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1.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi ; 37(11): 806-809, 2019 Nov 20.
Artigo em Chinês | MEDLINE | ID: mdl-31826542

RESUMO

Objective: To investigate the expression and role of LINC00052 during glycidyl methacrylate (GMA) -induced malignant transformation of 16HBE cells. Methods: Human bronchial epithelial (16HBE) cells were divided into GMA transformation group and corresponding DMSO control group, and the 10th, 20th and 30th generation cells of each group were collected LncRNA microarrays were used to analysis expression of LINC00052 in different stage of malignant transformation. Bioinformatics analysis was applied and the relative expression of LINC00052 and its potentially target genes was detected by real-time quantification PCR (qPCR) . Results: The results of microarray analysis showed that LINC00052 was up-regulated by 1.32-fold, down-regulated by 1.64-fold and down-regulated by 4.92-fold in the malignant transformation early (P10) , middle term (P20) and late (P30) , respectively, The results of qPCR showed that compared with the DMSO control group, the expression of LINC00052 was up-regulated by 1.55 times, down-regulated by 1.20 times and down-regulated by 2.35 times in P10, P20 and P30, respectively, and the difference was statistically significant (P<0.05) . There was a statistically significant difference in the relative expression of NTRK3 between the GMA transformation group of P10 and P30 generations with the corresponding DMSO control group (P<0.05) . Conclusion: LINC00052 is highly expressed in early time of GMA-induced malignant transformation of 16HBE, and down-regulated in the middle and last stage of malignant transformation and may play a protective role in GMA-induced malignant transformation of 16HBE by influencing the expression of its target gene NTRK3.


Assuntos
Transformação Celular Neoplásica , Células Epiteliais , Compostos de Epóxi , Regulação Neoplásica da Expressão Gênica , Metacrilatos , RNA Longo não Codificante , Brônquios/citologia , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Longo não Codificante/genética
2.
Eur J Histochem ; 63(3)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31505925

RESUMO

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.


Assuntos
Acetilgalactosamina/metabolismo , Brônquios/citologia , Células Epiteliais/metabolismo , Galactose/metabolismo , Alvéolos Pulmonares/citologia , Animais , Brônquios/crescimento & desenvolvimento , Feminino , Histocitoquímica/métodos , Peroxidase do Rábano Silvestre/química , Neuraminidase/química , Lectinas de Plantas/química , Gravidez , Alvéolos Pulmonares/crescimento & desenvolvimento , Ratos Wistar , Proteínas de Soja/química
3.
Artigo em Chinês | MEDLINE | ID: mdl-31495106

RESUMO

Objective: To study the effect of particulate matter 2.5 (PM(2.5)) on oncogene expression in human bronchial epithelial (HBE) cells. Methods: HBE cells were selected as the study subjects, and PM(2.5) treatment group (10 µg/ml and 50 µg/ml) , negative control group and positive control group (10 µmol/L Cr(6+)) were set. CCK8 assay was used to test the IC(50) value of PM(2.5). HBE cells were treated with PM(2.5) for 24 h at 10 µg/ml and 50 µg/ml, additionally, cells were treated with blank as negative control, 10 µmol/L Cr(6+) as a positive control for 24 h. After the treatment, mRNA expression of oncogenes including c-myc, c-fos, k-ras and p53 were detected by fluorescent quantitative RT-PCR, the protein expression of oncogenes were detected with western blot. Results: The IC(50) value of PM(2.5) in HBE cells is 70.12 µg/ml. The qRT-PCR data showed that compared with the control group, the expression level of c-myc gene increased by respectively 500.1%、780.7%、305.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; c-fos gene increased respectively 34.0%、76.7%、131.3% after exposure to 10、50 µg/ml PM(2.5) and positive control group; k-ras gene increased respectively 50.3%、107.0%、49.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 gene decreased by 28.3%、28.7%、59.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group. The western blot results showed that compared with the control group, c-myc protein increased respectively 29.7%、77.3% after exposure to 50 µg/ml PM(2.5) and positive control group; c-fos protein increased respectively 200.3%、137.0% after exposure to 50 µg/ml PM(2.5) and positive control group; k-ras protein increased respectively 106.3%、130.3%、116.7% after exposure to 10、50 µg/ml PM(2.5) and positive control group; p53 protein decreased by 43.7%、53.3%、52.1% after exposure to 10、50 µg/ml PM(2.5) and positive control group. Conclusion: PM(2.5) could promote the expression of oncogenes in HBE cells, the carcinogenicity of haze might be related to promotion of oncogenes expression induced by PM(2.5).


