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1.
Life Sci ; 269: 119090, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33465393

RESUMO

AIMS: Pyroptosis and inflammation are involved in the development of chronic obstructive pulmonary disease (COPD). However, the cigarette smoke-mediated mechanism of COPD remains unclear. In this study, we aimed to investigate the role of nucleotide-binding domain-like receptor protein-3 (NLRP3) inflammasome-mediated pyroptosis in the death of human bronchial epithelial (HBE) cells after cigarette smoke extract (CSE) exposure. MAIN METHODS: The protein level of NLRP3 in lung tissue was measured after cigarette smoke exposure in vivo. In vitro, HBE cells were treated with CSE. Subsequently, the activity of caspase-1, lactate dehydrogenase (LDH) release, release of interleukin (IL)-1ß and NLRP3 expression levels were measured. The involvement of reactive oxygen species (ROS) was also explored. KEY FINDINGS: After exposure to CSE, increased release of LDH, the transcriptional and translational upregulation of NLRP3, the caspase-1 activity levels, and enhanced IL-1ß and IL-18 release were observed in 16HBE cells. In addition, NLRP3 was required to activate the caspase-1. Our results suggested that pre-stimulated of 16HBE with a caspase-1 inhibitor, or using NLRP3 siRNA to silence NLRP3 expression, also caused the decrease of IL-1ß release and pyroptosis. SIGNIFICANCES: CSE induced inflammation and contributed to pyroptosis through the ROS/NLRP3/caspase-1 pathway in 16HBE cells. The NLRP3 inflammasome participates in CSE-induced HBE cell damage and pyroptosis, which could provide new insights into COPD.


Assuntos
Brônquios/patologia , Caspase 1/metabolismo , Células Epiteliais/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , Animais , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Caspase 1/genética , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais , Tabaco
2.
Ecotoxicol Environ Saf ; 208: 111609, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396129

RESUMO

With the wide application of neodymium oxide nanoparticles (NPs-Nd2O3) in various fields, their health hazards have aroused public concern in recent years. However, data regarding the cytotoxicity of NPs-Nd2O3 is limited. In this study, we investigated the function and mechanism of long-chain non-coding RNAs (lncRNAs) in NPs-Nd2O3-induced airway inflammation. Treatment with NPs-Nd2O3 induced an inflammatory response in human bronchial epithelial cells (16HBE) by upregulating the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8). The levels of LDH and intracellular ROS in the cells treated by various doses of NPs-Nd2O3 also increased significantly. After treatment with 10 µg/ml NPs-Nd2O3, RNA microarray and real-time quantitative polymerase chain reaction (qRT-PCR) showed a significant upregulation of lncRNA loc105377478. Functional experiments suggested lncRNA loc105377478 enhanced the expression of IL-6, IL-8 and ROS in NPs-Nd2O3-treated 16HBE cells, and it was further demonstrated that lncRNA loc105377478 promoted the activation of NF-κB by negatively regulating ADIPOR1 expression. Moreover, the expression of IL-6 and IL-8 in NPs-Nd2O3-treated 16HBE cells was regulated by lncRNA loc105377478, which was mediated by the NF-κB signaling pathway. In conclusion, lncRNA loc105377478 promotes NF-κB activation by negatively regulating ADIPOR1 expression, thereby upregulating the expression of IL-6 and IL-8 in 16HBE cells treated with NPs-Nd2O3.


Assuntos
Células Epiteliais/efeitos dos fármacos , NF-kappa B/metabolismo , Nanopartículas/química , Nanopartículas/toxicidade , Neodímio/toxicidade , Óxidos/toxicidade , RNA Longo não Codificante/genética , Receptores de Adiponectina/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Inflamação , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neodímio/química , Óxidos/química , Transdução de Sinais , Regulação para Cima
3.
Toxicol Lett ; 339: 70-77, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33370592

