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Aim To explore the mechanism of pediatric asthma based on network pharmacology and molecular docking technology. Methods Through TCMSP database, the chemical information and the targets of TCM chemical components and pediatric asthma targets in PubChem, SwissTargetPrediction and GeneCards were collected, and the intersection gene, namely the target gene of pediatric asthma was used. The “Drug-active ingredient-target” map was plotted with the Cytosacape 3.7.2 software. Protein interaction network maps were constructed based on the String database and analyzed by Cytoscape 3.7.2. GO and KEGG pathway analysis of acting targets using the Metascape database and bubbles were plotted on the Omicshare platform. Results A total of 238 active components and 11 corresponding 697 main targets were selected, 1 052 pediatric asthma disease target targets and 242 common targets were selected. Enrichment analysis found that common targets were primarily involved in biological processes such as MAPK cascade regulation and inflammatory response, as well as calcium signaling pathway, cAMP signaling pathway, AGE-RAGE signaling pathway, cGMP-PKG signaling pathway, etc. Molecular docking results showed that the active components of Astragalus(5'hydroxyiso-muronulatol-2',5'-di-O-Glucoside)docked well with the SRC, TP53, and IL-6 targets. We proved that anti-resistant drinking could down-regulate the expression of IL-6, SRC and TP53. Conclusions Gubenfangxiaoyin drinking may be involved in the regulation of the STAT3, SRC, AKT1, TP53, TNF, MAPK3, TP53,TNF,MAPK3 and IL6 and other targets, involved in the regulation of calcium-CAMP signaling pathway in childhood asthma, AGE-RAGE signaling pathway, cGMP-PKG signaling pathway, reduce the MAPK cascade, inflammatory response, etc, and play a role in the prevention and treatment of asthma.
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Seven nucleoside compounds were isolated from the Oenothera biennis L. by various chromatographic techniques such as Diaion HP-20, silica gel, Sephadex LH-20, MCI and semi-preparative HPLC. Their structures were identified by analysis of physicochemical properties and spectral data, and determined as 9-(3′-carbonyl methyl)hydroxypurine (1), 1-(3′-carbonyl methyl)purine-6,8-dione (2), N-methyl-2-pyridone-5-carboxamide (3), uracil (4), uridine (5), thymidine (6) and 2′-Ο-methoxy luridine (7). Compound 1 is a new nucleoside and compounds 2-7 were newly isolated from the Oenothera biennis L. Compounds 1-2 can significantly increase the viability of BEAS-2B cells induced by TGF-β1, showing potent anti-pulmonary fibrosis activity.
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Cigarette smoking is a major risk factor for chronic respiratory inflammatory diseases. Nicotine is the most important ingredient in tobacco smoke. The incidence rate of chronic respiratory diseases associated with nicotine is increasing rapidly. Therefore, it is urgent to find potential targets for nicotine-related chronic respiratory inflammatory diseases. This article hereby aims to investigate the effect of nicotine on apoptosis of BEAS-2B cells and its potential mechanism. The expressions of apoptosis, autophagy and PI3K/ Akt/ mTOR pathway-related proteins were detected by Western blotting. Flow cytometry and CCK-8 were used to detect cell apoptosis rate and cell viability. The results showed that nicotine induced apoptosis of BEAS-2B cells at 1, 2 and 4 mmol/ L, and the cell viability decreased with the increase of concentration. Compared with the control group, the expression of autophagy-related protein LC3Ⅱ and P62 increased significantly after nicotine treatment (P0. 05). Compared with the nicotine group, rapamycin pretreatment significantly decreased cell apoptosis and increased cell activity (P<0. 05). Compared with the nicotine group, the expression of p-Akt and p-mTOR decreased significantly and apoptosis decreased significantly after LY294002 pretreatment (P<0. 05). Furthermore, nicotine induces apoptosis of BEAS-2B cells by inhibiting autophagy may be via PI3K/ Akt/ mTOR pathway, which may be a potential target for the prevention and treatment of chronic respiratory inflammatory diseases.
