转录因子Nrf2促进内皮细胞存活和肺稳态的机制研究

Mechanism of Nrf2 promoting endothelial cell survival and lung homeostasis

目的探究转录因子Nrf2通过在复氧过程中保护线粒体功能和DNA促进内皮细胞存活和肺稳态的机制。方法根据实验要求,将实验小鼠分为Nrf2基因敲除组(Nrf2-/-)和野生型组(Nrf2+/+),每组10只,培养的细胞为Nrf2 siRNA组。使用改良方法分离小鼠肺血管内皮细胞(pulmonary microvascular endothelial cell,MLVEC)。麻醉后,向小鼠右心室灌注磷酸缓冲盐溶液,以清除肺部血液。将周围的胸膜下肺组织切成小块,并在含有20%胎牛血清,25 mmol/L HEPES,3.7 g/L NaHCO3、5 mg/ml肝素,1 mg/ml氢化可的松,80 mg/ml内皮细胞生长补充剂的改良伊格尔培养基中培养内皮细胞。酶标仪检测活性氧(reactive oxygen species,ROS)含量,活性氧分析试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)水平,酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)试剂盒测定谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)活性;蛋白印迹分小鼠血红素加氧酶-1(heme oxygenase-1,HO-1)、醌氧化还原酶1(quinone oxidoreductase 1,Nqo1)的蛋白表达;萤光素-萤光素酶测定法检测腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)释放,细胞色素C免疫测定试剂盒测量线粒体中的细胞色素C水平;MTT法比较细胞增殖情况。结果Nrf2基因敲除组较野生型组ROS水平升高(P<0.05),SOD、GPx水平降低(P<0.05),HO-1、Nqo1的蛋白表达降低(P<0.05),细胞色素C水平升高(P<0.05),ATP合成量、线粒体膜电位(mitochondrial membrane potential,MMP)电位水平降低(P<0.05)。Nrf2 siRNA组较正常培养细胞组细胞凋亡率升高(P<0.05),细胞组细胞活力降低(P<0.05)。结论Nrf2在机体复氧过程中可降低氧化应激反应,抑制线粒体功能受损,促进内皮细胞的生存从而保护肺稳态。

Objective To investigate the action mechanism by which the transcription factor Nrf2 can promote endothelial cell survival and lung homeostasis by protecting mitochondrial function and DNA during reoxygenation.Methods The wild-type(Nrf2+/+)and Nrf2 deficient(Nrf2-/-)mouse CD-1/ICR strains were used in this study.The mouse pulmonary vascular endothelial cells(MLVEC)were isolated by using a modified method.The endothelial cells were cultured in DMEM supplemented medium with endothelial cell growth supplements.Nrf2 siRNA was transfected with cultured siRNA by using Xfect siRNA transfection reagent.The ROS levels were detected by ELISA,and SOD levels were detected by a reactive oxygen analyzer kit,and the GPx activity was determined by ELISA.Moreover the expression levels of mouse HO-1 and Nqo1 protein were detected by Western BLOT,and the ATP release was detected by luciferin-luciferase assay.The cytochrome C immunoassay kit was used to measure cytochrome C levels in mitochondria,and the cell proliferation was detected by MTT assay.Results The ROS levels in Nrf2 knockout group were significantly higher than those in wild type group(P<0.05),however,the levels of SOD and GPx as well as the expression levels of HO-1 and Nqo1 protein in Nrf2 knockout group were significantly lower than those in wild type group(P<0.05).The levels of cytochrome C,and the ATP synthesis and MMP potential levels as well as cell apoptosis rate in Nrf2 knockout group were significantly higher than those in wild type group(P<0.05).However the cell viability in Nrf2 knockout group was significantly lower than that in wild type group(P<0.05).Conclusion Nrf2 can reduce oxidative stress,inhibit mitochondrial function,promote endothelial cell survival,so as to protect lung homeostasis during reoxygenation.