RESUMO

Hexavalent chromium (Cr(VI)) is a well-established human carcinogen with DNA damaging effects. Recently we established a Cr(VI)-induced malignant transformation model from a human bronchial epithelial (16HBE) cell line, and in the transformed (16HBE-T) cells reduced levels of 53BP1 (critical for DNA repair) and the acetylated histone H3K18/27 (H3K18/27ac) were observed. In 16HBE-T cells SET (a multifunctional protein) was elevated by Cr(VI) through quantitative proteomics analysis. In the present study, we further explore the involvement of SET in the H3K18/27ac/53BP1 cascade in the 16HBE-T model, primarily by knockdown of SET. Bioinformatic analysis of the differentially expressed proteins indicated enrichment in histone modifications, in which SET was a major regulator. In 16HBE cells SET expression was enhanced by Cr(VI) in a concentration- and exposure duration-dependent manner. In 16HBE-T cells, SET knockdown showed the following effects: reversal of H3K18/27ac and 53BP1 levels, enhanced enrichment H3K18/27ac in 53BP1's promotor region, increase rate of apoptosis and cell cycle G0/G1 arrest (with or without Cr(VI) treatment), and reduced colony-forming efficiency. Finally, In comparison with benzo(a)pyrene-transformed (malignant, 16HBE-B) cells from 16HBE where no changes in H3K18/27ac, 53BP1 or SET were observed, while the H3K18/27ac/53BP1 cascade was downregulated and SET upregulated in 16HBE-T cells, as compared with the parental 16HBE cells; thus the changes in 16HBE-T might be a specific effect of Cr(VI). In conclusion, our results suggest that SET may be involved in the malignant cell transformation, through inhibiting the H3K18/27ac/53BP1 cascade, at least in the 16HBE cell model.


Assuntos
Brônquios/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Cromo/farmacologia , Células Epiteliais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/farmacocinética , Cromo/uso terapêutico , Humanos , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/uso terapêutico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/toxicidade
4.
Mol Pharmacol ; 99(3): 197-216, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33376135

RESUMO

In 2019, the Global Initiative for Asthma treatment guidelines were updated to recommend that inhaled corticosteroid (ICS)/long-acting ß 2-adrenoceptor agonist (LABA) combination therapy should be a first-in-line treatment option for asthma. Although clinically superior to ICS, mechanisms underlying the efficacy of this combination therapy remain unclear. We hypothesized the existence of transcriptomic interactions, an effect that was tested in BEAS-2B and primary human bronchial epithelial cells (pHBECs) using formoterol and budesonide as representative LABA and ICS, respectively. In BEAS-2B cells, formoterol produced 267 (212 induced; 55 repressed) gene expression changes (≥2/≤0.5-fold) that were dominated by rapidly (1 to 2 hours) upregulated transcripts. Conversely, budesonide induced 370 and repressed 413 mRNAs, which occurred predominantly at 6-18 hours and was preceded by transcripts enriched in transcriptional regulators. Significantly, genes regulated by both formoterol and budesonide were over-represented in the genome; moreover, budesonide plus formoterol induced and repressed 609 and 577 mRNAs, respectively, of which ∼one-third failed the cutoff criterion for either treatment alone. Although induction of many mRNAs by budesonide plus formoterol was supra-additive, the dominant (and potentially beneficial) effect of budesonide on formoterol-induced transcripts, including those encoding many proinflammatory proteins, was repression. Gene ontology analysis of the budesonide-modulated transcriptome returned enriched terms for transcription, apoptosis, proliferation, differentiation, development, and migration. This "functional" ICS signature was augmented in the presence of formoterol. Thus, LABAs modulate glucocorticoid action, and comparable transcriptome-wide interactions in pHBECs imply that such effects may be extrapolated to individuals with asthma taking combination therapy. Although repression of formoterol-induced proinflammatory mRNAs should be beneficial, the pathophysiological consequences of other interactions require investigation. SIGNIFICANCE STATEMENT: In human bronchial epithelial cells, formoterol, a long-acting ß 2-adrenoceptor agonist (LABA), enhanced the expression of inflammatory genes, and many of these changes were reduced by the glucocorticoid budesonide. Conversely, the ability of formoterol to enhance both gene induction and repression by budesonide provides mechanistic insight as to how adding a LABA to an inhaled corticosteroid may improve clinical outcomes in asthma.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Brônquios/citologia , Budesonida/farmacologia , Fumarato de Formoterol/farmacologia , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Glucocorticoides/farmacologia , Administração por Inalação , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Sinergismo Farmacológico , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
5.
Biomed Environ Sci ; 33(9): 680-689, 2020 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-33106213

