Dendrobium Polysaccharides Mitigate CSE-induced Inflammatory Damage of Human Bronchial Epithelial Cells via TLR4/NF-κB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Dendrobium polysaccharides on the secretion of inflammatory cytokines and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB pathway in 16HBE cells exposed to cigarette smoke extract （CSE）. MethodThe 16HBE cells were classified into the control， CSE， and CSE+ Dendrobium polysaccharides （100， 200， 400 mg·L-1） groups. The cell-counting kit-8 （CCK-8） assay was employed to measure the cell viability， and a microscope was used to observe the cell morphology. The enzyme-linked immunosorbent assay was employed to measure the levels of interleukin （IL）-8， IL-1β， IL-4， IL-13， and transforming growth factor （TGF）-β in cell culture supernatants. Real-time PCR was carried out to determine the mRNA levels of Toll-like receptor 4 （TLR4）， nuclear factor-κB （NF-κB）， and IL-4. Western blot was employed to determine the protein levels of interleukin-4 receptor （IL-4R）， TLR4， myeloid differentiation primary response protein 88 （MyD88）， NF-κB， phosphorylated nuclear factor-κB （p-NF-κB）， and nucleoproteins nuclear factor-κB （NEs-NF-κB）. The immunofluorescence assay was employed to measure the nuclear translocation of NF-κB. ResultCompared with the control group， the CSE group showed elevated levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β in the cell culture supernatants （P<0.05， P<0.01）， up-regulated expression levels of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.01）， and significant nuclear translocation of NF-κB. Compared with the CSE group， Dendrobium polysaccharides increased the cell survival rate， recovered the cell activity， lowered the levels of IL-8， IL-1β， IL-4， IL-13， and TGF-β， down-regulated the expression of TLR4， MyD88， NF-κB， p-NF-κB， NEs-NF-κB， and IL-4 （P<0.05， P<0.01）， and reduced the nuclear translocation of NF-κB. ConclusionDendrobium polysaccharides showed significant protective effects on the 16HBE cells exposed to CSE by inhibiting the TLR4/NF-κB signaling pathway.

Bufei Yishen formula attenuates cigarette smoke extract-induced airway mucus hypersecretion by regulating Notch signaling pathway / 中华危重病急救医学

Objective:To explore the mechanism of Bufei Yishen formula (BYF) on attenuating cigarette smoke extract (CSE)-induced airway mucus hypersecretion by regulating Notch signaling pathway.Methods:The human airway epithelial cell 16HBE was cultured in vitro, and the cells in logarithmic growth phase were used for the experiments. ① Intervention condition screening experiment: the 16HBE cells were grouped, methylthiazolyldiphenyl-tetrazolium (MTT) method and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of different concentrations of CSE (2.5%, 5%, 10%, 20%, 40%), different concentrations of BYF drug-containing serum (5%, 10%, 20%, 40%), and different concentrations of Notch signal pathway blocker DAPT (5, 10, 20, 40 μmol/L) on cell activity and secretion of mucin 5AC (MUC5AC) levels. In addition, a blank control group was set up to screen out the best conditions for preparing CSE-induced cell mucus hypersecretion model and BYF and DAPT intervention. ② Intervention experiment: the 16HBE cells were divided into four groups. The blank control group was not given any treatment; the 16HBE cells were induced by 10% CSE for 24 hours to prepare mucus hypersecretion model in the CSE model group; the cells in the CSE+BYF group and CSE+DAPT group were given 10% BYF or 20 μmol/L DAPT, respectively, for intervention at the same time for 24 hours. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to detect the mRNA expressions of MUC5AC, Notch3 and hairy and enhancer of split 1 (HES1) in the cells. Western blotting was used to detect the protein expressions of Notch3 and HES1 in the cells. Results:① Results of the screening experiment of intervention conditions: compared with the blank control group, 10% CSE induction for 24 hours was the best condition for establishing cell mucus hypersecretion model that neither affected cell viability nor increased the secretion of MUC5AC; while 10% BYF and 20 μmol/L DAPT was the optimal intervention condition. ② Intervention experiment results: compared with the blank control group, the mRNA expressions of MUC5AC, Notch3, and HES1 and the protein expressions of Notch3 and HES1 in the CSE model group were significantly increased, indicating that CSE activated Notch3 and HES1 signal activation and induced 16HBE cells to secrete mucus protein. Compared with the CSE model group, BYF and DAPT could significantly down-regulate the mRNA and protein expressions of MUC5AC, Notch3, and HES1 in cells [MUC5AC mRNA (2 -ΔΔCT): 1.03±0.13, 0.96±0.05 vs. 1.35±0.07, Notch3 mRNA (2 -ΔΔCT): 1.10±0.14, 1.10±0.02 vs. 1.31±0.15, HES1 mRNA (2 -ΔΔCT): 1.26±0.10, 1.14±0.15 vs. 1.45±0.08, Notch3 protein (Notch3/GAPDH): 0.10±0.03, 0.16±0.03 vs. 0.31±0.09, HES1 protein (HES1/GAPDH): 0.37±0.06, 0.34±0.08 vs. 0.50±0.05, all P < 0.05]. Conclusion:The mechanism of BYF attenuating mucus hypersecretion of 16HBE cells induced by CSE was associated with the inhibition of Notch signaling pathway activation.

