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1.
Ecotoxicol Environ Saf ; 210: 111857, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33421718

RESUMO

OBJECTIVES: Benzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression. METHODS: The human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus. RESULTS: We found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation. CONCLUSION: Here, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.


Assuntos
Poluentes Atmosféricos/toxicidade , Benzo(a)pireno/toxicidade , Células Epiteliais/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mucina-5AC/genética , Mucina-5AC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Sistema Respiratório/citologia , Transdução de Sinais/efeitos dos fármacos
2.
Mar Drugs ; 18(12)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327522

RESUMO

The mucus layer of the nasopharynx and bronchial epithelium has a barrier function against inhaled pathogens such as the coronavirus SARS-CoV-2. We recently found that inorganic polyphosphate (polyP), a physiological, metabolic energy (ATP)-providing polymer released from blood platelets, blocks the binding of the receptor binding domain (RBD) to the cellular ACE2 receptor in vitro. PolyP is a marine natural product and is abundantly present in marine bacteria. Now, we have approached the in vivo situation by studying the effect of polyP on the human alveolar basal epithelial A549 cells in a mucus-like mucin environment. These cells express mucins as well as the ectoenzymes alkaline phosphatase (ALP) and adenylate kinase (ADK), which are involved in the extracellular production of ATP from polyP. Mucin, integrated into a collagen-based hydrogel, stimulated cell growth and attachment. The addition of polyP to the hydrogel significantly increased cell attachment and also the expression of the membrane-tethered mucin MUC1 and the secreted mucin MUC5AC. The increased synthesis of MUC1 was also confirmed by immunostaining. This morphogenetic effect of polyP was associated with a rise in extracellular ATP level. We conclude that the nontoxic and non-immunogenic polymer polyP could possibly also exert a protective effect against SARS-CoV-2-cell attachment; first, by stimulating the innate antiviral response by strengthening the mucin barrier with its antimicrobial proteins, and second, by inhibiting virus attachment to the cells, as deduced from the reduction in the strength of binding between the viral RBD and the cellular ACE2 receptor.


Assuntos
Organismos Aquáticos/metabolismo , Produtos Biológicos/farmacologia , Polifosfatos/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Células A549 , Bactérias/metabolismo , Produtos Biológicos/uso terapêutico , Humanos , Imunidade Inata/efeitos dos fármacos , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Polifosfatos/metabolismo , Polifosfatos/uso terapêutico , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , /patogenicidade , Metabolismo Secundário , Ligação Viral/efeitos dos fármacos
3.
J Pharmacol Sci ; 144(4): 189-196, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33070837

RESUMO

Pneumonia is a common illness that continues to be the major killer of remaining to be a significant source of morbidity and mortality in the patient population. Many microorganisms cause pneumonia, and now concern is turning to the importance of the cause the new therapies for viral pneumonia. In the current study, we report the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on poly I: C-induced pneumonia. Andrographolide sulfonate was administrated through intraperitoneal injection to mice with poly I: C-induced pneumonia. Recruitment of airway inflammatory cells, alteration of lung histological induced by Poly I: C were significantly ameliorated by andrographolide sulfonate. The protein levels of pro-inflammatory cytokines in bronchoalveolar fluid (BALF) and serum were reduced by andrographolide sulfonate treatment. The levels of MUC5AC and MUC5B in lung tissue were also suppressed. These results reveal that andrographolide sulfate remarkably alleviated pneumonia induced by poly I:C in mice. Moreover, andrographolide sulfonate markedly inhibited the activation of nuclear factor-κB (NF-κB). Taken together, we demonstrated that andrographolide sulfonate ameliorated poly I: C-induced pneumonia in mice, suggesting the possible use of andrographolide sulfonate for virus-induced pneumonia in clinical.


Assuntos
Diterpenos/administração & dosagem , Diterpenos/farmacologia , NF-kappa B/metabolismo , Fitoterapia , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Poli I-C/efeitos adversos , Animais , Citocinas/sangue , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos C57BL , Mucina-5AC/metabolismo , Mucina-5B/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/patologia , Pneumonia Viral/tratamento farmacológico
4.
Viruses ; 12(6)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599823

RESUMO

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host-pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Junções Intercelulares , Células Madin Darby de Rim Canino , Modelos Biológicos , Mucina-5AC/metabolismo , Pandemias , Cultura de Vírus
5.
Clin Sci (Lond) ; 134(10): 1107-1125, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32400877