RESUMO

Objective: To investigate the effect of c-fos gene silencing on differentially expressed proteins (DEPs) in human bronchial epithelial (HBE) cells after exposure to fine particulate matter (PM 2.5). Methods: HBE cells and c-fos-silenced HBE cells were exposed to 50 µg/mL PM 2.5, LC-MS/MS and tandem mass tag (TMT) labeling methods were combined with bioinformatics methods, and DEPs and interaction networks were identified. Results: In the HBE group, 414 DEPs were screened, of which 227 were up-regulated and 187 down-regulated. In the c-fos silenced HBE group, 480 DEPs were screened, including 240 up-regulated proteins and 240 down-regulated proteins. KEGG annotations showed that DEPs in the HBE group are mainly concentrated in the glycolysis/gluconeogenesis pathway and those in the c-fos silenced group are concentrated mainly in endoplasmic reticulum and the processing of proteins. Additionally, the abnormal expression of GPRC5C, DKK4, and UBE2C was identified in top 15 DEPs. After constructing the protein interaction network, 20 Hub proteins including HNRNPA2B1, HNRNPL, RPS15A, and RPS25 were screened from the HBE group and the c-fos silenced HBE group. Conclusion: c-fos gene affected the expression of cancer-related proteins. Our results provided a scientific basis for further study of PM 2.5-induced carcinogenesis mechanism.


Assuntos
Poluentes Atmosféricos/toxicidade , Brônquios/efeitos dos fármacos , Regulação da Expressão Gênica , Inativação Gênica , Genes fos/genética , Material Particulado/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Humanos , Proteômica , Mucosa Respiratória/metabolismo
6.
Environ Health Prev Med ; 25(1): 56, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-32979924

RESUMO

BACKGROUND: We previously demonstrated that continuous exposure to nitrous acid gas (HONO) for 4 weeks, at a concentration of 3.6 parts per million (ppm), induced pulmonary emphysema-like alterations in guinea pigs. In addition, we found that HONO affected asthma symptoms, based on the measurement of respiratory function in rats exposed to 5.8 ppm HONO. This study aimed to investigate the dose-response effects of HONO exposure on the histopathological alterations in the respiratory tract of guinea pigs to determine the lowest observed adverse effect level (LOAEL) of HONO. METHODS: We continuously exposed male Hartley guinea pigs (n = 5) to four different concentrations of HONO (0.0, 0.1, 0.4, and 1.7 ppm) for 4 weeks (24 h/day). We performed histopathological analysis by observing lung tissue samples. We examined samples from three guinea pigs in each group under a light microscope and measured the alveolar mean linear intercept (Lm) and the thickness of the bronchial smooth muscle layer. We further examined samples from two guinea pigs in each group under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: We observed the following dose-dependent changes: pulmonary emphysema-like alterations in the centriacinar regions of alveolar ducts, significant increase in Lm in the 1.7 ppm HONO-exposure group, tendency for hyperplasia and pseudostratification of bronchial epithelial cells, and extension of the bronchial epithelial cells and smooth muscle cells in the alveolar duct regions. CONCLUSIONS: These histopathological findings suggest that the LOAEL of HONO is < 0.1 ppm.