Assuntos
Células Epiteliais/efeitos dos fármacos , Oncogenes , Material Particulado/toxicidade , Brônquios/citologia , Células Cultivadas , Humanos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética
4.
Mol Pharmacol ; 96(4): 515-525, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31427400

RESUMO

ORKAMBI, a combination of the corrector, lumacaftor, and the potentiator, ivacaftor, partially rescues the defective processing and anion channel activity conferred by the major cystic fibrosis-causing mutation, F508del, in in vitro studies. Clinically, the improvement in lung function after ORKAMBI treatment is modest and variable, prompting the search for complementary interventions. As our previous work identified a positive effect of arginine-dependent nitric oxide signaling on residual F508del-Cftr function in murine intestinal epithelium, we were prompted to determine whether strategies aimed at increasing arginine would enhance F508del-cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in patient-derived airway epithelia. Now, we show that the addition of arginine together with inhibition of intracellular arginase activity increased cytosolic nitric oxide and enhanced the rescue effect of ORKAMBI on F508del-CFTR-mediated chloride conductance at the cell surface of patient-derived bronchial and nasal epithelial cultures. Interestingly, arginine addition plus arginase inhibition also enhanced ORKAMBI-mediated increases in ciliary beat frequency and mucociliary movement, two in vitro CF phenotypes that are downstream of the channel defect. This work suggests that strategies to manipulate the arginine-nitric oxide pathway in combination with CFTR modulators may lead to improved clinical outcomes. SIGNIFICANCE STATEMENT: These proof-of-concept studies highlight the potential to boost the response to cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, lumacaftor and ivacaftor, in patient-derived airway tissues expressing the major CF-causing mutant, F508del-CFTR, by enhancing other regulatory pathways. In this case, we observed enhancement of pharmacologically rescued F508del-CFTR by arginine-dependent, nitric oxide signaling through inhibition of endogenous arginase activity.


Assuntos
Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Arginase/antagonistas & inibidores , Arginina/metabolismo , Benzodioxóis/farmacologia , Fibrose Cística/metabolismo , Óxido Nítrico/metabolismo , Quinolonas/farmacologia , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Citosol/metabolismo , Combinação de Medicamentos , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Mutação , Nariz/citologia , Nariz/efeitos dos fármacos
5.
Eur J Pharmacol ; 859: 172548, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31323224

RESUMO

Due to the radiosensitivity of the airway epithelium, radiation-induced sinusitis or bronchitis is not uncommon, and makes mitigation of resulting inflammatory airway diseases a principal goal of many investigations. This study examined whether Ovatodiolide (Ova) sensitizes the human metastatic nasopharyngeal cancer (NPC) cell line, NPC-BM2, to irradiation using viability, clonogenicity and Western blot assays. Concurrently, we used varying concentrations of histamine and/or Ova to determine the anti-inflammatory potential of Ovatodiolide on normal bronchus epithelial BEAS-2B cells, as well as on the subcellular distribution of Aquaporin 5 (AQP5) and expression levels of p-CREB, AQP5, p38 MAPK, NF-κB, PI3K, Akt and ERK proteins. We demonstrated that Ova in synergism with irradiation inhibited NPC-BM2 cell viability and suppressed their clonogenicity. Immunofluorescence analysis revealed low-dose (≤ 2.5 µM) Ova reversed histamine-induced suppression of AQP5 expression, and abrogated histamine-enhanced NF-κB nuclear translocation, indicating Ova modulates the p38 MAPK/NF-κB signaling pathway and elicits p-CREB/AQP5-mediated antihistamine effects. Similarly, Ova deregulates the PI3K/Akt/ERK signaling in BEAS-2B cells, suggesting its cytoprotective potential. In conclusion, this study highlights the radio-sensitizing anticancer efficacy of Ova in human metastatic NPC cells, as well as its putative cytoprotective role in normal bronchial cells, for airway surface liquid maintenance and homeostasis during or after radiotherapy.