RESUMO

There is little information on mucins versus potential regulatory factors in the peripheral airway lumen of long-term smokers with (LTS+) and without (LTS-) chronic obstructive pulmonary disease (COPD). We explored these matters in bronchoalveolar lavage (BAL) samples from two study materials, both including LTS+ and LTS- with a very similar historic exposure to tobacco smoke, and healthy non-smokers (HNSs; n=4-20/group). Utilizing slot blot and immunodetection of processed (filtered and centrifuged), as well as unprocessed BAL samples from one of the materials, we compared the quantity and fraction of large complexes of mucins. All LTS displayed an enhanced (median) level of MUC5AC compared with HNS. LTS- displayed a higher level of large MUC5AC complexes than HNS while LTS+ displayed a similar trend. In all LTS, total MUC5AC correlated with blood leukocytes, BAL neutrophil elastase and net gelatinase activity. Large mucin complexes accounted for most MUC5B, without clear group differences. In all LTS, total MUC5B correlated with total MUC5AC and local bacteria. In the same groups, large MUC5B complexes correlated with serum cotinine. MUC1 was increased and correlated with BAL leukocytes in all LTS whereas MUC2 was very low and without clear group differences. Thus, the main part of MUC5AC and MUC5B is present as large complexes in the peripheral airway lumen and historic as well as current exposure to tobacco smoke emerge as potential regulatory factors, regardless of COPD per se. Bacteria, leukocytes and proteinases also constitute potential regulatory factors, of interest for future therapeutic strategies.


Assuntos
Pulmão/metabolismo , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Complexos Multiproteicos/metabolismo , Fumantes , Fumar/metabolismo , Bactérias/crescimento & desenvolvimento , Lavagem Broncoalveolar , Difusão , Feminino , Gases/metabolismo , Humanos , Pulmão/microbiologia , Masculino , Viabilidade Microbiana , Mucina-2/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fatores de Tempo
6.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L82-L90, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32401676

RESUMO

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.


Assuntos
Células Caliciformes/metabolismo , Mucina-5AC/metabolismo , Mucina-1/metabolismo , Fumar/efeitos adversos , Animais , Linhagem Celular Tumoral , Polaridade Celular , Células Epiteliais/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Receptores ErbB/metabolismo , Células Caliciformes/patologia , Metaplasia , Fosforilação , Ratos Sprague-Dawley
7.
PLoS One ; 15(4): e0231990, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320453

RESUMO

Environmental mold (fungus) exposure poses a significant threat to public health by causing illnesses ranging from invasive fungal diseases in immune compromised individuals to allergic hypertensive diseases such as asthma and asthma exacerbation in otherwise healthy people. However, the molecular pathogenesis has not been completely understood, and treatment options are limited. Due to its thermo-tolerance to the normal human body temperature, Aspergillus. fumigatus (A.fumigatus) is one of the most important human pathogens to cause different lung fungal diseases including fungal asthma. Airway obstruction and hyperresponsiveness caused by mucus overproduction are the hallmarks of many A.fumigatus induced lung diseases. To understand the underlying molecular mechanism, we have utilized a well-established A.fumigatus extracts (AFE) model to elucidate downstream signal pathways that mediate A.fumigatus induced mucin production in airway epithelial cells. AFE was found to stimulate time- and dose-dependent increase of major airway mucin gene expression (MUC5AC and MUC5B) partly via the elevation of their promoter activities. We also demonstrated that EGFR was required but not sufficient for AFE-induced mucin expression, filling the paradoxical gap from a previous study using the same model. Furthermore, we showed that fungal proteases in AFE were responsible for mucin induction by activating a Ras/Raf1/ERK signaling pathway. Ca2+ signaling, but ROS, both of which were stimulated by fungal proteases, was an indispensable determinant for ERK activation and mucin induction. The discovery of this novel pathway likely contributes to our understanding of the pathogenesis of fungal sensitization in allergic diseases such as fungal asthma.


Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/toxicidade , Interações Hospedeiro-Patógeno/fisiologia , Mucinas/metabolismo , Peptídeo Hidrolases/toxicidade , Aspergillus fumigatus/patogenicidade , Cálcio/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Pulmão/citologia , Sistema de Sinalização das MAP Quinases , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , Mucinas/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas ras/metabolismo
8.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1270-L1279, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348677

RESUMO

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b-/-, Muc5ac-/-, and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In Muc5ac-/- mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b-/- mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.


Assuntos
Mucina-5B/metabolismo , Muco/metabolismo , Sistema Respiratório/metabolismo , Animais , Transporte Biológico , Fluorescência , Células Caliciformes/metabolismo , Camundongos Endogâmicos C57BL , Mucina-5AC/metabolismo , Membrana Mucosa/metabolismo , Traqueia/metabolismo
9.
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
10.
Am J Physiol Lung Cell Mol Physiol ; 318(5): L1063-L1073, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32208929

RESUMO

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.


