Airway epithelial cells increase macrophage chemotaxis and inflammatory cytokine secretion under hypoxic conditions / 中南大学学报(医学版)

To investigate the effects of airway epithelial cells on macrophages chemotaxis and inflammatory cytokine expression under hypoxic conditions.
 Methods: Human bronchial epithelial cells (HBE) treated with different concentrations (0, 100, 200, 400, 800 μmol/L) of CoCl2 or transfected with HIF-1α siRNA were co-cultured with THP-1-derived M1 macrophages or M2 macrophages. The chemotactic effects on macrophages were analyzed by Transwell assay. The levels of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were detected by ELISA, and HIF-1α or Cav-1 mRNA expression in HBE or macrophages was detected by RT-qPCR.
 Results: HBE cells promoted macrophages chemotaxis in a time- and concentration-dependent manner. Compared to un-transfected group, the chemotactic ability of HBE transfected with HIF-1α siRNA was significantly weakened (P<0.01). Under the same culture conditions, the chemotaxis of M2 macrophages was greater than that in THP1-derived M1 macrophages. The concentrations of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were increased in a time-and concentration-dependent manner. The concentrations of TNF-α and IFN-γ were increased further after co-culturing for 8 and 12 h; while IL-4, IL-13 and IL-10 concentrations were increased further during 24 h of co-culture. The levels of cytokines in the supernatants of macrophages co-cultured with HBE and transfected with HIF-1α siRNA were significantly lower than those in un-transfected cells (P<0.05 or P<0.01). The reduction of TNF-α or IFN-γ was more obvious. The expression of HIF-1α or Cav-1 mRNA in HBE or macrophages was increased in a concentration-dependent manner after 8 or 12 h co-culture, which was significantly reduced when HBE was transfected with HIF-1α siRNA.
 Conclusion: Airway epithelial cells can enhance macrophages chemotaxis and pro-inflammatory cytokines expressions under hypoxic condition. HIF-1α and Cav-1 may be the important mediators in these processes.

Heterogeneity of airway macrophage in different clinical phenotypes of COPD patients with frequent or infrequent exacerbations / 中南大学学报(医学版)

To compare the clinical features and the heterogeneity of macrophages in different clinical phenotypes of chronic obstructive pulmonary disease (COPD) patients with frequent or infrequent exacerbations.
 Methods: Clinical characteristics of eighty COPD patients with chronic bronchitis (CB), emphysema (EM) or asthma-COPD overlap (ACO) phenotypes suffered from acute exacerbation were analyzed. The expressions of CCL3 and CD163 in sputum macrophages were detected by flow cytometry. The expressions of HIF-1α and Cav-1 in sputum macrophages were detected by quantitative PCR (qPCR).
 Results: The age, forced expiratory volume in one second (FEV1)/forced vital capacity (FVC), sputum bacteria positive rate, COPD Assessment Test (CAT) score, and Modified Medical Research Council (mMRC) score between the patients with FE and iFE were significantly different (P0.05), while CD163 was slightly raised (P>0.05). Meanwhile, HIF-1α levels were slightly elevated (P>0.05), while Cav-1 expression was significantly increased (P0.05). At the same time, the expression of HIF-1α (P<0.01) and Cav-1(P<0.05) also increased significantly. There was a significant negative correlation between CCL3 and HIF-1α or Cav-1 in all FE and FE patients with CB phenotype. CD163 was only positively correlated with HIF-1α in those patients and FE patients with EM phenotype. There was a significant negative correlation between CCL3 and HIF-1α in FE patients with ACO phenotype, while CD163 was significantly positively correlated with HIF-1α.
 Conclusion: The clinical features of FE or iFE patients with CB, EM or ACO phenotype are different, and M2 in induced sputum from FE patients are dominant. HIF-1α may play a key role in the polarization process.

Analysis of clinical features and inflammatory factors in induced sputum in common phenotypes of COPD with frequent exacerbations / 中国综合临床