Assuntos
Aquaporina 5/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diterpenos/farmacologia , Células Epiteliais/efeitos dos fármacos , Carcinoma Nasofaríngeo/patologia , Tolerância a Radiação/efeitos dos fármacos , Brônquios/citologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/radioterapia , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Vet Microbiol ; 235: 80-85, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282382

RESUMO

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.


Assuntos
Coinfecção/veterinária , Células Epiteliais , Interações Microbianas , Infecções por Pasteurella/veterinária , Infecções por Vírus Respiratório Sincicial/veterinária , Animais , Complexo Respiratório Bovino/microbiologia , Complexo Respiratório Bovino/virologia , Brônquios/citologia , Brônquios/microbiologia , Brônquios/virologia , Bovinos , Linhagem Celular , Células Cultivadas , Coinfecção/microbiologia , Coinfecção/virologia , Citocinas/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Pulmão/citologia , Pulmão/microbiologia , Pulmão/virologia , Infecções por Pasteurella/virologia , Pasteurella multocida/genética , Pasteurella multocida/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/microbiologia , Vírus Sincicial Respiratório Bovino/genética , Vírus Sincicial Respiratório Bovino/isolamento & purificação , Traqueia/citologia , Traqueia/microbiologia , Traqueia/virologia
7.
Int J Pharm ; 567: 118500, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31288056

RESUMO

The development of new antibacterial molecules is essential in view of the emergence of pathogenic strains resistant to multiple antibiotics. Among the infectious pathologies, pulmonary infections are particularly difficult to treat due to the complexity of lung anatomy and the presence of natural barriers such as mucus. At present, the aerosol delivery of antibacterial compounds is still poorly employed. Furthermore, the presence of bacteria in lungs negatively affects aerosolized Cystic Fibrosis gene therapy efficiency. A multi-functional formulation (antibacterial and transfection activities) could increase the therapeutic effect. This work reports the synthesis of new N-heterocyclic carbene silver complexes (Ag-NHC) featuring a lipid chain and the evaluation of their antibacterial potency, especially when delivered following aerosolization. When formulated alone in water, these Ag-NHC displayed remarkable antibacterial activities against some Staphyloccocus aureus strains and Pseudomonas aeruginosa clinical strains. Moreover, combined with cationic lipid and DNA (ternary combination), they could be used to deliver therapeutic genes via aerosolization in infected lungs. Altogether, the data reported herein support n-alkyl chain Ag-NHC as a possible alternative to conventional antibiotics for treating respiratory infections and to combat the emergence of multi-resistant bacteria.


Assuntos
Antibacterianos/administração & dosagem , DNA/administração & dosagem , Metano/análogos & derivados , Prata/administração & dosagem , Transfecção/métodos , Aerossóis , Brônquios/citologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Luciferases/genética , Metano/administração & dosagem , Plasmídeos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
8.
Nutrients ; 11(6)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181780

RESUMO

Allium genus plants, such as leek (Allium porrum), are rich sources of anti-inflammatory and anti-oxidant secondary metabolites; this is of interest because it demonstrates their suitability as pharmacological alternatives for inflammatory processes, including allergy treatment. The composition of methanolic leek extract (LE) was analyzed by GC-MS and LC-IT/MS, and the total phenolic content and antioxidant capacity were quantified by colorimetric methods. Its pharmacological potential was analyzed in human bronchial epithelial Calu-3 cells, human mast cells LAD2, and humanized rat basophiles RBL-2H3. LE exhibited a cytotoxic effect on Calu-3 cells and HumRBL-2H3 cells only at high concentrations and in a dose-dependent manner. Moreover, LE decreased the degranulation of LAD2 and HumRBL-2H3 cells. LE treatment also significantly prevented alterations in transepithelial electrical resistance values and mRNA levels of glutathione-S-transferase (GST), c-Jun, and NFκB after treatment with H2O2 in ALI-cultured Calu-3 cells. Finally, ALI-cultured Calu-3 cells treated with LE showed lower permeability to Ole e 1 compared to untreated cells. A reduction in IL-6 secretion in ALI-cultured Calu-3 cells treated with LE was also observed. In summary, the results obtained in this work suggest that A. porrum extract may have potential anti-allergic effects due to its antioxidant and anti-inflammatory properties. This study provides several important insights into how LE can protect against allergy.