Assuntos
Meios de Cultura/farmacologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Ar , Brônquios/citologia , Brônquios/metabolismo , Diferenciação Celular/efeitos dos fármacos , Quimiocina CCL26/genética , Quimiocina CCL26/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Meios de Cultura/química , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Voluntários Saudáveis , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Modelos Biológicos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Cultura Primária de Células , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo
11.
Sci Rep ; 10(1): 2933, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076085

RESUMO

To compare goblet cell (GC) number and area in the covered superior (SB) versus exposed temporal (TB) bulbar conjunctiva in control versus aqueous tear deficient eyes (ATD) and evaluate correlation with tear MUC5AC protein. SB and TB impression cytology performed on control eyes, Sjögren syndrome (SS) ATD, and non-SS ATD was stained with period acid Schiff. GC number and area were measured with image analysis software. Protein-normalized MUC5AC level was measured in Schirmer strip-collected tears. Compared to control conjunctiva, GC number and area were significantly lower in SS, non-SS, and combined ATD groups in exposed TB, and were also significantly lower in SS and combined ATD groups in covered SB. In all ATD, GC number and area were significantly correlated, but differences between SB and TB were non-significant. Normalized tear MUC5AC protein was lower in all ATD groups versus control eyes, and correlated only with GC area. GCs are significantly decreased in the covered and exposed conjunctiva in SS. GC area may be a better disease measure than number for ATD. Correlation between tear MUC5AC concentration and GC area suggests tear MUC5AC mucin can be used as a disease-relevant biomarker for conjunctiva GC health.


Assuntos
Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Mucina-5AC/metabolismo , Lágrimas/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
J Immunol ; 204(6): 1650-1660, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32060135

RESUMO

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.


Assuntos
Fibrose Cística/imunologia , Interleucina-33/metabolismo , Pulmão/patologia , Mucina-5AC/metabolismo , Células Th2/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Fibrose Cística/patologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/metabolismo , Canais Epiteliais de Sódio/genética , Humanos , Interleucina-33/genética , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Muco/imunologia , Muco/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Células Th2/metabolismo
13.
Int J Mol Sci ; 21(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033337

RESUMO

This study investigated the effect of dexamethasone (DEX) on intracellular calcium levels and the expressions of transient receptor potential cation channel subcomponent V member 6 (TRPV6), sodium-calcium exchanger 1 (NCX1), and plasma membrane calcium ATPase 1 (PMCA1) in A549 cells. The intracellular calcium level, by using the calcium indicator pGP-CMV-GCaMP6f, increased following DEX treatment for 6, 12, and 24 h in A549 cells. In addition, Rhod-4 assay after DEX treatment for 24 h showed that DEX increased the level of intracellular calcium. The expression of the calcium influx TRPV6 gene significantly increased, whereas the expressions of the calcium outflow NCX1 and PMCA1 genes significantly decreased with DEX treatment. The mRNA levels of surfactant protein genes SFTPA1, SFTPB, SFTPC, and SFTPD and the secreted airway mucin genes MUC1 and MUC5AC were investigated by treating cells with DEX. The DEX treatment decreased the mRNA levels of SFTPA1 and SFTPB but increased the mRNA levels of SFTPC and SFTPD. The MUC1 mRNA level was increased by DEX treatment, whereas MUC5AC mRNA was significantly decreased. These results indicate that DEX influences the intracellular calcium level through TRPV6, and affects pulmonary surfactant genes and secreted airway mucin genes in A549 cells.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/análise , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Canais de Cátion TRPV/metabolismo , Células A549 , Canais de Cálcio/genética , Linhagem Celular , Humanos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo , RNA Mensageiro/genética , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPV/genética
14.
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
15.
Virology ; 540: 195-206, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31929001

RESUMO

Respiratory syncytial virus (RSV) infection can cause mucus overproduction and bronchiolitis in infants leading to severe disease and hospitalization. As a therapeutic strategy, immune modulatory agents may help prevent RSV-driven immune responses that cause severe airway disease. We developed a high throughput screen to identify compounds that reduced RSV-driven mucin 5AC (Muc5AC) expression and identified dexamethasone. Despite leading to a pronounced reduction in RSV-driven Muc5AC, dexamethasone increased RSV infection in vitro and delayed viral clearance in mice. This correlated with reduced expression of a subset of immune response genes and reduced lymphocyte infiltration in vivo. Interestingly, dexamethasone increased RSV infection levels without altering antiviral interferon signaling. In summary, the immunosuppressive activities of dexamethasone had favorable inhibitory effects on RSV-driven mucus production yet prevented immune defense activities that limit RSV infection in vitro and in vivo. These findings offer an explanation for the lack of efficacy of glucocorticoids in RSV-infected patients.


