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.
【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To investigate the effects of bursopentin ( BP5) on expression of extracellular matrix in human lung fibroblasts ( HLFs) and its mechanism.Methods HLFs were cultured in vitro and divided into five groups. The cells in the control group were cultured in DMEMwithout TGF-β1 or BP5. The cells in TGB-β1 treatment group were cultured in DMEMcontaining 5 μg/L TGF-β1 . While in three TGF-β1 + BP5 treatment groups, the cells were cultured in DMEM containing 5 μg/L TGF-β1 and simultaneously intervened with BP5 at three different concentrations ( 2. 5 μg/mL, 5 μg/mL, and 10 μg/mL respectively) . The expression of α-SMA was detected using a fluorescent-labeling strategy. The expressions of Collagen-Ⅰ, p-Smad2/3, p-Smad3, and Smad7 proteins were measured by Western blot. Results The cells in the TGF-β1 treatment group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In the TGF-β1 treatment group, the expressions of collagen-Ⅰ( 1. 402 ±0. 158 vs. 0. 605 ±0. 367) , p-Smad2/3 ( 1. 457 ±0. 111 vs. 0. 815 ±0. 039) , and p-Smad3 ( 1. 320 ±0. 147 vs. 0. 623 ±0. 128) increased with statistical significance ( P lt; 0. 01) . Meanwhile the expression of Smad7 reduced ( 0. 614 ±0. 107 vs. 0. 865 ±0. 063, P lt;0. 05) . But in the TGF-β1 + BP5 treatment groups, over-expressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by BP5, especially at the BP5 concentration of 10 μg/mL ( collagen-Ⅰ: 0. 718 ±0. 049 vs. 1. 402 ±0. 158; p-Smad2 /3: 0. 696 ±0. 031 vs. 1. 457 ±0. 111; p-Smad3: 0. 766 ±0. 006 vs. 1. 320 ±0. 147; all P lt; 0. 01) . Otherwise, the up-regulation of Smad7 ( 1. 237 ±0. 173 vs. 0. 614 ±0. 107) was found.Conclusions Bursopentin can reduce the expressions of collagen-Ⅰ and α-SMA protein of fibroblast stimulated by TGF-β1 , maybe through inhibiting TGF-β1 /Smads transduction pathway. It is suggested that bursopentin may have intervention effect on pulmonary fibrosis.
Objective To investigate the effects of curcumin on oxidative stress in the co-culture system including human fetal lung fibroblasts and A549 cells, and discuss the potential and protective mechanism of the prophylactic effect of curcumin on pulmonary fibrosis. Methods The human fetal lung fibroblasts co-cultured with A549 cells were divided into five groups. The cells in the control group were cultured in DMEM without TGF-β1 or curcumin. The cells in the TGF-β1 group were cultured in DMEM containing 5 ng/mL TGF-β1 . In three TGF-β1 + cucurmin treatment groups, the cells were cultured in DMEM containing 5 ng/mL TGF-β1 and three different concentration of curcumin( 5, 10, 20 μmol /L, respectively) . ELISA was used to analyze the content of TNF-α. Serum level of MDA and SOD were tested by spectrophotometric analysis. Intracellular ROS production was detected by flow cytometry. NF-κB was measured by western blot. Results The serum MDA, intracellular ROS, the content of TNF-αand NF-κB protein expression in the TGF-β1 group were significantly increased while the activity of SOD was significantly decreased( P lt; 0. 01) , suggesting that the oxidative level of human fetal lung fibroblasts was obviously increased after TGF-β1 stimulation. After intervening by different concentration of curcumin, the serum MDA, intracellular ROS, content of TNF-αand NF-κB were significantly decreased while the activity of SOD was obviously increased( P lt;0.01) . Conclusion Low concentration of curcumin can reduce the oxidative level of human fetal lung fibroblasts co-cultured with A549 after TGF-β1 stimulation, and significantly increase the level of SOD, implying that curcumin may intervene pulmonary fibrosis by reduce oxidative level.
Objective To explore the prognostic value of preoperative pulmonary ventilation function for postoperative survival of patients with non-small cell lung cancer ( NSCLC) . Methods 146 NSCLC patients who underwent cured lung surgical resection between January 1, 2003 and December 31,2008 in Nanjing Drum Tower Hospital were recruited in the study. Pulmonary ventilation function was obtained preoperatively for each patient, including vital capacity ( VC) , forced vital capacity ( FVC) , forcedexpiratory volume in 1 second ( FEV1 ) , FEV1 /FVC, and peak expiratory flow ( PEF) . The effects of the above lung function variables on postoperative survival were evaluated by both univariate and multivariate Cox proportional hazard models. Kaplan-Meier method was used to assess the survival probabilities betweendifferent groups.Results The median survival time after surgery was 31. 0 months ( 95% CI 22. 55-39. 45) . VC% pred, FVC% pred and FEV1% pred showed significant associations with the risk of mortality in the NSCLC patients after surgery ( hazard ratios 0. 979-0. 981, P lt; 0. 05) . The survival time after surgery was significantly shorter in the patients with VC ≤ 80% predicted compared to those with VC gt; 80% predicted ( median survival time: 31. 0 months vs. 34. 0 months) . The same difference could be found between the patients with FVC≤80% predicted and those with FVC gt; 80% predicted ( median survival time: 27. 0 months vs. 43. 0 months) . There was also significant difference in median survival between the patients with FEV1 ≤80% predicted and those with FEV1 gt; 80% predicted ( median survival time: 17. 0 months vs. 44. 0 months) . Conclusion Preoperative pulmonary ventilation function parameters may be used to informclinical decisions and indicate the prognosis of NSCLC patients after surgery.