Effects of high copper on Wnt/β-catenin signaling pathway in hepatocytes

目的研究高铜对于Wnt/β-catenin信号通路及氧化应激的影响。方法不同剂量的CuSO_4培养BRL-3A细胞;用MTT法检测不同剂量CuSO_4作用不同时间点对BRL-3A细胞生长和增殖的影响;用流式细胞仪检测细胞内活性氧的产生和线粒体膜电位的变化;用酶标仪检测丙二醛的含量和超氧化物歧化酶的活性;Western Blot法检测Wnt/β-catenin信号通路相关蛋白表达情况。结果 (1)MTT实验结果显示CuSO_4在一定时间和浓度下对BRL-3A细胞生长和增殖有明显抑制作用(P<0.01);(2)流式细胞仪检测实验结果显示CuSO_4可诱导BRL-3A细胞内产生大量的活性氧和显著降低线粒体膜电位(P<0.01);(3)酶标仪检测实验结果显示CuSO_4可诱导MDA含量上升和SOD活性降低(P<0.05);(4)Western Blot法检测实验结果显示CuSO_4可显著降低BRL-3A细胞内总蛋白β-catenin、p-Ser 9-GSK-3β的表达(P<0.01),增加细胞内总蛋白GSK-3β、DKK1、Dishevelled3的表达,以及降低细胞核内蛋白p-(S33+S37)-β-catenin、c-Myc的表达(P<0.01)。结论高铜能诱导肝细胞内线粒体氧化应激并且引起Wnt/β-catenin信号通路失活,从而诱导肝细胞损伤的发生。

Objective To study the effect of high copper on Wnt /β-eatenin signaling pathway and oxidative stress. Methods BRL-3A cells were incubated with different concentrations of CuSO4. The cell growth and proliferation wre assessed using MRR method. The production of reactive oxygen species （ROS） and mitochondrial membrane potential （JC-1） were examined usingf flow cytometry. The content of malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） were detected by microplate reader. The expression of protein was detected by Western Blot. Results （1）The results of MTT showed that CuSO4 inhibited the growth and proliferation of BRL-3A cells in a time- and concentration-dependent manner （P〈0.01）. （2） Flow eytometry results showed that CuSO4 induced a large amount of ROS and significantly decreased the fluorescence intensity of JC-1 in BRL-3A cells （P〈0.01）. （3） Microplate reader results showed that CuSO4 increased the content of MDA and decreased the activity of SOD （P〈0.05）. （4） Western blotting assay showed that CuSO4 significantly decreased the total expression levels of β-catenin and p-Ser 9-GSK-313 protein as well as nuclear levels of p-（S33＋S37）-β-catenin and c-Myc （P〈0.01） and increased expression levels of GSK-3β,DKK1, Dishevelled3 protein in BRL-3A cells. Conclusion High copper can induce oxidative stress and induce Wnt /β- catenin signaling pathway to deactivate liver cells, leading to hepatocellular injury.