Objective To compare the clinical characteristics of chronic bronchitis ( CB),emphysema (EM ), asthma - chronic obstructive pulmonary disease overlapping syndrome ( ACOS ) with frequent exacerbations ( FE ) or infrequent exacerbations ( iFE ) and induced sputum inflammatory cells and the heterogeneity of the transmitter. Methods Ninety-one cases of chronic obstructive pulmonary disease( COPD) with acute exacerbation were divided into CB,EM or ACOS phenotype,among which 44 were frequent,and 47 were non frequent. The clinical data,induced sputum inflammatory cells,interferon-γ(IFN-γ),tumor necrosis factor-α ( TNF-α ), interleukin ( IL )-4, IL-13 were analyzed. Results The FEV1% was ( 47 ± 13. 1 )%, significantly lower than that of non frequent episodes (( 56. 2 ± 10. 2)%),and the difference was statistically significant(P＝0.049).The FEV1/FVC% was (54.3±9.3)%,significantly lower than that of non frequent episodes (60. 1±7. 3)%,and there was a significant difference between them ( P＝0. 001) . The proportion of patients with GOLD III and IV,the percentage of neutrophils in induced sputum,tumor necrosis factor -α(TNF-α) and interferon-γin the patients with frequent episodes were significantly higher than those with non frequent episodes (P＜0. 05). Among them,FEV1/FVC% and TNF-αwere independent risk factors for COPD patients (P＝0. 032, 0. 021) . The FEV1% of patients with CB phenotypic frequent episodes were ( 47. 9 ± 14. 9 )%, significantly lower than that of non frequent episodes ((57. 2±10. 9)%)(P＝0. 000),and FEV1/FVC% was (53. 4± 9. 5)% in patients with CB frequent episodes,significantly lower than that of non frequent episodes ((60. 3±6. 9)%),and the difference was statistically significant (P＝0. 022),while the level of N%,TNF-α in induced sputum were significantly higher in CB phenotype subjects with FE than those in subjects with iFE(P＜0. 01). Patients with frequent episodes of emphysema had longer duration of disease (P＜0. 05),lower FEV1%and FEV1/FVC%(P＜0. 05),the proportion of GOLD III patients and the induced sputum TNF-αwere higher, but there was no significant difference in the number and proportion of phlegm inflammatory cells,interferon-γ, interleukin 4 and interleukin 3. The level of GOLD III and the IL-13 level of induced sputum in patients with frequent ACOS phenotype were significantly higher than those in patients with non frequent episodes (P＜0. 05) . Conclusion The lung function,the severity of the disease,the course of the disease,and the percentage of sputum neutrophils,tumor necrosis factor-α,or interleukin 13 are helpful in diagnosing patients with high risk of frequent episodes.

ERCC1-shRNA inhibits the proliferation and promotes the apoptosis of lung cancer A549/DDP cells / 医学研究生学报

Objective Studies show that the ERCC1 gene may be involved in secondary cisplatin resistance.This article aims to investigate the effects of shRNA targeting silencing excision repair cross-complementation group 1 (ERCC1-shRNA) on the proliferation and apoptosis of lung cancer A549/DDP cells treated with different concentrations of cisplatin.Methods Lung cancer A549/DDP cells were divided into a negative control, a blank control, an ERCC1-shRNA1, and an ERCC1-shRNA2 group.Human interfering RNA (RNAi) targeting the human ERCC1 gene was constructed and transfected into the A549/DDP cells using Lipofectamine 2000.The mRNA and protein expressions of ERCC1 in the A549/DDP cells were detected by real-time PCR and Western blot respectively, the proliferation-inhibition rate was assessed by MTT, and their cell cycle and apoptosis were determined by flow cytometry.Results ERCC1-shRNA was successfully constructed and transfected into the A549/DDP cells.Both the mRNA and protein expressions of ERCC1 were significantly lower in the ERCC1-shRNA1 (0.20±0.04 and 0.24±0.10) and ERCC1-shRNA2 (0.47±0.28 and 0.37±0.11) than in the negative control (0.96±0.12 and 1.32±0.13) and blank control groups (0.84±0.07 and 1.45±0.23) (P<0.01).Compared with the negative and blank control groups, the ERCC1-shRNA1 group showed a significantly decreased IC50 value (16.71±2.33 and 16.69±1.69 vs 7.78±0.54, P<0.01) and an increased proportion of G0/G1 phase cells ([72.87±3.23] and [71.75±4.56] vs [82.99±4.23]%, P<0.05), with the cell cycle arrested in the G0/G1 phase.The apoptosis rate of the cells in the ERCC1-shRNA1 group was remarkably lower after treated with cisplatin at the concentrations of 6.25 and 12.5 μg/mL than at 0 μg/mL ([8.17±0.65] and [11.91±1.41] vs [29.97±3.14]%, P<0.05).Conclusion ERCC1-shRNA can inhibit the proliferation and enhance the apoptosis of A549/DDP cells by silencing the expression of the ERCC1 gene.

Imaging Features of Lung Adenocarcinoma with Epidermal Growth Factor Receptor Gene Mutation / 沈阳医学院学报

With the development of molecular biology,lung cancer targeted therapy for the gene mutation of lung adenocarcinoma brought the gospel,for patients with lung adenocarcinoma who are not for EGFR gene detection,to predict whether there is a genetic mutation by noninvasive methods such as imaging features,and is the prerequisite for the treatment of EGFR-TKI,so as to provide a basis for diagnosis and treatment,in order to improve the survival quality and prognosis of patients with lung cancer.