Assuntos
Dexametasona/farmacologia , Interferons/metabolismo , Muco/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Camundongos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/genética
16.
Am J Respir Cell Mol Biol ; 62(4): 513-523, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922915

RESUMO

In asthma, goblet cell numbers are increased within the airway epithelium, perpetuating the production of mucus that is more difficult to clear and results in airway mucus plugging. Notch1, Notch2, or Notch3, or a combination of these has been shown to influence the differentiation of airway epithelial cells. How the expression of specific Notch isoforms differs in fully differentiated adult asthmatic epithelium and whether Notch influences mucin production after differentiation is currently unknown. We aimed to quantify different Notch isoforms in the airway epithelium of individuals with severe asthma and to examine the impact of Notch signaling on mucin MUC5AC. Human lung sections and primary bronchial epithelial cells from individuals with and without asthma were used in this study. Primary bronchial epithelial cells were differentiated at the air-liquid interface for 28 days. Notch isoform expression was analyzed by Taqman quantitative PCR. Immunohistochemistry was used to localize and quantify Notch isoforms in human airway sections. Notch signaling was inhibited in vitro using dibenzazepine or Notch3-specific siRNA, followed by analysis of MUC5AC. NOTCH3 was highly expressed in asthmatic airway epithelium compared with nonasthmatic epithelium. Dibenzazepine significantly reduced MUC5AC production in air-liquid interface cultures of primary bronchial epithelial cells concomitantly with suppression of NOTCH3 intracellular domain protein. Specific knockdown using NOTCH3 siRNA recapitulated the dibenzazepine-induced reduction in MUC5AC. We demonstrate that NOTCH3 is a regulator of MUC5AC production. Increased NOTCH3 signaling in the asthmatic airway epithelium may therefore be an underlying driver of excess MUC5AC production.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Mucina-5AC/metabolismo , Receptor Notch3/metabolismo , Transdução de Sinais/fisiologia , Idoso , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Células Caliciformes/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/metabolismo
17.
J Ethnopharmacol ; 249: 112425, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31765763

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Mucus hypersecretion (MH) is recognized as a key pathophysiological and clinical feature of many airway inflammatory diseases. MUC5AC is a major component of airway mucus. Tanreqing injection (TRQ) is a widely used herbal formula for the treatment of respiratory inflammations for years in China. However, a holistic network pharmacology approach to understanding its therapeutic mechanisms against MH has not been pursued. AIM OF THE STUDY: This study aimed to explore the systems-level potential active compounds and therapeutic mechanisms of TRQ in the treatment of MH. MATERIALS AND METHODS: We established systems pharmacology-based strategies comprising compound screenings, target predictions, and pathway identifications to speculate the potential active compounds and therapeutic targets of TRQ. We also applied compound-target and target-disease network analyses to evaluate the possible action mechanisms of TRQ. Then, lipopolysaccharide (LPS)-induced Sprague-Dawley (SD) rat model was constructed to assess the effect of TRQ in the treatment of MH and to validate the possible molecular mechanisms as predicted in systems pharmacology approach. RESULTS: The comprehensive compound collection successfully generated 55 compound candidates from TRQ. Among them, 11 compounds with high relevance to the potential targets were defined as representative and potential active ingredients in TRQ formula. Target identification revealed 172 potential targets, including pro-inflammatory cytokines of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-8. Pathway analyses uncovered the possible action of TRQ in the regulation of IL-17 signaling pathway and its downstream protein MUC5AC. Then in vivo experiment indicated that TRQ could significantly inhibit LPS stimulated MUC5AC over-production as well as the expression of TNF-α, IL-6, IL-8, and IL-17A, in both protein and mRNA levels. CONCLUSIONS: Based on the systems pharmacology method and in vivo experiment, our work provided a general knowledge on the potential active compounds and possible therapeutic targets of TRQ formula in its anti-MH process. This work might suggest directions for further research on TRQ and provide more insight into better understanding the chemical and pharmacological mechanisms of complex herbal prescriptions in a network perspective.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Etnofarmacologia/métodos , Muco/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Mucosa Respiratória/efeitos dos fármacos , Animais , Análise de Dados , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Mucina-5AC/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos , Ratos Sprague-Dawley , Mucosa Respiratória/patologia , Software , Máquina de Vetores de Suporte
18.
Rhinology ; 58(1): 66-73, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31680128