Comparison of volatile organic compounds between cigarette smoke condensate (CSC) and extract (CSE) samples

Cigarette smoke is a major risk factor for several diseases, including chronic obstructive pulmonary and cardiovascular diseases. The toxicity of the cigarette smoke can be determined in vitro. The cytotoxicity test of the cigarette smoke is commonly conducted using the cigarette smoke condensate (CSC) and cigarette smoke extract (CSE). The CSC and CSE methods are well known for sampling of the particles and water-soluble compounds in the cigarette smoke, respectively. In this study, the CSC and CSE were analyzed by using a gas chromatography-mass spectrometry (GC-MS) system equipped with a wax column for separation of the volatile organic compounds. The cytotoxic effect of the CSC and CSE were evaluated thoroughly by comparing the analytical results of the CSC and CSE samples. The total concentration of the volatile organic compounds detected in the CSC sample was similar to that in the CSE sample based on the peak area. Except for the dimethyl sulfoxide solvent, nicotine had the highest concentration in the CSC sample, while acetonitrile had the highest concentration in the CSE sample. The compositions were as follows: (1) CSC sample: 55.8% nicotine, 18.0% nicotyrine, 3.20% 1,2,3-propanetriol, triacetate, 1.28% ethyl chloride, 1.22% phenol, etc. and (2) CSE sample: 18.7% acetonitrile, 18.0% acetone, 12.5% 2-hydroxy-2-methyl-propanenitrile, 8.98% nicotine, 5.86% nicotyrine, etc. In this manner, to accurately examine the cytotoxicity of the cigarette smoke using CSC or CSE, the components and their concentrations in the CSC and CSE samples should be considered.

Comparison of volatile organic compounds between cigarette smoke condensate (CSC) and extract (CSE) samples

Cigarette smoke is a major risk factor for several diseases, including chronic obstructive pulmonary and cardiovascular diseases. The toxicity of the cigarette smoke can be determined in vitro. The cytotoxicity test of the cigarette smoke is commonly conducted using the cigarette smoke condensate (CSC) and cigarette smoke extract (CSE). The CSC and CSE methods are well known for sampling of the particles and water-soluble compounds in the cigarette smoke, respectively. In this study, the CSC and CSE were analyzed by using a gas chromatography-mass spectrometry (GC-MS) system equipped with a wax column for separation of the volatile organic compounds. The cytotoxic effect of the CSC and CSE were evaluated thoroughly by comparing the analytical results of the CSC and CSE samples. The total concentration of the volatile organic compounds detected in the CSC sample was similar to that in the CSE sample based on the peak area. Except for the dimethyl sulfoxide solvent, nicotine had the highest concentration in the CSC sample, while acetonitrile had the highest concentration in the CSE sample. The compositions were as follows: (1) CSC sample: 55.8% nicotine, 18.0% nicotyrine, 3.20% 1,2,3-propanetriol, triacetate, 1.28% ethyl chloride, 1.22% phenol, etc. and (2) CSE sample: 18.7% acetonitrile, 18.0% acetone, 12.5% 2-hydroxy-2-methyl-propanenitrile, 8.98% nicotine, 5.86% nicotyrine, etc. In this manner, to accurately examine the cytotoxicity of the cigarette smoke using CSC or CSE, the components and their concentrations in the CSC and CSE samples should be considered.