Assuntos
Antialérgicos/farmacologia , Brônquios/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Cebolas/química , Fenóis/uso terapêutico , Animais , Antialérgicos/análise , Antialérgicos/uso terapêutico , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/análise , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Humanos , Hipersensibilidade/metabolismo , Hipersensibilidade/prevenção & controle , Inflamação/metabolismo , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Fenóis/análise , Fenóis/farmacologia , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos
9.
Vet Microbiol ; 234: 119-127, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213267

RESUMO

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.


Assuntos
Orthoreovirus/fisiologia , Tropismo Viral , Replicação Viral , Células Epiteliais Alveolares/virologia , Animais , Vírus Bluetongue/fisiologia , Brônquios/citologia , Brônquios/virologia , Cinética , Técnicas de Cultura de Órgãos , Ovinos , Suínos
10.
Cell Physiol Biochem ; 53(1): 49-61, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169991

RESUMO

BACKGROUND/AIMS: The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. METHODS: Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. RESULTS: Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. CONCLUSION: Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.


Assuntos
Fusobacterium nucleatum/patogenicidade , Interleucina-6/metabolismo , Sistema Respiratório/microbiologia , Animais , Brônquios/citologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-8/sangue , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Respiratório/metabolismo , Streptococcus pneumoniae/patogenicidade
11.
Artif Cells Nanomed Biotechnol ; 47(1): 2293-2297, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31172816

RESUMO

Objective: To study the effect of formaldehyde on the proliferation of human bronchial epithelial cells 16HBE and to explore its mechanism. Methods: MTT assay was used to detect the inhibition rate of formaldehyde-treated 16HBE cells; FCOH + miR-375 group (transfected miR-375 mimics), FCOH + miR-con group (transfected miR-con), FCOH + si-KLF4 group (transfected si-KLF4) and FCOH + si-con group (transfected si-con), were transfected into 16HBE cells by liposome method, then treated with formaldehyde 200 µmol/L for 24 h; qRT-PCR was used to detect the expression of miR-375 in each group; the protein expression of KLF4 in each group was detected by Western blot. The fluorescence activity of each group was detected by dual-fluorescein gene detection assay. Results: Compared with 16HBE cells in Control group, the expression of miR-375 was significantly decreased in FCOH group, cell proliferation was significantly decreased, and KLF4 expression was significantly increased (p < .05). Overexpression of miR-375 and KLF4 knockdown could reverse the inhibition effect of formaldehyde on proliferation of 16HBE cells; KLF4 is a target of miR-375. KLF4 could reverse the promotion of miR-375 on the proliferation of formaldehyde-treated 16HBE cells. Conclusion: Formaldehyde can inhibit the proliferation of human bronchial epithelial cells. The mechanism may be related to the down-regulation of miR-375 targeting KLF4, which will provide support for the treatment of chronic respiratory diseases.


Assuntos
Brônquios/citologia , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Formaldeído/farmacologia , MicroRNAs/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética
12.
J Pharmacol Exp Ther ; 370(1): 104-110, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068382

RESUMO

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.