RESUMO

BACKGROUND: Airway inflammation and excessive mucin production are pathophysiological characteristics of airway diseases. Fipronil, a pesticide, is being extensively used in agriculture and veterinary medicine worldwide. However, this compound impairs immune function in non-target organisms. The present study aimed to evaluate the effect of fipronil on pro-inflammatory cytokine and mucus production and signalling pathways in human primary nasal METHODOLOGY: The effect of fipronil on pro-inflammatory cytokine and MUC5AC expression and the signalling pathway of fipronil were investigated using real-time PCR, enzyme immunoassays, immunofluorescence, and immunoblot analysis with specific inhibitors and small interfering RNA. RESULTS: Fipronil treatment increased pro-inflammatory cytokine interleukin (IL)-1beta, IL-6, IL-8, and MUC5AC expression in human primary nasal epithelial cells. It also induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) mitogenactivated protein kinase (MAPK), p38 MAPK, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). MAPK and NF-kB inhibitor treatment significantly inhibited increases in IL-1beta, IL-6, IL-8, and MUC5AC expression. Ex vivo data confirmed that fipronil-induced MUC5AC expression occurs through ERK1/2, p38, and NF-kB signalling pathways in nasal inferior turbinate tissue. CONCLUSIONS: Fipronil induced pro-inflammatory cytokine IL-1beta, IL-6, IL-8, and MUC5AC expression via ERK1/2 MAPK, p38 MAPK, and NF-kB in human primary nasal epithelial cells.


Assuntos
Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Mucina-5AC/metabolismo , Pirazóis/farmacologia , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , NF-kappa B/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
FEBS Lett ; 594(1): 153-160, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31365127

RESUMO

Bromhexine was reported to relieve the symptoms of Sjogren Syndrome at an early stage. However, the underlying mechanism remains unclear. Here, we administered bromhexine at low doses in human primary conjunctival fornix epithelial cells, and found it stimulated MUC5AC secretion and lipid droplet production. Expression of the metabolism-related gene CHML was also upregulated by bromhexine treatment, and REP2, the protein produced by the CHML gene, was induced. These results suggest that bromhexine is a potential candidate eye drop drug for the treatment of multiple types of dry eye disease, not only limited to the treatment of dry eyes in Sjogren Syndrome.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bromoexina/farmacologia , Túnica Conjuntiva/efeitos dos fármacos , Expectorantes/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Células Cultivadas , Túnica Conjuntiva/citologia , Túnica Conjuntiva/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Mucina-5AC/genética , Mucina-5AC/metabolismo
20.
Chemosphere ; 241: 125087, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31622892

RESUMO

Brominated flame-retardant (BFRs) exposure promotes multiple adverse health outcomes involved in oxidative stress, inflammation, and tissues damage. We investigated BFR effects, known as polybrominated diphenyl ethers (PBDEs) (47, 99 and 209) in an air-liquid-interface (ALI) airway tissue derived from A549 cell line, and compared with ALI culture of primary human bronchial epithelial cells (pHBEC). The cells, exposed to PBDEs (47, 99 and 209) (0.01-1 µM) for 24 h, were studied for IL-8, Muc5AC and Muc5B (mRNAs and proteins) production, as well as NOX-4 (mRNA) expression. Furthermore, we evaluated tight junction (TJ) integrity by Trans-Epithelial Electrical Resistance (TEER) measurements, and zonula occludens-1 (ZO-1) expression in the cells, and pH variations and rheological properties (elastic G', and viscous G″, moduli) in apical washes of ALI cultures. N-acetylcysteine (NAC) (10 mM) effects were tested in our experimental model of A549 cells. PBDEs (47, 99 and 209) exposure decreased TEER, ZO-1 and pH values, and increased IL-8, Muc5AC, Muc5B (mRNAs and proteins), NOX-4 (mRNA), and rheological parameters (G', G″) in ALI cultures of A549 cell line and pHBEC. NAC inhibited PBDE effects in A549 cells. PBDE inhalation might impairs human health of the lungs inducing oxidative stress, inflammatory response, loss of barrier integrity, unchecked mucus production, as well as altered physicochemical and biological properties of the fluids in airway epithelium. The treatment with anti-oxidants restored the negative effects of PBDEs in epithelial cells.


Assuntos
Brônquios/citologia , Éteres Difenil Halogenados/toxicidade , Pneumopatias/induzido quimicamente , Células A549 , Idoso , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Retardadores de Chama/toxicidade , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Pneumopatias/fisiopatologia , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/fisiologia
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