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Objective@#To evaluate the viability of gasoline engine exhaust (GEE) with different particle sizes on human lung cell line BEAS-2B in vitro by air-liquid interface (ALI) .@*Methods@#GEE were collected with a Tedlar bag and their particulate matter (PM) number, surface and mass concentration in three kind of GEE (filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter) were measured by two type of particle size spectrometer including TSI-3321 and SMPS-3938. Five groups were included, which divided into blank control group, clean air group, filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group. Except the blank control group, BEAS-2B cells, cultured on the surface of Transwells, were treated with clean air or GEE by ALI method at a flow rate of 25 ml/min, 37 ℃ for 60 min in vitro. CCK-8 cytotoxicity test kit was used to determine the cell relative viability of BEAS-2B cells.@*Results@#In the filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter, high concentrations of fine particles can be detected, but the coarse particles only accounted for a small proportion, and the sequence of PM concentration was motorcycle exhaust without three-way catalytic converter group> non-filtered automobile exhaust group> filtered automobile exhaust group (P<0.001) . Compared with the clean air group, the cell relative viability in the 3 GEE-exposed groups were significantly lower (P<0.001) . Among the comparisons of GEE exposure groups with different particle size spectra, the sequence of the cell relative viability was filtered automobile exhaust group >non-filtered automobile exhaust group> motorcycle exhaust without three-way catalytic converter group (P<0.001) . When took the clean air control group as a reference, the mean of the cell relative viability in the filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group, was decreased by 26.34%, 36.00% and 49.59%, respectively.@*Conclusion@#GEE with different particle size spectra could induce different levels of toxic effects to the human lung cells BEAS-2B by ALI. After lowering the concentration of particles in the GEE and using the three-way catalytic converter could obviously improve the survival rate of lung cells.
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This study was designed to investigate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glycoside(TSG)on hypoxia/reoxygenation(H/R)-induced oxidative stress injury and its potential mechanism in human bronchial epithelial cell(BEAS-2B)cells. BEAS-2B cells were exposed to H/R treatment. Level of intracellular ROS was detected using DCFH-DA probe and fluorescence microplate reader. Production of MDA and activity of SOD were evaluated with MDA and SOD kits. Nucleus was shaped by DAPI staining. Translocation of Bax to mitochondria was observed in MCF-7/GFP-Bax cells. Change in mitochondrial membrane potential was detected by JC-1 staining. Release of cytochrome C from mitochondria was detected by immunofluorescence. Expressions of mitochondrial/cytoplasmic Bax and cytochrome C, caspase-9, caspase-3, phosphorylated MAPK, HIF-1α and phosphorylated p53(p-p53)were determined by Western blotting. TSG significantly improved cell viability and reduced H/R-induced ROS production in BEAS-2B cells, while significantly decreased MDA production. It inhibited Bax translocation and nucleus fracture, reversed the decrease in mitochondrial membrane potential and inhibited the release of cytochrome C and following activation of caspase-9/caspase-3. Simultaneously, TSG down-regulated the signals of SAPK JNK1/2 and p38 MAPK without an impact in ERK1/2. It attenuated expression of HIF-1α and phosphorylation of p53. This study suggests that TSG could protect BEAS-2B against H/R-induced apoptosis, perhaps through the MAPK, HIF-1α and p53 pathways.
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Objective A large number of studies have shown that the exosome is closely related to the occurrence and devel -opment of tumor cells , but the specific mechanism is still unknown .The study was to investigate the effects of A 549-derived exosome on tight junction protein and cytoskeleton remodeling in normal lung epithelial BEAS-2B cells. Methods A549-derived exosome were isolated by ultracentrifugation , the morphology of which was observed by transmission electron microscope .Western blot analysis was used to examine the surface markers of CD 9 and CD63.Immunofluorescence assay, western blot assay and qPCR assay were applied to detect the effects of exosome on tight junction protein ZO-1 and occludin mRNA expression in BEAS-2B cell.Phalloidin-FITC staining experiment was used to detect the effects of exosome on the cytoskeleton remodeling of BEAS-2B cells.The invasiveness of A549 to BEAS-2B was detected by Transwell invasion test . Results Typical vesicles were observed under electron microscope and exosome markers CD 9 and CD63 expression were detected.The expression levels of ZO-1, occludin pro-tain (P<0.05) and mRNA (P<0.01) were decreased in exosome-treated BEAS-2B cells, and the cytoskeleton remodelled .Transwell invasion test showed that the number of A 549 cells passing through the microporous membrane (22±4) after exosome treatment BEAS-2B was significantly higher than that of the control group (10.6±4.5) (P<0.05). Conclusion A549-derived exosome can promote the cytoskeleton remodeling of BEAS-2B cells by down-regulating the expression of ZO-1 and occludin in BEAS-2B cells, aiming to de-stroy the barrier of BEAS-2B cell to tumor cell A549.