Clinical Analysis of 25 Cases of Cryptogenic Organizing Pneumonia / 沈阳医学院学报

Objective:To analyze clinical features and imaging characteristics of cryptogenic organizing pneumonia (COP) confirmed by pathological examination, and improve clinicians understanding of the disease and reduce the disease misdiagnosis. Methods:General information, clinical manifestations,laboratory examination and radiographic features of 25 patients with COP confirmed by pathological examination of lung biopsy were collected, and a comprehensive analysis of these data was made. Results:There were 15 males and 10 females in 25 patients.The mean age was (61.4±13.1) years.And 6 cases (24??0%) had smoking history. All patients had no obvious allergic and industrial dust related exposure history. The clinical presentations were nonspecific, and the main clinical symptoms were cough with a small amount of sputum. The most common laboratory tests was counting normal white blood cells and faster blood sedimentation.The main features of chest CT were space occupying lesions and patchy consolidation or ground-glass opacification.Conclusion:The clinical manifestation and laboratory examination of COP lack of characteristic, and the chest CT performance is similar to tumor and lung infection, and the diagnosis of COP depends on pathological examination.In order to reduce misdiagnosis,clinical doctors should improve the awareness and vigilance of the disease.

Hypoxia-induced caveolin-1 up-regulation is involved in migration and in-vasion of lung adenocarcinoma A549 cells / 中国病理生理杂志

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride ( CoCl2 ) on cave-olin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcino-ma A549 cells.METHODS:The concentrations of Cav-1 and hypoxia-inducible factor ( HIF)-1αin pleural effusion of the patients with lung cancer ( MPE) or tuberculous pleurisy ( TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1αinhibitor YC-1.The concentrations of Cav-1 and HIF-1αin the cell supernatants were measured by ELISA.The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respec-tively.RESULTS:The levels of Cav-1 and HIF-1αin MPE were significantly higher than those in TBPE.There was a highly positive correlation between Cav-1 and HIF-1αlevels in the pleural effusion.CoCl2 induced the generation of Cav-1 and HIF-1αin A549 cells in a concentration-and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration-or time-dependent inhibition was ob-served.HIF-1αinhibitor YC-1 concentration-dependently inhibited the generation of HIF-1αand Cav-1 induced by CoCl2 in A549 cells.CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1.CONCLUSION:The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells.A possible role for HIF-1αis indicated in Cav-1 generation.

Effects of simvastatin on inflammation in patients with chronic obstructive pulmonary diseases / 中国综合临床

Objective To investigate the influence of simvastatin on inflammatory indices in nasal lavage,sputum and blood and clinical index in patients with chronic obstructive pulmonary diseases (COPD). Methods Thirty-seven stable COPD patients were randomly divided into simvastatin-treatment group (n=17),orally given simvastatin tablets for 4 weeks in addition to basic therapy,40 mg,qd) and control group (n=20),given usual med-ication). Total cell counts,percentage of leukocytes (N%) and levels of interleukin IL-8,IL-6 in nasal lavage and sputum at pre-post-treatment were compared;Serum C-reactive protein (CRP),total cholesterol (TC),low-density lipoprotein-cholesterol (LDL-C) as well as IL-8,IL-6 concentrations were measured,the variation of lung function,Sino-Nasal Outcome Test 20(SNOT-20) and St George's Respiratory Questionnaire(SGRQ) score were analyzed. Results After the treatment,the nasal lavage and sputum total cell counts,N%,IL-8 and IL-6 levels[nasal lavage: (0.7±0.3)×107/L,(41.1±10.9)%,(105.8±74.5) ng/L,(3.8±1.6) ng/L;sputum: (0.8±0.3)×109/L,(56.6±9.6) %,(2565.5±831.9) ng/L,(109.8±42.3) ng/L] dropped slightly in the simvastatin group com-pared with that at pretreatment [nasal lavage: (0.8±0.3)×107/L,(43.2±10.8) %,(107.6±86.3) ng/L,(4.1±1.9)ng/L;sputum: (0.8±0.3)×109/L,(58.1±9.3)% ,(2659.4±885.2) ng/L,(111.8±46.6) ng/L] (P>0.05) ;There were significant decreases in serum CRP [(4.3±3.7) mg/L vs (2.6±1.8) mg/L],IL-6 [(4.8±2.0)ng/L vs(4.7±1.9)ng/L] ,TC[(4.2±1.0) mmol/L vs(3.7±0.8)mmol/L] ,LDL-C[(2.4±0.5) mmol/L vs (2.2±0.5)mmol/L] (P>0.05) ;IL-8 concentrations in serum were lower gently[(6.2±1.8) ng/L vs (6.4±1.9) ng/L] (P>0.05). Significant change of simvastatin treatment on SGRQ was only reflected in the symp-tom score [pre-post-treatment:39.6±10. 8 vs 32.3±11.6,P<0.05,respectively],while other observation items (SNOT-20,FEV1%,FEV1/FVC) changed not notably (P>0.05). No marked changes in inflammatory markers and quality of life scores,lung function were observed in control group (P>0.05). Conclusion Simvastatin may be as-sociated with the potential to alleviate systemic inflammation and relieve symptoms in COPD patients.