Curcumin suppresses cigarette smoke extract-induced oxidative stress through PPARγ/ NF-κB pathway in human bronchial epithelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of curcumin against cigarette smoke extract (CSE)- induced oxidative stress in human bronchial epithelial cells and explore the underlying mechanism.</p><p><b>METHODS</b>Human bronchial epithelial cell line 16HBE was treated for 24 h with curcumin, CSE, CSE + curcumin, and CSE + curcumin with transfection by a short hairpin RNA targeting PPARγ (shPPARγ). MTT assay was used to observe the changes in the cell viability after the treatments. Quantitative real-time PCR was performed to detect the mRNA expressions of tumor necrosis factor- (TNF-), iNOS and PPARγ in the cells, and the protein expressions of iNOS, PPARγ and the phosphorylation of NF-κB p65 were detected using Western blotting.</p><p><b>RESULTS</b>The treatments did not cause significant changes in the cell viability. Exposure to CSE for 24 h significantly lowered PPARγ expression and increased TNF- and iNOS expressions and phosphorylation of NF-κB p65 in the cells. The effects of CSE were significantly suppressed by curcumin, but transfection of the cells with shRNA-PPARγ obviously abrogated the suppressive effects of curcumin.</p><p><b>CONCLUSIONS</b>Curcumin suppresses CSE-induced oxidative stress and inflammation via the PPARγ/NF-κB signaling pathway in 16HBE cells, suggesting the potential of curcumin in the treatment of chronic obstructive pulmonary disease.</p>

Autophagy is involved in pulmonary artery endothelial cell apoptosis induced by cigarette smoke extract / 中国病理生理杂志

AIM: To investigate the role of autophagy in the apoptosis of human pulmonary artery endothelial cells (HPAECs) induced by cigarette smoke extract (CSE).METHODS: HPAECs were cultured routinely.HPAECs were treated with CSE at different concentrations, and the cell viability was detected by MTT assay.HPAECs were divided into control group, CSE group, 3-methyladenine (3-MA) group and 3-MA+CSE group.The autophagy was observed under fluorescence microscope with monodansylcadaverine (MDC) staining.Annexin V/propidium iodide staining and Hoechst 33342 staining were employed to detect apoptosis.In addition, the protein levels of LC3, beclin-1 and cleaved caspase-3 were determined by Western blot.RESULTS: MDC staining showed the increased production of autophagic vacuoles was observed in CSE group.The results of Western blot showed that the expression levels of autophagy-related proteins LC3 and beclin-1 were increased, while 3-MA pretreatment inhibited the expression of these proteins and the production of autophagic vacuoles.Observation with Annexin V/propidium iodide staining and Hoechst 33342 staining showed that the apoptotic rate in CSE group was significantly higher than that in control group, and pretreatment with 3-MA induced further increase in the cell apoptosis.The protein level of cleaved caspase-3 in CSE group was significantly higher than that in control group (P<0.05), and 3-MA+CSE treatment induced the further increase in the protein level of cleaved caspase-3.CONCLUSION: CSE induces autophagy and apoptosis in the HPAECs.Inhibition of autophagy promotes the apoptosis induced by CSE in HPAECs, which can be achieved through activation of caspase-3.

Shp2 regulates cigarette smoke extract-induced epithelial-mesenchymal transition / 中国药理学通报

Aim To explore the effects of Shp2 on cig-arette smoke extract (CSE)-induced epithelial-mesen-chymal transition (EMT). Methods The effects of CSE on TGF-β1 levels in epithelial cells were meas-ured by Q-PCR and ELISA. Immunofluorescent stai-ning was used to assess the expressions of CSE-induced EMT-related markers. The activation of CSE-induced Shp2,Smad2 was investigated by Western blot. Re-sults CSE induced Shp2 phosphorylation in a concen-tration-dependent manner in A549 cells. PHPS1 inhib-ited the increase in mRNA and protein expression of TGF-β1 induced by CSE. PHPS1 regulated the expres-sions of CSE-induced EMT markers (down-regulation of E-cadherin,up-regulation expression of Vimentin and α-SMA). The inhibition of either Shp2 inhibitor or Shp2 siRNA decreased Smad2 phosphorylation induced by CSE. Conclusions CSE initiates EMT through the Shp2 / Smad2 signaling pathway,which is activated by CSE through TGF-β1 generation. It is suggested that Shp2 might be a possible new target for COPD and lung cancer therapy.

Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract / 华中科技大学学报（医学）（英德文版）

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE).Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups:CSE-stimulated group,CSE-stimulated with 4-PBA group,and negative control group.HUVECs were cultured and stimulated with CSE at concentrations of 5％,10％ and 20％,respectively,mRNA of CXCL-8 and GRP78 was detected by real-time PCR.ELISA was performed to test the expression of CXCL-8 protein,and neutrophils migration was detected by Transwell board test.The NF-κB,ERK,p38MAPK and transforming growth factor beta (TGF-β) were detected by flow cytometry.The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P＜0.05).Furthermore,it was concentration-dependent.4-PBA significantly reduced the expression of CXCL-8 protein (P＜0.05) and neutrophil migration (P＜0.05).The TGF-β,rather than the NF-κB,ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE.CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells.ER stress might play a role in the effect of neutrophils migration stimulated with CSE,and TGF-β pathway may contribute to the ER stress in HUVECs.

Effect of PLTP on CSE-induced IL-8 production in A549 cells / 中国免疫学杂志

Objective:To investigate the effect of PLTP gene on CSE-induced IL-8 production in human alveolar Type Ⅱcells ( Adenocarcinomic human alveolar epithelial cells , A549 ) .Methods: The different concentrations of CSE co-cultured with human alveolar epithelial cell line ( A549 ) for 24 hours.MTT assay was performed to study the effect of CSE on human alveolar epithelial cell line(A549) growth.Expression levels of PLTP mRNA and IL-8 mRNA were examined by RT-PCR,protein of PLTP were examined by Western blot ,and protein of IL-8 was examined by ELISA .Results: MTT assay showed that the proliferation of A 549 cell line were stimulated by the 0.125%CSE,while the proliferation of A549 cell tends to decrease at high concentrations of CSE (2.0% CSE and 4.0%CSE),and in this middle concentrations of CSE (0.25%CSE ,0.5%CSE and 1.0%CSE),the proliferation of A549 cell was not significantly affected .Our studys suggested that PLTP and IL-8 release were induced by CSE in a concentration-dependent and time-dependent manner ,and expression levels of IL-8 obviously increased after silence PLTP gene .Conclusion:PLTP siRNA can increased CSE-induced IL-8 production in human alveolar epithelial cells (A549).

Effect of PLTP on CSE-induced IL-8 production in human bronchial epithelial cells / 中国免疫学杂志

Objective:To investigate the effect of PLTP gene on CSE-induced IL-8 production in human bronchial epithelial cell line (HBECs). Methods:Wistar rats were exposed to air or cigarette smoke for 6 hours/day on 3 consecutive days,then the lungs were sectioned and examined. The number of total white blood cell and differential white blood cells in BALF were counted. The different concentrations of CSE co-cultured with HBECs for 24 hours. Cells growth was detected by MTT assay. Expression levels of PLTP mRNA and IL-8 mRNA were examined by RT-PCR,protein of PLTP was investigated by Western blot,and production of IL-8 ex-amined by ELISA. Results:The number of white blood cells in BALF was significantly increased compared with controls. Enhanced ex-pression level of PLTP and IL-8 were observed in CS-exposure group. Proliferation of HBECs tends to decrease at high concentrations of CSE(2. 0% CSE and 4. 0% CSE). The results suggested that the production of IL-8 induced by CSE in a time- and concentration-dependent manner,while the expression of PLTP induced by CSE in a dose-dependent manner. Furthermore,expression levels of IL-8 significantly increased after silence PLTP gene. Conclusion:PLTP siRNA could increase CSE-induced IL-8 production in HBECs.