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Objective A large number of studies have shown that the exosome is closely related to the occurrence and devel -opment of tumor cells , but the specific mechanism is still unknown .The study was to investigate the effects of A 549-derived exosome on tight junction protein and cytoskeleton remodeling in normal lung epithelial BEAS-2B cells. Methods A549-derived exosome were isolated by ultracentrifugation , the morphology of which was observed by transmission electron microscope .Western blot analysis was used to examine the surface markers of CD 9 and CD63.Immunofluorescence assay, western blot assay and qPCR assay were applied to detect the effects of exosome on tight junction protein ZO-1 and occludin mRNA expression in BEAS-2B cell.Phalloidin-FITC staining experiment was used to detect the effects of exosome on the cytoskeleton remodeling of BEAS-2B cells.The invasiveness of A549 to BEAS-2B was detected by Transwell invasion test . Results Typical vesicles were observed under electron microscope and exosome markers CD 9 and CD63 expression were detected.The expression levels of ZO-1, occludin pro-tain (P<0.05) and mRNA (P<0.01) were decreased in exosome-treated BEAS-2B cells, and the cytoskeleton remodelled .Transwell invasion test showed that the number of A 549 cells passing through the microporous membrane (22±4) after exosome treatment BEAS-2B was significantly higher than that of the control group (10.6±4.5) (P<0.05). Conclusion A549-derived exosome can promote the cytoskeleton remodeling of BEAS-2B cells by down-regulating the expression of ZO-1 and occludin in BEAS-2B cells, aiming to de-stroy the barrier of BEAS-2B cell to tumor cell A549.
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Sulforaphane (SFN) is a naturally occurring compound which is known to induce the phase II antioxidant genes via Nrf2 activation, although the underlying mechanism has not been fully elucidated. In this study, we investigated Nrf2 induction in response to SFN in human bronchial epithelial BEAS-2B cells and determined the signaling pathways involved in this process. SFN treatment reduced cell viability. Prior to cell death, intracellular reactive oxygen species (ROS) were generated at a high rate within a minute of commencing SFN treatment. Pretreatment with antioxidant N-acetylcysteine (NAC) blocked SFN-induced decrease in cell growth. Erk1/2 was activated within 30 min of SFN addition, whereas Akt phosphorylation did not significantly change until the first 8 hr after SFN treatment but then became substantially low until 48 hr. Inhibition of Erk1/2 phosphorylation attenuated SFN-induced loss of cell viability. Nrf2 protein levels in both nuclear and whole cell lysates were increased by SFN treatment, which was dependent on ROS production. Knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation. Induction of the Nrf2/HO-1 after SFN treatment was potently suppressed by pretreatment with NAC. Overall, our results indicate that SFN mediates antioxidative and antiproliferative responses by generating ROS in BEAS-2B cells.
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Humanos , Acetilcisteína/farmacología , Anticarcinógenos/farmacología , Antioxidantes/farmacología , Bronquios/citología , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Depuradores de Radicales Libres/farmacología , Hemo-Oxigenasa 1/biosíntesis , Factor 2 Relacionado con NF-E2/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Mucosa Respiratoria/citología , Transducción de Señal/efectos de los fármacos , Tiocianatos/farmacologíaRESUMEN
Objective To establish a model of malignant transformation of human cells in vitro to study the lung cancer induced by radon and cigarette smoke. Methods The immortalized human bronchial epithelial cells BEAS-2B were divided into control group( C ), radon group ( Rn), cigarette smoke group (Sm) and combined group (Rn-Sm). Cells were planted onto transwell membrane one day before exposure and were directly exposed to radon and cigarette smoke pumped in a gas inhalation box. After the exposure cells were trypsinized into dishes for further growth and malignancy transformation phenotype was detected in order to compare the effects due to radon and cigarette smoke exposure. Results BEAS-2B cells showed malignantly transformed phenotype by exposure to radon and cigarette smoke. A series of sequential steps emerged among transformed cells, including altered growth kinetics, resistance to serum has changed from 0. 31 ± 0. 18 to 1.92 ± 0. 27,2. 03 ± 0. 14,2.95 ± 0. 60, and anchorage-independence growth increased from (0.01 ±0.02)% to (4.89 ±0.30)%,(8.36 ±0.50)%,(11.74 ±0.69)%.After being subculture for 20 generations, cell apoptosis of the fifth generation cells exposed to radon,cigarette smoke and both was significant decreased from ( 11.76 ± 0. 17 ) % to (4. 62 ± 0. 42 ) %、 ( 8.63 ±0. 15 )%、 (3.68 ± 0. 33 )%. Conclusions BEAS-2B cells could be malignancy transformed by radon and cigarette smokein vitro, which could be used as a cell model in lung bronchial carcinogenesis.
