Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.
Objective To investigate the expression of transcriptional factors zinc finger Krüppel like transcription factor 2 ( Klf2) and NF-E2 related factor 2 ( Nrf2) /BTB CNC homology 1 ( Bach1) in rat bronchial epithelial cells stimulated by cigarette smoke extract ( CSE) , and explore the regulatingmechanism of γ-glutamylcysteine synthetase ( γ-GCS) expression in the oxidative condition. Methods Rat bronchial epithelial cells were harvested using enzyme digestion method, and intervened by 10% CSE for 6 hours. Then γ-GCS activity was detected by two enzymes method, and the nuclear transfer of Nrf2 /Bach1 in cells was detected by immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction ( RT-PCR) techniques were used for detecting the protein and mRNA expressions of Klf2, Nrf2, Bach1, and γ-GCS in the cells. Results The γ-GCS activity was elevated in the CSE group. Immunohistochemical results showed that Nrf2 translocated from cytoplasm to nucleus in response to stimulation by CSE. On the contrary, Bach1 translocated from nucleus to cytoplasm in the same condition. Western blot results showed that protein levels of Klf2, Nrf2, Bach1, and γ-GCS were higher in the CSE group than those in the control group ( Plt;0.05) . RT-PCR results were the same as the Western blot results ( Plt;0.05) . Linear correlation analysis showed that γ-GCS expression was positively correlated with Klf2, Nrf2, and Bach1 ( Plt;0. 05) . Conclusion CSE might regulate the expression of γ-GCS through the transcription factors of Klf2, Nrf2, and Bach1.
ObjectiveTo explore the effect of naringenin on the expression of vascular endothelial growth factor (VEGF) released by human fetal lung fibroblasts. MethodsHuman fetal lung fibroblast cells were divided into a control group,a cigarette smoke extract (CSE) group and a naringenin group. Cells in the naringenin group were incubated with different doses of naringenin for 2h. Then the naringenin group and the CSE group were incubated with CSE to adjust the final concentration of naringenin (5 μmol/L,10 μmol/L,and 20 μmol/L) and of CSE(5%). The concentration of VEGF was measured in human fetal lung fibroblasts after cultured for 24h,48h and 72h by enzyme-linked immunoabsorbent assay. Smad3 and p-Smad3 levels were detected by Western blot. ResultsELISA results showed that the CSE can significantly increase the VEGF expression,and naringenin can inhibit the increasing of the VEGF expression. Western blot results showed that the CSE can increase the p-Smad3 expression,and the naringenin can inhibit the increasing of the p-Smad3 expression. ConclusionNaringenin can inhibit phosphorylation of Smad3,and decrease the expression of VEGF released by human fetal lung fibroblasts.
ObjectiveTo investagte the effects of single immunoglobin IL-1 receptor related protein (SIGIRR) on inflammation induced by cigarette smoke extract (CSE) in A549 cells derived from mouse alveolar epithelial cells. MethodsA549 cells were divided into a control group and an over-expressed SIGIRR group. Eukaryotic expression vectors pcDNA3.1(+) constructed with SIGIRR cDNA were transiently transfected into A549 cells, in which SIGIRR was forced to be over-expressed. The expression level of SIGIRR after transfection was detected with Western blot and RT-PCR method. After stimulated by CSE in both groups, the protein level of IL-6 was detected by ELISA, the transcriptional activity of cyclooxygenase-2(COX-2) was detected by dual-luciferase reporter assay system, and the release of reactive oxygen species (ROS) was measured by chemiluminescence method. ResultsThe expression level of SIGIRR increased significantly in A549 cells transfected with SIGIRR vectors. The COX-2 expression and the levels of ROS and IL-6 were significantly increased in the control group after CSE stimulation. Nevertheless, in the over-expressed SIGIRR group, the COX-2 expression and the release of ROS was reduced while the protein level of IL-6 was down-regulated compared with the control group(P < 0.05). ConclusionsUp-regulated SIGIRR expression can suppress the levels of ROS, COX-2 and IL-6 in A549 cells stimulated by CSE. It suggests that SIGIRR can inhibit airway inflammation caused by smoking.