Effects of tobacco extract on proliferation of human airway smooth muscle cells / 天津医药

Objective To explore the effects and mechanism of cigarette smoke extract (CSE) on the proliferation of air?way smooth muscle cells (ASMCs) and the expression of CCAAT/enhancer-binding protein (CEBPα) and calreticulin. Meth?ods (1) The ASMCs were stimulated with different concentrations of CSE for twenty-four hours. According to the concentra?tions of CSE,the cells were divided into control group, 2.5%CSE group, 5%CSE group and 10%CSE group. The prolifera?tion of ASMCs was measured by MTT colrimetric method. The CEBPαmRNA was analyzed by RT-PCR. Western bloting as?say was performed to detect the levels of CRT and CEBPαprotein. (2) In 10%CSE group, transfection of the siRNA respec?tively for negative control or calreticulin was performed in accordance with instructions. The cell proliferation and the expres?sion of calreticulin and CEBPαwere compared in negative control siRNA group and calreticulin siRNA group. Results (1) With the increasing of the concentrations of CSE, the protein expression of CEBPαdecreased gradually (P0.05). (2) Under the 10%CSE, the expression of CEBPαwas significantly higher in CRT siRNA group than that in negative control group (P<0.05),but the cell proliferation and CRT were significantly lower in the calreticulin siRNA group than those in negative control siRNA group (P<0.05). Conclusion The CSE exposure contributes to the expression of calreticulin protein,and then inhibits the translation of CEBPαmRNA,thus promotes the proliferation of ASMCs.

Cigarette smoke extract promotes senescence by reducing expression of HDAC2 in C2C12 myoblasts / 中国病理生理杂志

AIM:To investigate whether the cigarette smoke extract (CSE) causes senescence of C2C12 myo-blasts and the relationship between senescence and histone deacetylase 2(HDAC2).METHODS: Murine C2C12 cells were induced to differentiate into myoblasts.The HDAC2 activator and inhibitor were used to investigate the effects of CSE in the myoblasts on cell senescence and the expression of HDAC2.The expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blot, respectively, and the positive cell rate of β-galactosidase staining for cell senescence was also detected.RESULTS:The optimal concentration of CSE was 60 mL/L and the intervention time was 24 h.After the intervention of CSE, the positive cell rate ofβ-galactosidase staining was increased, accompanied with the reduction of HDAC2 expression at mRNA and protein levels.The expression of HDAC2 at mRNA and protein levels was increased by 4, 5, 6, 7-tetrabromobenzotriazole (TBB), accompanied with the reduction of positive cell rate ofβ-galacto-sidase staining.Furthermore, when HDAC2 expression at mRNA and protein levels was reduced by HDAC2 inhibitor valp-roic acid, the positive cell rate of β-galactosidase staining was increased.CONCLUSION: CSE promotes the senescence by reducing the expression of HDAC2 in C2C12 myoblasts.

Protein kinase C-nuclear factor-erythroid 2-related factor 2 regulating the expression of heme oxygenase-1 in rat airway epithelial cells / 中南大学学报(医学版)

Objective:To observe the effect of the signaling pathway of protein kinase C (PKC)-nuclear factor-erythroid 2-related factor 2 (Nrf2) on the expression of heme oxygenase -1 (HO-1) induced by cigarette smoke extract in rat airway epithelial cells.Methods:The airway epithelial cells of 25 male SD rats were randomly divided into a control group, a CSE3h group, a RO318220 group (PKC inhibitor), a Nrf2 siRNA group and a Nrf2 siRNA+RO318220 group, 5 rats in each group. hTe control group was incubated with DMEM/F12 alone. hTe CSE3h group was treated with 10% CSE for 3 h. hTe RO318220 group was pretreated with 3 μmol/L RO318220 for 0.5 h and subsequently treated with 10% CSE for 3 h. hTe Nrf2 siRNA group was pretreated with Nrf2 siRNA, and then treated with 10% CSE for 3 h. hTe Nrf2 siRNA+RO318220 group was pretreated with Nrf2 siRNA and 3 μmol/L RO318220 for 0.5 h, and then treated with 10% CSE for 3 h. hTe protein levels of Nrf2 in the nucleus and cytoplasm, and HO-1 and PKC in the whole cells were semi-quantified by Western blot. The protein expression of HO-1 was measured by immunocytochemistry. HO-1 mRNA expression was detected by RT-PCR. Immunolfuorescence staining was used to observe the nuclear translocatin of Nrf2. Results: CSE markedly induced Nrf2 nuclear translocation in the rat airway epithelial cells, and RO318220 pretreatment blocked the CSE induced Nrf2 nuclear translocation. Immunocytochemistry showed that HO-1 protein expression was strongly positive in the CSE3h group, weakly positive in the other 4 groups. hTe expression of PKC protein, HO-1 mRNA and protein signiifcantly increased in the CSE3h group, and HO-1 activity markedly improved in the CSE group (P0.05). Conclusion: CSE induces the nuclear translocation of Nrf2 by PKC signaling pathway, thus upregulating the HO-1 expression in the rat airway epithelial cells.