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Objective To observe the oxidative damage in human bronchial epithelial cells(BEAS-2B) induced by depleted uranium(DU)and protection of DMSO.Methods The measurement of extracellular superoxide anions(O2-·)was based on the reduction of ferricytochrome C.Quantitative analysis of extracellular hydrogen peroxides(H2O2)was used by the horseradish peroxidase-dependent oxidation of phenol red.The determination of extracellular hydroxyl radicals(·OH)was based on discoloration of safranine T.Ethidium bromide and 2,7'-dichlorofluorescein,fluorescent products of the membrane-permeable dyes-hydroethineand 2,7'-dichloroflurescin diacetate were used to monitor the intracellular production of O2-·and H2O2 by fluorometric method.The enzyme activity of SOD and GSH were measured by chemiluminescence and spectrophotometric method,respectively.Results The ROS production,including H2O2,O2-·and·OH,increased remarkably which induced by DU in BEAs-2B cells.The enzyme activity of SOD and GSH was descended remarkedly.These changes could be effectively inhibited by 0.5% of DMSO.Conclusions DU causes oxidative damage to BEAS-2B cells.Through removing active oxygen,DMSO can inhibit oxidative damage of DU.
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@#Objective To reconstruct tissue-engineered 3D bronchial model using human bronchial epithelial cells and human embryo lung fibroblast as seeding cells, and liquid collagen mixed Matrigel as scaffold. Methods Human bronchial epithelial cells and human embryo lung fibroblast were mixed with liquid collagen supplementing with matrigel and casted in 12-wells plate to reconstruct cells-collagen sheet. Macroscopic observation, phase-contrast microscopy observation, routine HE staining and immunohistochemistry staining(CK ets) were employed to assess the engineered 3D model. Results We reconstructed engineered 3D bronchial model successfully in vitro by tissue engineering techniques and exerted static stretch onto the collagen sheet. From Macroscopic observation, we gained contracted well sheet. We also observed network structure in phase-contrast microscopy meanwhile the viability of cells was fine. HE staining showed the formation of 3D network structure. The immunohistochemistry staining of CK and Vimentin were positive.Conclusion We reconstructed engineered 3D bronchial model successfully in vitro and seeding cells could implement polarity growing in the scaffold materials then gained the network structure.
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BACKGROUND: Cigarette smoking is an important risk factor for chronic bronchitis and COPD. Airway epithelial cells exposed to cigarette smoke components such as nicotine, cotinine and benzopyrene can generate reactive oxygen species (ROS) and be subject to oxidative stress. This oxidative stress can induce the inflammatory response in the lung by the oxidant itself or by the release of proinflammatory cytokines. It has been reported that nicotine stimulates ROS, which are associated with NF-kappaB. METHODS: Beas2B cells were treated with nicotine, cotinine and benzopyrene. RT PCR was used to measure the expression of several antioxidant factors using the total RNA from the Beas2B cells. The level of superoxide dismutase(CuZnSOD), thioredoxin, glutathione reductase expression was examined. RESULTS: 0.5 to 4 hours after the benzopyrene, nicotine and cotinine theatments, the level of thioredoxin and glutathione reductase expression decreased. Longer exposure to these compounds for 24 to 72 hours inhibited the expression of most of these antioxidant factors. CONCLUSION: During exposure to smoke compounds, thioredoxin and glutathione reductase are the key antioxidant factors induced sensitively between 0.5 and 4 hours but the levels these antioxidants decrease between 24 hour and 72hours.