Assuntos
Enfisema/induzido quimicamente , Hiperplasia/induzido quimicamente , Exposição por Inalação/efeitos adversos , Pulmão/patologia , Ácido Nitroso/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Brônquios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Cobaias , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Miócitos de Músculo Liso/efeitos dos fármacos
7.
Mutat Res ; 856-857: 503217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32928365

RESUMO

We studied the genotoxicity and cellular uptake of nanosized (<50 nm) and fine (<10 µm) copper oxide (CuO) particles in vitro in human bronchial epithelial (BEAS-2B) cells. In addition, the effect of dispersing the particles using bovine serum albumin (BSA) on DNA damage induction was investigated. DNA damage was assessed by the alkaline comet (single cell gel electrophoresis) assay after 3-h, 6-h and 24-h exposures. The cytokinesis-block micronucleus assay was applied to study chromosome damage. Both fine- and nanosized CuO particles induced a dose-dependent increase in DNA damage at all timepoints tested. However, nanosized CuO damaged DNA at lower doses and higher levels compared with fine CuO. Dispersing the nanoparticles in the presence of BSA (0.6 mg/mL) resulted in a small and inconsistent decrease in DNA damage compared with dispersions in serum-free cell culture medium only. CuO nanoparticles induced a clear dose-dependent increase in micronucleated cells at doses that strongly increased cytostasis and were markedly cytotoxic at 24 and 48 h. Fine CuO showed a slight induction of micronuclei. Hyperspectral microscopy indicated a substantial cellular uptake of both types of particles after a 3-h exposure to a dose of 20 µg/cm2. The number of particles internalized by the cells was higher for nanosized than fine CuO, as quantified by the frequency of spectral matches in the total cell area and by the number of spectrally matched visible particles or agglomerates per cell. The particle uptake was limited by particle size. The stronger genotoxic activity of nanosized than fine CuO particles is likely to derive from the higher cellular uptake and more effective intracellular dissolution of nanoparticles.


Assuntos
Brônquios/efeitos dos fármacos , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Humanos , Nanopartículas Metálicas/química , Mutagênicos/química , Mutagênicos/farmacologia , Tamanho da Partícula , Análise de Célula Única
8.
Biomed Environ Sci ; 33(8): 583-592, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32933610

RESUMO

Objective: To screen the differentially expressed proteins (DEPs) in human bronchial epithelial cells (HBE) treated with atmospheric fine particulate matter (PM 2.5). Methods: HBE cells were treated with PM 2.5 samples from Shenzhen and Taiyuan for 24 h. To detect overall protein expression, the Q Exactive mass spectrometer was used. Gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and Perseus software were used to screen DEPs. Results: Overall, 67 DEPs were screened in the Shenzhen sample-treated group, of which 46 were upregulated and 21 were downregulated. In total, 252 DEPs were screened in the Taiyuan sample-treated group, of which 134 were upregulated and 118 were downregulated. KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PM 2.5 samples-treated group. The GO analysis demonstrated that Shenzhen sample-induced DEPs were mainly involved in the biological process for absorption of various metal ions and cell components. The Taiyuan PM 2.5-induced DEPs were mainly involved in biological processes of protein aggregation regulation and molecular function of oxidase activity. Additionally, three important DEPs, including ANXA2, DIABLO, and AIMP1, were screened. Conclusion: Our findings provide a valuable basis for further evaluation of PM 2.5-associated carcinogenesis.


Assuntos
Poluentes Atmosféricos/análise , Brônquios/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Material Particulado/análise , Brônquios/efeitos dos fármacos , Biologia Computacional , Células Epiteliais/efeitos dos fármacos , Humanos , Espectrometria de Massas , Tamanho da Partícula , Proteômica
9.
Am J Physiol Lung Cell Mol Physiol ; 319(3): L481-L496, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32640839

