KLF2对吸烟相关大鼠气道上皮细胞氧化应激反应的调控

Regulation of KLF2 on oxidative stress in airway epithelial cells of smoking related rats

目的 通过研究转录因子锌指Krüppel样转录因子2 (KLF2)及红系衍生核因子相关因子2 (Nrf2)在吸烟相关大鼠气道上皮细胞中的表达情况,明确其感受氧化应激对靶基因γ-谷氨酰半胱氨酸合酶 (γ-GCS-HS)的调控作用;观察 KLF2质粒转染细胞后分别对 Nrf2和γ-GCS-HS表达水平的影响。方法 将22只雄性SD大鼠按随机数字表法分为对照组和慢性阻塞性肺疾病 (COPD)模型组,每组11只,采用每日熏香烟和2次气管内滴入脂多糖 (LPS)复制COPD大鼠模型。逆转录-聚合酶链反应 (RT-PCR)检测肺组织 KLF2、Nrf2、γ-GCS-HSmRNA 的表达;蛋白质免疫印迹(Westernblot)分别检测KLF2、Nrf2、γ-GCS-HS蛋白表达水平。给予KLF2质粒转染细胞使基因过表达,分为4组:空白质粒组、空白质粒+香烟烟雾提取物 (CSE)组、KLF2质粒组、KLF2质粒+CSE组,采用RT-PCR、Westernblot观察细胞中 KLF2、Nrf2、γ-GCS-HS的 mRNA及蛋白表达变化。结果 大鼠肺功能指标 [第0 3秒用力呼气容积 (FEV0 3)、FEV0 3/用力肺活量、呼气峰流速]在COPD组较对照组明显降低 (P 值均<0.01),COPD组大鼠肺部病理改变符合COPD的形态学改变。RT-PCR及 Westernblot结果显示:COPD组 KLF2、γ-GCS-HSmRNA水平均显著高于对照组 (P值均<0.05),但2组Nrf2mRNA水平比较,差异无统计学意义。Westernblot结果显示:COPD组KLF2、Nrf2及γ-GCS-HS蛋白质的相对表达量均明显高于对照组 (P 值均<0.05)。KLF2过表达质粒成功转染细胞后,大鼠气道上皮细胞中Nrf2、γ-GCS-HSmRNA及蛋白质表达水平均显著高于转染空白质粒组 (P 值均<0.01)。空白质粒+CSE组中Nrf2、γ-GCS-HSmRNA与蛋白质水平均明显高于空白质粒组 (P 值均<0.05)。KLF2质粒+CSE组中Nrf2、γ-GCSmRNA与蛋白质水平均明显高于KLF2质粒组 (P 值均<0.05)。KLF2质粒+CSE组中 Nrf2、γ-GCS-HSmRNA与蛋白质水平均明显高于空白质粒+CSE组 (P 值均<0.05)。结论 吸烟可以诱发氧化应激反应,KLF2参与氧化应激反应并通过增加抗氧化因子Nrf2、γ-GCS-HS的表达对抗氧化应激。

Objective To research the expresston of transcription factors kruppel-like transcription factor 2 (KLF2)and nuclear factor-erythroid-related factor 2 (Nrf2)in ratsr airway epithelial cells related by smoking,and define the regulated function of target genes 7-glutamylcysteine synthetase (7-GCS-HS) feeling of the oxidative stress,and observe the influence of Nrf2and 7-GCS-HS expression level by transfected KLF2 plasmid into cells.Methods 22 SD adult male rats were randomly divided into control group and chronic obstructive pulmonary diseases (COPD)group,and each group contained 11 rats.The COPD model was established by cigarette smoking and intratracheal instillation of lipopolysaccharide (LPS)twice.Reverse transcription-polymerase chain reaction (RT-PCR),and Western blot techniques were used to detect the mRNA and protein expressions of KLF2,Nrf2,7-GCS-HS in the rats lung.To transfect KLF2 plasmid into cells in order to its gene overexpression,they were divided into four groups : blank plasmid group,blank pIasmidq-cigarette smoke extract (CSE)group,KLF2 plasmid group,KLF2 piasmid+CSE group.Western blot and RT-PCR techniques were used for research protein and its mRNA expressions of KLF2,Nrf2,7-GCS-HS in these cells.Results The results of rats pulmonary function showed:forced expiratory volume in first 0.3 second (FEV0 3 ),percentage of forced expiratory volume in first 0.3 second/forced vital capacity (FEV0 3/FVC),and peak expiratory flow (PEF)were all obviously decreased in COPD rats compared with control rats (all P <0.01).Lung pathological changes in COPD model group conformed morphological character of COPD.RT-PCR and Western blot results showed that the mRNA and protein levelsexpressions of KLF2,7-GCS-HS mRNA in COPD group were conspicuously higher than those in control group (all P <0.05).Western blot results showed that lung tissue KLF2, Nrf2 ,γ-GCS-HS protein levels were higher in COPD rats than that in control group (all P <0.05).After KLF2 overexpression plasmid successed transfecting ceils,the mRNA and protein levels of Nrf2 and γ-GCS-HS were observably increased compared with blank plasmid group in ratsr airway epithelial cells (all P <0.01).In blank plasmid+CSE group,mRNA and protein levels of Nrf2 and γ-GCS-HS were all increased compared with blank plasmid (all P<0.05).In KLF2 plasmid+CSE group,mRNA and protein levels of Nrf2,γ-GCS-HS were all markedly elevated compared with KLF2 plasmid group (all P d0.05). mRNA and protein levels of Nrf2,γ-GCS-HS in KLF2 plasmld+CSE group were all higher than those in blank plasmid+CSE group (all P <0.05).Conclusions Smoking can induce oxidative stress,KLF2 participates in oxidative stress and protects against oxidative stress by increasing the expression of antioxidant factors Nrf2 and 7-GCS-HS.