[Effect of Krüppel like zinc finger transcription factor 2 on γ-glutamylcysteine synthetase in bronchial epithelial cells of chronic obstructive pulmonary disease].

Objective: To research the regulation effects of Krüppel like zinc finger transcription factor 2 (KLF2) on γ-glutamylcysteine synthetase (γ-GCS) in airway epithelial cells of chronic obstructive pulmonary disease (COPD). Methods: (1) Human specimen experiment: lung tissue of pulmonary lobectomy patients with lung cancer with or without COPD was collected from Department of Thoracic Surgery of Hunan Cancer Hospital from December 2008 to December 2009. The patients were divided into COPD group and control group without COPD. The levels of KLF2, γ-GCS mRNA and protein expression in lung tissues were measured by immunohistochemistry and in situ hybridization (ISH). Then, the correlation between KLF2 and γ-GCS mRNA and protein expression levels were analyzed, as well as the correlation between KLF2 or γ-GCS protein and smoking index, percentage of forced expiratory volume in one second to predicted value (FEV1 %), percentage of forced expiratory volume in one second to forced vital capacity (FVC/FEV1 ). (2) Animal experiment: the primary bronchial epithelial cells of rats were extracted by enzyme digestion. After 6 hours of incubation with 10% tobacco smoke extract (TSE), cellular glutathione (GSH) was measured by enzyme linked immunosorbent assay (ELISA) method. The cells were transfected by specific inhibitor of KLF2 through the liposom, which inhibited the protein expression of KLF2. Then, the cells were divided into KB group (blank control group without any treatment), KB+ TSE group (treated with TSE), NC group (control group transfected with miRNA), NC+ TSE group (treated with miRNA and TSE), 92a group (transfected with KLF2 inhibitor), 92a+ TSE group (treated with KLF2 inhibitor transfection and TSE) based in the treatment. After that, the changes of KLF2 and γ-GCS mRNA and protein expression in the cells of each group were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method. Results: (1) Human specimen experiment: The expressions of KLF2 mRNA, protein and γ-GCS mRNA, protein in the lung tissue of COPD patients were strong positive and higher than those in control group (0.32±0.04 vs 0.19±0.03, 0.35±0.05 vs 0.22±0.03; 0.28±0.03 vs 0.16±0.03, 0.31±0.05 vs 0.21±0.03; all P <0.01). Linear correlation analysis showed that KLF2 mRNA and protein were positively correlated with γ-GCS mRNA and protein ( r =0.705, 0.722; both P <0.01). The KLF2 and γ-GCS protein were positively correlated with smoking index, FEV1 % and FEV1 /FVC ( r =0.552, 0.728, 0.670, and r =0.631, 0.727, 0.657; all P <0.01). (2) Animal experiment: The level of GSH in KB+ TSE group was significantly higher than that in KB group[(28.05±2.04) vs (7.27±0.33) nmol/mg, P <0.01]. The KLF2 mRNA, protein and γ-GCS mRNA, protein in KB+ TSE group (1.715±0.026, 1.842±0.028 and 2.117±0.067, 1.879±0.065) were higher than those in KB group (1.130±0.017, 1.177±0.033 and 1.378±0.053, 1.177±0.042; all P <0.05), and those in 92a group (0.472±0.028, 0.634±0.025 and 0.582±0.025, 0.554±0.021) were significantly lower than those in KB group, NC group (1.047±0.056, 1.092±0.045 and 1.303±0.037, 1.252±0.037), and those in TSE+ 92a group (0.262±0.017, 0.288±0.017 and 0.337±0.022, 0.321±0.022) were significantly lower than those in KB+ TSE group, 92a group and NC+ TSE group (1.576±0.036, 1.646±0.066 and 1.948±0.093, 1.843±0.078) (all P <0.05). Conclusion: KLF2 exerts antioxidative effect by regulating the expression of γ-GCS in the bronchial epithelial cells of chronic obstructive pulmonary disease.