Effect of cigarette smoke extract on the interaction between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy / 中国药理学通报

Aim To study the effect of CSE ( cigarette smoke extract ) on the single-molecule interactional force between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy. Methods CSE was prepared by a previously reported method. The plasmid of TM-GFP was constructed and transfect-ed in COS-7 cells. The expression of TM-GFP was de-tected by fluorescence microscopy and laser scanning confocal microscopy. The transfected COS-7 cells were grouped ( 1 ) GFP -thrombin group ( 2 ) TM-thrombin group ( 3 ) CSE-TM-thrombin group ( 4 ) CSE- GFP-thrombin group. Force measurements with the thrombin modified AFM tips on the living cell surface were car-ried out on PicoSPM II with a Pico-Scan 3000 control-ler and a larger scanner. The force curves measured in living cells were recorded by PicoScan 5 software and analyzed by MATLAB R2009aMetlab. Results The single-molecule binding force of thrombomodulin and thrombin ( TM-Thr ) was determined ( 60. 90 ± 0. 82 ) pN. The binding probability for TM-Thr was about (22. 58 ± 3. 95)%. Antibody blocking binding proba-bility for TM-Thr was ( 2. 58 ± 2. 0 )%. The binding probabilities for GFP-Thr group, CSE-TM-Thr group and CSE-GFP-Thr group were significantly decreased compared with TM-Thr group ( P<0. 05 ) . The mean value of the most probable single molecular interaction force of thrombin/TM-ECD was determined as ( 45. 30 ± 1. 37 ) pN, the binding probability of thrombin and TM-ECD was ( 23. 25 ± 7. 02 )%. When the binding was blocked with the TM-MAb solution, the binding probability decreased to ( 4. 64 ± 2. 31 )%. The bind-ing probability was ( 8. 31 ± 1. 06 )% in the CSE-TM-thr-S group. When further blocked with TM-MAb, the binding probability was ( 5. 17 ± 2. 96 )%. Conclusion CSE significantly decreases the binding probability for TM-Thr to induce intravascular thrombosis.

Cigarette smoke extract regulates expression of heme oxygenase-1 through signaling pathway of protein kinase C-nuclear factor-erythroid 2-related factor 2 in rat airway epithelial cells / 中国病理生理杂志

[ABSTRACT]AIM:ToobservetheeffectofthesignalingpathwayofproteinkinaseC(PKC)-nuclearfactor-ery-throid 2-related factor 2 (Nrf2) on the expression of heme oxygenase-1 (HO-1) induced by cigarette smoke extract (CSE) in the rat airway epithelial cells .METHODS:After exposed to CSE , the airway epithelial cells of male SD rats were di-vided into control group , CSE3 group, RO318220 group, Nrf2 siRNA group and RO318220+Nrf2 siRNA group.The pro-tein levels of HO-1, Nrf2 and PKC were semi-quantified by Western blotting .The protein expression of HO-1 was assessed by immunocytochemistry .The mRNA expression of HO-1 was detected by reverse transcription-polymerase chain reaction ( RT-PCR) .Nrf2 protein location was observed by immunofluorescence staining .HO-1 activity was also determined .RE-SULTS:After exposure to CSE for 3 h, the Nrf2 protein was mainly located in the nucleus , and the expression level of Nrf2 protein in the nucleus was stronger than that in the control cells .CSE also significantly enhanced the levels of p-PKC protein, and HO-1 mRNA, protein and activity .Pretreatment with RO318220 significantly decreased the levels of PKC pro-tein, Nrf2 protein, and HO-1 mRNA, protein and activity.Meanwhile, Nrf2 protein significantly decreased , and the activ-ity, mRNA and protein levels of HO-1 were also significantly attenuated by pretreatment with siRNA to knock down Nrf2. Pretreatment with RO318220 and Nrf2 siRNA significantly decreased the PKC protein , Nrf2 protein, and HO-1 mRNA, protein and activity .CONCLUSION:CSE up-regulates the HO-1 expression through PKC-induced nuclear translocation of Nrf2 in the rat airway epithelial cells .

Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-alpha secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-alpha secretion and NF-kappaB activation. CSE induced an increase in intracellular Ca2+ concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.

Effects of Cigarette Smoke Extract on the Proliferation and Secretion of Hydrogen Peroxide in Human Lung Fibroblasts Induced by Transforming Growth Factor-β1 / 中国呼吸与危重监护杂志

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 faetor-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.H2O2release 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 < 0.01 or 0.05), while 10% CSE inhibited cell proliferation (P < 0.01).In the normal group, both low and high concentration of CSE inhibited cell proliferation (P < 0.01 or P < 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 < 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.

Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21WAF1 / 华中科技大学学报（医学）（英德文版）

Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

Effect of Cigarette Smoke Extract on Cell Biological Activities and Collagen Synthesis of Primary Culture of Skin Fibroblast / 中国康复理论与实践

@#Objective To study the biological changes and the collagen synthesis of the primary cultured skin fibroblast treated with Cigarette Smoke Extract (CSE). Methods The morphological changes of fibroblasts after 24 hours' treatment with CSE were observed with invert microscope. The inhibitory effect at different concentrations of CSE on fibroblast activities was determined by the tetrazolium dye colorimetric test (MTT Test). The growth curves of fibroblasts treated with CSE were drawn with MTT method. Cell aging was observed with β-galactosidase, which was the biological marker of senescence. Flow cytometry (FCM) was used to estimate cell cycle phases after the fibroblasts were treated at different concentrations of CSE and different time. The mRNA expression of type Ⅰ procollagen was detected by RT-PCR. Results After the treatment, the fibroblasts displayed morphological changes and the growth of fibroblasts was apparently slowed down by CSE. The positive β-galactosidase staining was observed in the treated fibroblasts, which were affected by CSE for 5 passages. FCM analysis demonstrated that CSE decreased the cells in S phase and increased the cells in G1 and G2 phase. The result of RT-PCR showed that type Ⅰ procollagen was decreased after the treatment with CSE. Conclusion CSE can not only inhibit the growth and proliferation of the skin fibroblasts, but also decrease collagen synthesis of dermal fibroblast which is very important to the skin health.

Mechanism of Bovine Coronary Artery Endothelial Cells Damage Induced by Cigarette Smoke Extract / 环境与健康杂志

Objective To study the bovine coronary artery endothelial cells(BCAEC)damage induced by cigarette abstracts and further clarify the relationship between smoking and cardiovascular diseases.Methods BCAEC were treated with nicotine, mainstream smoke extract(MSW)and sidestream smoke extract(SSW)which had the normal concentration(1.0?10~(-5),0.8?10~(-5), 0.9?10~(-5)mol/L)of nicotine in smoker.The morphological changes of BCAEC were recorded by microscope digital image system. The quantification of apoptotic BCAEC cells was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole and trypan blue exclusion assay was used to examine the percentage of necrotic BCAEC.The caspase activity assay was employed to discuss the mechanism of BCAEC apoptosis.Results BCAEC exposed to nicotine and MSW appeared the typical morphological alteration of apoptosis and necrotic morphological alteration were observed after BCAWC were treated with SSW. 5.89% and 11.94% apoptotic ceils were found after BCAEC were exposed to nicotine and MSW for 24 hours.The level of BCAEC necrosis after treated with SSW was 62.84%.Caspase-3 activity was induced by nicotine and MSW.Conclusion Cigarette smoke extract can induce the cell death of BCAEC.Nicotine and MSW can induce caspase-3 activity increase.Even in the presence of a non-cytotoxic concentration of nicotine and mainstream smoke solution,protease-induced apoptosis of BCAEC can be significantly increased.Sidestream smoke solution may cause BCAEC necrosis instead of apoptosis.Caspase-3 activation is probably the mechanism of BCAEC apoptosis.