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Humanos , Antioxidantes , Bronquitis Crónica , Cotinina , Citocinas , Células Epiteliales , Glutatión Reductasa , Pulmón , FN-kappa B , Nicotina , Estrés Oxidativo , Reacción en Cadena de la Polimerasa , Enfermedad Pulmonar Obstructiva Crónica , Especies Reactivas de Oxígeno , Factores de Riesgo , ARN , Humo , Fumar , Superóxidos , Tiorredoxinas , Productos de TabacoRESUMEN
Objective To investigate chromosome aberration during malignant transformation of immortalized human bronchial epithelial cells ( BEAS - 2B) induced by tobacco specific nitrosamine ( NNK) , and explore those significance of early - warning and mass screening of lung cancer in high - risk smoking population. Methods Immortalized human bronchial epithelial cells ( BEAS - 2B) were induced to malignant transformation cells ( BEAS - 2BNNK ) by NNK, and chromosome aberration were detected in the different passage of BEAS - 2BNNK and BEAS - 2B cells by analysis of metaphase chromosome. Results 1. Model of malignant transformation of BEAS -2B cells induced by NNK was created (1)The serum resistance was significantly increased in the 5th passage BEAS - 2BNNK cells.(2) The anchorage independent growth was appeared in the 15th passage. (3)The ultrastructure of the 20th passage BEAS - 2BNNK cells shown obvious heteromorphy characterization. (4)The 25th passage BEAS -2BNNK cells formed tumors in nude mice, and tumors were overdifferentiation squamous cell carcinoma confirmed by histopathological examination. 2. Chromosome aberration (1) BEAS -2B cells hold relatively stable diploid karyotype which ratio was 91%-97% up to the 25th passage. Different passage BEAS -2BNNK cells gradually lost the normal diploid karyotype and the ratio of diploid cells decreased from 83% to 54% , the proportion of polyploid and aneu-ploid cells increased in process of passing generation. (2)The ratio of structure aberration neared to 2% ~ 4% in BEAS - 2BNNK and BE-AS -2B cells but the 25th passage of BEAS -2BNNK cells was 11%. Conclusion The model of malignant transformation of BEAS - 2B cells induced by NNK could be created successfully and provided for investigating the molecular biological mechanism of lung cancer, especially smoking - related cases. The polyploid and aneuploid chromosomes could be the early event in the process of malignant transformation of BEAS - 2B cells induced by NNK. In a word, the chromosome aberration might be useful markers for the early warning and mass screening of lung cancer, especially in high - risk smoking population.
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BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection in infants and young children, but the pathogenesis of RSV-induced inflammation is not well defined. MATERIAL AND METHOD: In order to examine the potential interactions between virus-infected airway epithelial cells and neutrophils, we studied the ability of neutrophils to adhere to yirus-infected airway epithelial cell monolayers by myeloperoxidase assay. Also we measured the ability of airway epithelial cells to secrete interleukin-8(IL-8) and inter-cellular adhesion molecule-1(ICAM-1) in virus-infected airway epithelial cell cultures by enzyme-linked immunosorbent assay(ELISA). The degree of IL-8 and ICAM-1 gene expression in the RSV-infected BEAS-2B cell cultures were analyzed by reverse transcription-polymerase chain reaction(RT-PCR). RESULTS: The RSV-infected BEAS-2B cell resulted in significantly enhanced level of neutrophil adherence compared to the uninfected control(p (0.001). IL-8 and ICAM-1 production significantly increased by RSV infection(p<0.05). There was a significant positive correlation between neutrophil adherence and IL-8 level(r=0.73, p=0.002), and ICAM-1 level (r=0.843, p=0.001) in RSV-infected cells. The degree of both IL-8 and ICAM-1 mRNA expression increased in the RSV-infected cells compared with the uninfected ones. CONCLUSION: These results suggest that RSV infection significantly enhances the production of IL-8 and ICAM-1 in airway epithelial cells which then results in increased neutrophil adherence.
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Niño , Humanos , Lactante , Técnicas de Cultivo de Célula , Células Epiteliales , Expresión Génica , Inflamación , Molécula 1 de Adhesión Intercelular , Interleucina-8 , Neutrófilos , Peroxidasa , Virus Sincitiales Respiratorios , ARN MensajeroRESUMEN
Objective To study the antagonism of DMSO against the toxicity of cooking oil fume condensation to BEAS-2B cell.Methods The comet assay,micronucleus test and multinucleated cells test were used to research the genotoxicity induced by cooking oil fume condensation(COFC)and the antagonism of DMSO.Results COFC induced DNA broken,the tail area,rate of comet occurrence,tail length,tail moment,olive tailmoment increased significantly,the frequencies of micronucleus and multinucleated cells were significantly increased and the damage of cells could be inhibited effectively by DMSO.Conclusion The antagonistic effects of DMSO on the toxicity of COFC was significant in BEAS-2B cell.