Assuntos
Adenilil Ciclases/metabolismo , Brônquios/citologia , AMP Cíclico/biossíntese , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/citologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Humanos , Isoproterenol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos
13.
Chemosphere ; 229: 284-294, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078885

RESUMO

Hexavalent chromium (Cr(VI)) is a well-known human carcinogen and a strong oxidizer that causes severe DNA damage. However, the associations between epigenetic dysregulation and DNA damage have not been well-characterized. In this study, we evaluated the effects of short-term and long-term exposure to Cr(VI) in human bronchial epithelial (16HBE) cells. Then, we explored the role of epigenetic modification in Cr(VI)-induced DNA damage. We found that short- and long-term exposure to Cr(VI) induced DNA damage and reduced the expression 53BP1, but increased the expression of other DNA repair mediators. Short- and long-term exposure to Cr(VI) reduced the levels of H3K18ac and H3K27ac and reduced their enrichment at the promoter of 53BP1. Long-term Cr(VI) exposure resulted in multiple malignant characteristics including cell invasion, migration, and tumorgenicity. These data demonstrated that reduced H3K18ac and H3K27ac following Cr(VI) treatment contributed to the suppression of 53BP1. Our study demonstrated that epigenetic changes and DNA damage responses are involved in short-term toxicity and long-term carcinogenesis induced by Cr(VI).


Assuntos
Cromo/farmacologia , Exposição Ambiental/efeitos adversos , Epigênese Genética , Células Epiteliais/efeitos dos fármacos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Brônquios/citologia , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Humanos , Fatores de Tempo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/efeitos dos fármacos
14.
J Vis Exp ; (145)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30958479

RESUMO

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.


Assuntos
Técnicas de Cultura de Células/instrumentação , Brônquios/citologia , Contagem de Células , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Géis , Humanos , Imagem Molecular
15.
Toxicol In Vitro ; 59: 228-237, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31002973

RESUMO

Biosoluble AES wools are increasingly used since considered not hazardous, however, few toxicity studies are available. We evaluated cytotoxic, genotoxic-oxidative and inflammatory effects of two differently soluble AES wools, AES1 (high MgO percentage) and AES2 (high CaO percentage), on alveolar (A549) and bronchial (BEAS-2B) cells. Fiber dimensions and dissolution in cell media were evaluated by SEM analysis. Cell viability, LDH release, direct/oxidative DNA damage (fpg-comet assay) and IL-6, IL-8 and TNF-α release (ELISA), were analysed after 24 h exposure to 2-200 µg/ml. On A549 cells AES1 induced LDH release, slight direct DNA damage and oxidative DNA damage with very high IL-6 release at 100 µg/ml; AES2 induced higher DNA damage than AES1 and slight oxidative DNA damage. On BEAS-2B cells we found direct DNA damage (higher for AES1) and slight oxidative DNA damage (associated to slight increased IL-6 and IL-8 release for AES1). The higher genotoxicity of more soluble AES2 on A549 cells could be explained by higher respirable fibers % and fiber number/µg found after 24 h in RPMI-medium at 100 µg/ml. The higher membrane damage, oxidative DNA damage and inflammation induced by AES1 in A549 cells could be due to the higher DLG and silica percentage. These findings suggest further investigations on AES toxicity.


Assuntos
Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Alvéolos Pulmonares/citologia , Silicatos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Citocinas/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Humanos , Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos
16.
Environ Pollut ; 250: 650-661, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31035147

