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

Abstract

Objective To research the expression of transcription factors kruppel-like transcription factor 2 (KLF2) and nuclear factor-erythroid-related factor 2 (Nrf2) in rats′ airway epithelial cells related by smoking, and define the regulated function of target genes γ-glutamylcysteine synthetase (γ-GCS-HS) feeling of the oxidative stress, and observe the influence of Nrf2and γ-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, γ-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 plasmid+ cigarette smoke extract (CSE) group, KLF2 plasmid group, KLF2 plasmid+ CSE group.Western blot and RT-PCR techniques were used for research protein and its mRNA expressions of KLF2, Nrf2, γ-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、γ-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 cells, the mRNA and protein levels of Nrf2 and γ-GCS-HS were observably increased compared with blank plasmid group in rats′ 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<0.05). mRNA and protein levels of Nrf2, γ-GCS-HS in KLF2 plasmid+ 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 γ-GCS-HS. Key words: Cigarette smoke extract; Kruppel-like transcription factor 2; Nuclear factor-erythroid-related factor 2; γ-glutamylcysteine synthetase