Signal mechanism of endothelin-1-mediated activation of airway fibroblasts induced by injured airway epithelial cells / 中国病理生理杂志

AIM: To explore the effects of p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt on interleukin (IL)-6, the endothelin (ET)-1-mediated process of airway fibroblast activation induced by injured human bronchial epithelial cells (HBE). METHODS: Human primary cultured airway fibroblasts were co-cultured with HBE pre-treated with or without poly-L-arginine (PLA). The procedure was also performed in the presence or absence of p38 MAPK selective inhibitor SB203580, PI3K selective inhibitor LY294002 or ETA receptor blocker BQ123, respectively. Immunostaining, Western blotting or ELISA were used for detecting α-smooth muscle actin (α-SMA) expression, the activities of p38 MAPK and Akt in fibroblasts or IL-6 levels in supernatants of fibroblasts. In addition, fibroblasts were mixed with soluble collagen and cultured with HBE treated as the same mentioned above, the gel contraction was measured by serial area measurements. RESULTS: ET-1 and IL-6 levels [(13.69±1.36) ng/L, (56.7±10.7) ng/L] in the supernatants of fibroblasts cultured with injured HBE were significantly higher than those in the supernatants of fibroblasts cultured with HBE [(3.79±0.64) ng/L, (15.5±3.2) ng/L]. BQ123, SB203580 or LY294002 decreased IL-6 levels [(27.2±3.1) ng/L, (31.5±3.6) ng/L, (41.3±3.2) ng/L] differently in the supernatants of fibroblasts induced by injured HBE. Activation of p38 MAPK preceded Akt in fibroblasts cultured with injured HBE. BQ123 reduced the phosphorylation levels of p38 MAPK and Akt. SB203580 concentration-dependently attenuated Akt phosphorylation, while LY294002 had little effect on p38 MAPK phosphorylation. Fibroblasts expressed more α-SMA after cultured with injured HBE and showed significant increase in the gel contraction compared to fibroblasts cultured with HBE [percentage of gel contraction: (61.2±2.7)% vs (15.4±7.3)%], all these effects were diminished or inhibited by BQ123, SB203580 or LY294002. Furthermore, the effects of BQ123 and SB203580 on decreased gel contraction were stronger than the effect of LY294002. CONCLUSION: ET-1 exerts a key role in the airway fibroblasts activation induced by injured HBE through activating p38 MAPK, PI3K/Akt signaling and promoting IL-6 expression.

Effects of damage on endothelin production in epithelial cells and sub-epithelial fibroblasts / 中国病理生理杂志

AIM: To investigate the level of ET-1 produced by cultured human bronchial epithelial cells (HBECs) under injury and the effects of injured HBECs on ET-1 production in sub-epithelial fibroblasts. The interaction between ET-1 and matrix metalloproteinase-9(MMP-9) was detected in HBECs under damage. The purpose of the study is to evaluate the effect of injured HBECs related to ET-1 release on airway remodeling in asthma. METHODS: ET-1 level was detected in supernatants from cultured HBECs 12 h after being treated with either mechanical scraping or LPS stimulation or mechanical scraping plus LPS, as well as from subepithelial fibroblasts cocultured with mechanical damaged HBECs. It was also measured in the supernatant from HBECs transfected with MMP-9 expression plasmid. MMP-9 activity was assessed in supernatants from HBECs stably transfected with pEGFPc1 -antisense-ET-1 converting enzyme(ECE) RNA. RESULTS: It was found that there was an increase in ET-1 level in supernatants from HBECs either treated with mechanical scraping plus LPS or transiently transfected with MMP-9 plasmid, as well as from sub-epithelial fibroblasts cocultured with mechanical scraping HBECs compared with that in controls. Gelatin zymography showed a obviously attenuated gelatinolytic activity of MMP-9 in conditioned media of HBECs expressing antisense ECE RNA after mechanical damage. CONCLUSIONS: Airway epithelial cells under injury are able to overproduce ET-1 as well as initiate ET-1 release from sub-epithelial fibroblasts, MMP-9 produced by injured bronchial epithelial cells may also increase ET-1 processing leading to ET-1 production further. The interaction between ET-1 and MMP-9, both of which enhanced in damaged HBECs, may play an important role in airway inflammation related to airway remodeling in asthma.