RESUMO

Aldehydes are well-known air pollutants and often studied alone, while co-exposure to aldehyde mixtures is more common than single aldehydes. Unfortunately, it has been very little known about the (mechanism of) combined toxicity of aldehyde mixtures. Here, formaldehyde and acrolein were selected as the typical representatives of common aldehydes, and were used to explore to get in-depth insight into the mechanism of combined toxicity of aldehyde mixtures. The NOECs (non-observed effect concentrations) are 60 µmoL/L for formaldehyde, and 0.5 µmoL/L for acrolein, so acrolein is more toxic than formaldehyde. Formaldehyde and acrolein mixtures showed significant cytotoxicity and synergistic effects in a concentration/time-dependent way on BEAS-2B cells based on acute and chronic cytotoxicity assay. Acrolein was dominant in aldehyde mixtures in inducing cytotoxicity at environmentally relevant doses because of higher toxicity. Moreover, aldehyde mixtures significantly synergistically increased the intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) leakage, while caused an antagonistic effects on glutathione (GSH). Besides, formaldehyde could significantly potentiated the activation of environmental stress sensitive Nrf2 pathway induced by acrolein, even at doses at which formaldehyde treatment alone had no any response. Furthermore, as the downstream components of Nrf2 pathway, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) and heme oxygenase-1 (HO-1) were significantly synergistically induced by formaldehyde and acrolein mixtures. Antioxidants N-acetylcysteine and reduced glutathione could significantly suppress the acute and chronic combined cytotoxicity of acrolein and formaldehyde mixtures, and changed their interactions (synergism) on cytotoxicity. Taken together, aldehyde mixtures have higher toxicity than that expected for additivity based on single aldehydes even at environmentally relevant concentrations, and the combined cytotoxicity may be enhanced through oxidative stress and the related Nrf2 pathway. Prolonged exposure to pollutants containing aldehyde mixtures through inhalation may have more serious threat to respiratory system in animal and human.


Assuntos
Acroleína/toxicidade , Poluentes Atmosféricos/toxicidade , Brônquios/efeitos dos fármacos , Citotoxinas/toxicidade , Formaldeído/toxicidade , Antioxidantes/química , Brônquios/citologia , Catalase/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo
17.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30962401

RESUMO

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.


Assuntos
Células Epiteliais/microbiologia , Mannheimia haemolytica/patogenicidade , Pasteurelose Pneumônica/microbiologia , Animais , Brônquios/citologia , Brônquios/microbiologia , Bovinos , Mannheimia haemolytica/crescimento & desenvolvimento , Mannheimia haemolytica/fisiologia , Sistema Respiratório/microbiologia , Virulência
18.
Eur J Pharmacol ; 853: 229-235, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30935895

RESUMO

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.


Assuntos
Movimento Celular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Niclosamida/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Acetilcolina/farmacologia , Animais , Brônquios/citologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Potássio/farmacologia , Ratos , Traqueia/citologia , Vasoconstrição/efeitos dos fármacos
19.
Chem Biol Interact ; 305: 119-126, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30935901

RESUMO

Epidemiological and toxicological studies indicate that polyhexamethylene guanidine phosphate (PHMG-p) is a guanidine-based cationic disinfectant strongly associated with interstitial lung diseases. As individuals exposed to aerosolized PHMG-p complain of respiratory problems (asthma and rhinitis), whether PHMG-p can cause respiratory diseases other than interstitial fibrosis should be investigated. MUC5AC, the predominant mucin gene expressed in airways, is associated with obstructive respiratory disease pathogenesis. Therefore, in this study, we elucidated the relationship between PHMG-p and MUC5AC overexpression. First, in immunofluorescence studies, the bronchial epithelia of mice intratracheally administrated PHMG-p appeared to be sloughing and tethered by MUC5AC. Second, Calu-3 cells exposed to PHMG-p showed concentration-dependent increases in MUC5AC mRNA and protein expression. c-Jun N-terminal kinase (JNK), p38, and c-jun were phosphorylated in cells exposed to PHMG-p. SP600125 and SB203580, JNK and p38 inhibitors, respectively, reduced the upregulation of MUC5AC by PHMG-p in Calu-3 cells. When toll-like receptor (TLR)2, 4, and 6 were silenced, PHMG-p-induced JNK and p38 phosphorylation decreased. Furthermore, TLR2-, 4-, and 6-silenced cells showed reduced levels of MUC5AC mRNA and protein induced by PHMG-p, with TLR6 knockdown showing the greatest effect. In conclusion, PHMG-p induced MUC5AC overexpression via activation of the TLR-p38 MAPK and JNK axis.


Assuntos
Guanidinas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mucina-5AC/metabolismo , Receptores Toll-Like/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Brônquios/citologia , Brônquios/metabolismo , Brônquios/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC/genética , Muco/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
20.
Toxicol In Vitro ; 58: 60-68, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30898553

RESUMO

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 µg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Emissões de Veículos/toxicidade , Brônquios/citologia , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética
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