RESUMO

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide. While most develop a mild, self-limiting illness, some develop severe acute lower respiratory infection and persistent airway disease. Exposure to ambient particulate matter has been linked to asthma, bronchitis, and viral infection in multiple epidemiological studies. We hypothesized that coexposure to nanoparticles worsens RSV-induced airway epithelial barrier dysfunction. Bronchial epithelial cells were incubated with titanium dioxide nanoparticles (TiO2-NP) or a combination of TiO2-NP and RSV. Structure and function of epithelial cell barrier were analyzed. Viral titer and the role of reactive oxygen species (ROS) generation were evaluated. In vivo, mice were intranasally incubated with TiO2-NP, RSV, or a combination. Lungs and bronchoalveolar lavage (BAL) fluid were harvested for analysis of airway inflammation and apical junctional complex (AJC) disruption. RSV-induced AJC disruption was amplified by TiO2-NP. Nanoparticle exposure increased viral infection in epithelial cells. TiO2-NP induced generation of ROS, and pretreatment with antioxidant, N-acetylcysteine, reversed said barrier dysfunction. In vivo, RSV-induced injury and AJC disruption were augmented in the lungs of mice given TiO2-NP. Airway inflammation was exacerbated, as evidenced by increased white blood cell infiltration into the BAL, along with exaggeration of peribronchial inflammation and AJC disruption. These data demonstrate that TiO2-NP exposure exacerbates RSV-induced AJC dysfunction and increases inflammation by mechanisms involving generation of ROS. Further studies are required to determine whether NP exposure plays a role in the health disparities of asthma and other lung diseases, and why some children experience more severe airway disease with RSV infection.


Assuntos
Células Epiteliais/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/patogenicidade , Infecções Respiratórias/tratamento farmacológico , Titânio/farmacologia , Animais , Asma/tratamento farmacológico , Asma/etiologia , Brônquios/efeitos dos fármacos , Brônquios/virologia , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/virologia , Inflamação/complicações , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Vírus Sinciciais Respiratórios/efeitos dos fármacos
10.
Toxicol Lett ; 331: 218-226, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32562635

RESUMO

INTRODUCTION: The benchmark dose (BMD) is a dose that produces a predetermined change in the response rate of an adverse effect. This approach is increasingly utilized to analyze quantitative dose-response relationships. To proof this concept, statistical analysis was compared with the BMD approach in order to rank the sensitivity as well as the toxicity and to describe the mode of action. METHODS: Bronchial (BEAS-2B) and alveolar epithelial cells (A549) were exposed to a wide concentration range (0.4-100 µg/mL) of five metal oxide nanoparticles (CeO2, CuO, TiO2, ZnO, ZrO2). Eight toxicity endpoints were determined representing integrity of lysosomal and cell membrane, oxidative stress level, glutathione based detoxification (glutathione S-transferase), oxidative metabolism (cytochrome P450), alteration of the mitochondrial membrane potential, alteration of phase II antioxidative enzyme (NAD(P)H:quinone oxidoreductase), and de novo DNA synthesis. RESULTS: Based on the BMD calculated for the most sensitive test, the toxicity decreased in the following order: ZnO > CuO > TiO2>ZrO2>CeO2 in BEAS-2B. Both statistical evaluation methods revealed a higher sensitivity of BEAS-2B cells. The BMD-derived mode of action for CuO confirmed the existing hypotheses and provided insights into less known mechanisms. CONCLUSION: The findings proofed that BMD analysis is an effective tool to evaluate different aspects of risk assessment.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Células A549 , Benchmarking , Brônquios/citologia , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Humanos , Pulmão/citologia , Óxidos , Medição de Risco
11.
Sci Rep ; 10(1): 7865, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398725

RESUMO

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung-liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.


Assuntos
Aflatoxina B1/farmacologia , Técnicas de Cocultura/métodos , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Técnicas de Cultura de Órgãos/métodos , Esferoides Celulares/efeitos dos fármacos , Administração por Inalação , Apoptose/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Venenos/farmacologia , Substâncias Protetoras/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
12.
Toxicol Lett ; 326: 61-69, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32169443

RESUMO

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.


Assuntos
Apoptose/efeitos dos fármacos , Brônquios/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Receptores Notch/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Humanos
13.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L908-L920, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32159371

RESUMO

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.


Assuntos
AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Isoquinolinas/farmacologia , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Pirimidinonas/farmacologia , Células Epiteliais da Tireoide/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Colforsina/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Regulador de Condutância Transmembrana em Fibrose Cística/classificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Mutação , Cultura Primária de Células , Ratos , Ratos Endogâmicos F344 , Células Epiteliais da Tireoide/citologia , Células Epiteliais da Tireoide/metabolismo , Transgenes
14.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L873-L887, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32160007

RESUMO

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.


Assuntos
Ácido Acético/administração & dosagem , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/genética , Obstrução das Vias Respiratórias/induzido quimicamente , Fibrose Cística/induzido quimicamente , Diminazena/análogos & derivados , Canais Iônicos Sensíveis a Ácido/metabolismo , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/patologia , Animais , Animais Recém-Nascidos , Bicarbonatos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Cloretos/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Diminazena/farmacologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Suínos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Traqueia/patologia
15.
Rev Mal Respir ; 37(3): 201-204, 2020 Mar.
Artigo em Francês | MEDLINE | ID: mdl-32139106

RESUMO

The main purpose of this review is to highlight mitochondria as a new therapeutic target to prevent bronchial smooth muscle (BSM) remodeling in asthma. Severe asthmatic patients, representing 5-10% of all asthmatics, are characterized by an increased BSM mass which is highly correlated with the severity of the disease and the rate of exacerbations. None of the current asthma therapies are effective in reducing BSM remodelling. This review, based on the current literature, reports the role of mitochondria in BSM, particularly in calcium signaling.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Brônquios , Mitocôndrias Musculares/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Animais , Antiasmáticos/administração & dosagem , Asma/metabolismo , Asma/patologia , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/ultraestrutura , Sistemas de Liberação de Medicamentos/métodos , Metabolismo Energético/efeitos dos fármacos , Humanos , Mitocôndrias Musculares/metabolismo , Terapia de Alvo Molecular/tendências , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/ultraestrutura , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Terapias em Estudo/métodos , Terapias em Estudo/tendências
17.
Environ Toxicol ; 35(8): 861-866, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32198912

RESUMO

Radon and its progeny have been classified as human class I carcinogens by the IARC. However, the mechanisms by which radon induces lung and other cancers, especially the radon-induced Warburg effect, have not been fully elucidated. The aim of this study was to investigate the role of the succinate dehydrogenase subunit A (SDHA)-mediated Warburg effect in (human bronchial epithelial) BEAS-2B cells with malignant transformations induced by long-term radon exposure. Soft agar colony formation and MMP-9 were increased following radon-induced malignant transformation. Additionally, we observed the Warburg effect in BEAS-2B cells following long-term radon exposure, evidenced by increases in the levels of glucose uptake, lactate, and lactate dehydrogenase (LDH). Following radon exposure, the expression of SDHA was decreased, while the levels of HIF-1α and hexokinase-2 (HK2) were increased. Our findings suggested that the SDHA-associated pathway may be involved in mediating the Warburg effect in radon-induced malignant transformation of BEAS-2B.


Assuntos
Poluentes Radioativos do Ar/toxicidade , Radônio/toxicidade , Brônquios/efeitos dos fármacos , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Metaloproteinase 9 da Matriz/metabolismo , Testes de Toxicidade
18.
BMC Pulm Med ; 20(1): 26, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000730

RESUMO

BACKGROUND: IL-17A directly induces epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. It could coordinate with cigarette smoke extract (CSE) to promote proliferation of bronchial epithelial cells. In this study, we aim to explore the direct effect of IL-17A and CSE on EMT in bronchial epithelial cells. METHODS: Bronchial epithelial cells were isolated from C57BL/6 mice, and cocultured with CSE or/and IL-17A. E-cadherin and Vimentin expressions in cells were detected using immunofluorescence staining. IL-17R expression was detected using immunohistochemistry staining. NF-κB expression was assessed using western blotting. When NF-κB was inhibited by BAY 11-7821, expressions of NF-κB, E-cadherin and Vimentin were measured. RESULTS: The protein expression of E-cadherin in bronchial epithelial cells was lowest in CSE + IL-17A group, followed by CSE group. In contrast, the protein expression of Vimentin was highest in CSE + IL-17A group, followed by CSE group. Similarly, IL-17R and NF-κB expressions were highest in CSE + IL-17A group, followed by CSE group and IL-17A group. NF-κB inhibitor could inhibit the expressions of E-cadherin and Vimentin. CONCLUSIONS: Cigarette and IL-17A could synergistically induce EMT in bronchial epithelial cells through activating IL17R/NF-κB signaling. Our findings contribute to a better understanding in airway EMT and pathogenesis of respiratory diseases, which are involved IL-17A and cigarette smoking. Those will provide novel avenues in the immunotherapy of lung diseases.


Assuntos
Fumar Cigarros/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Interleucina-17/farmacologia , Transdução de Sinais , Produtos do Tabaco/toxicidade , Animais , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Caderinas/metabolismo , Fumar Cigarros/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Receptores de Interleucina-17/metabolismo , Produtos do Tabaco/análise
19.
Exp Cell Res ; 389(2): 111897, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035951

RESUMO

Mucins are major macromolecular components of lung mucus that are mainly responsible for the viscoelastic property of mucus. MUC5AC is a major mucin glycoprotein that is hypersecreted in asthmatic individuals. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Our previous studies indicate that VEGF upregulates MUC5AC expression by interacting with VEGF receptor 2 (VEGFR2). It has been shown that dexamethasone (Dex) downregulates MUC5AC expression; however, the underlying mechanisms have not been completely elucidated. Therefore, we sought to investigate the effect of Dex on MUC5AC expression induced by VEGF and study the underlying mechanisms. We tested the effects of Dex on VEGFR2 and RhoA activation, caveolin-1 expression, and the association of caveolin-1 and VEGFR2 in primary bronchial epithelial cells. Dex downregulated MUC5AC mRNA and protein levels in a dose- and time-dependent manner, and suppressed the activation of VEGFR2 and RhoA induced by VEGF. Additionally, Dex upregulated caveolin-1 protein levels in a dose- and time-dependent manner. Furthermore, phospho-VEGFR2 expression was decreased through overexpression of caveolin-1 and increased after caveolin-1 knockdown. Dex treatment attenuated the VEGF-decreased association of caveolin-1 and VEGFR2. Collectively, our findings suggest that Dex downregulates VEGF-induced MUC5AC expression by inactivating VEGFR2 and RhoA. Furthermore, decreased MUC5AC expression by Dex was related to the increased association of caveolin-1 with VEGFR2. Further studies characterizing these mechanisms are required to facilitate the development of improved treatment strategies for asthma.


Assuntos
Asma/patologia , Brônquios/metabolismo , Dexametasona/farmacologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucina-5AC/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Asma/metabolismo , Brônquios/citologia , Brônquios/efeitos dos fármacos , Caveolina 1/genética , Caveolina 1/metabolismo , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Mucina-5AC/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Environ Toxicol ; 35(5): 609-618, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31943712

RESUMO

Nickel is a common environmental pollutant that can impair the lung, but the underlying mechanisms have not yet been fully elucidated. Furthermore, natural products are generally used to inhibit cell damage induced by heavy metal. Resveratrol possesses wide biological activities, including anti-inflammation and antioxidative stress. This study was conducted to explore the toxicity of nickel on human bronchial epithelial (BEAS-2B) cells and evaluate the protective effect of resveratrol. The results showed that nickel could induce cell apoptosis, increase oxidative stress, and promote the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-8, C-reaction protein. Western blot analysis showed that nickel activated p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B, and nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 pathways, while resveratrol could reverse these effects. Our results suggested that resveratrol could protect BEAS-2B cells from nickel-induced cytotoxicity. Therefore, resveratrol is a potential chemopreventive agent against nickel-induced lung disease.


Assuntos
Antioxidantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Inflamassomos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Níquel/toxicidade , Resveratrol/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia
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