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1.
Respir Res ; 20(1): 285, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852500

RESUMO

BACKGROUND: ß2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear. METHODS: We established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement. RESULTS: We found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 µM, and ESI-09 promoted SOCE of ASMCs at 10 µM and 100 µM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365. CONCLUSIONS: Our results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Hipersensibilidade Respiratória/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Apoptose , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/fisiopatologia , Sinalização do Cálcio , Proliferação de Células , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Modelos Animais de Doenças , Feminino , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Hidrazonas/farmacologia , Mediadores da Inflamação/metabolismo , Isoxazóis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovalbumina , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Pneumonia/prevenção & controle , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/tratamento farmacológico , Hipersensibilidade Respiratória/fisiopatologia
2.
Pharmacol Rep ; 70(1): 22-28, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29306759

RESUMO

BACKGROUND: Circulating fibrocytes (CFs) have been shown to participate in subepithelial fibrosis of asthma with chronic airflow limitation by acting as an important source of fibroblasts deposited beneath airway epithelia. Serum amyloid P (SAP) is an innate inhibitor of fibrocytes differentiation. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx of non-excitable cells. In this study, the role of SOCE in the regulation of fibrocytes differentiation and the effects of Th2 cytokine IL-4 and SAP on SOCE of fibrocytes were investigated. METHODS: Peripheral blood mononuclear cells or monocytes were cultured in serum-free medium for 7days to differentiate into fibrocytes; the expression of SOC channels was determined with PCR, SOCE was measured with Ca2+ fluorescence imaging. RESULTS: IL-4 significantly promoted monocyte derived fibrocytes differentiation in vitro. It also increased both SOCE which was induced by thapsigargin or UTP and molecules STIM1 and Orai1 which were related to expression of SOC channels in fibrocytes. Fibrocytes differentiation induced by IL-4 and SOC channels activity could be inhibited by SOC channel blocker SKF-96365. As expected, SAP significantly inhibited IL-4-induced differentiation of fibrocytes, the activity of SOCE and the expression of STIM1 and Orai1 in IL-4-treated fibrocytes. CONCLUSION: IL-4 and SAP reversely regulates cultured fibrocytes differentiation in vitro by respectively promoting or inhibiting the expression and activity of SOC channels in fibrocytes.


Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Interleucina-4/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Proteína ORAI1/efeitos dos fármacos , Componente Amiloide P Sérico/farmacologia , Molécula 1 de Interação Estromal/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imidazóis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Fatores de Tempo
3.
J BUON ; 22(6): 1422-1427, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29332333

RESUMO

PURPOSE: Green tea (Camellia sinensis) is considered as a rich source of epigallocatechin gallate (EGCG) which has been shown to exert impressive pharmacological properties. The anticancer properties of EGCG have been extensively studied however, its anticancer activity has not been explored in lung cancer. The present study was therefore designed to evaluate the anticancer effects of EGCG against non-small cell lung cancer (NSCLC) cell line A-549 and normal human fibroblast FR-2 cells. METHODS: Cell viability was assessed by CCK8 assay, apoptosis by DAPI, annexin V/propidium iodide (PI) and flowcytometery and cell cycle analysis by flow cytometry. Cell migration capacity was investigated by wound-healing assay and protein expression was examined by Western blotting. RESULTS: The results revealed that EGCC could inhibit the proliferation of A-549 cells in a concentration-dependent manner and exhibited an IC50 of 25 µM against the IC50 of 100 µM against the normal human fibroblasts. Further evaluation revealed that EGCG exerts its anticancer effects via induction of apoptosis, modulation of Bax/blc-2 ratio and by triggering G2/M cell cycle arrest. Furthermore, EGCG could also inhibit the migration of A5-49 cells in a concentration-dependent manner. CONCLUSION: In conclusion, based on our results, we believe that EGCG could prove to be an important lead molecule for the treatment of lung cancer.


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Catequina/análogos & derivados , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Chá/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia
4.
Eur J Pharmacol ; 740: 733-41, 2014 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-24912144

RESUMO

Plasma membrane Ca2+-ATPase (PMCA) plays an important role in regulating intracellular Ca2+ homeostasis by extruding excessive Ca2+ to extracellular spaces. PMCA has four isoforms and is widely expressed in different tissues and cells including airway smooth muscle cells (ASMCs). In the present study, we investigated the role of PMCA in the maintenance of Ca2+ homeostasis and regulation of ASMCs proliferation. By using Ca2+ fluorescence, we found that inhibition of PMCA with LaCl3 or carboxyeosin (CE) decreased the decay rate of Ca2+ transient induced by bradykinin (BK). No obvious decay was observed when SERCA was inhibited by thapsigargin (TpG). LaCl3 and CE also induced a spontaneous [Ca2+]i increase in the presence of TpG even in Ca2+-free bath solution. Both LaCl3 and CE inhibited UTP-induced Ca2+ oscillations in ASMCs. PCR assay found that PMCA1 and PMCA4 mRNA were expressed in rat ASMCs. The expression of PMCA4 was downregulated in proliferating ASMCs when compared to resting cells. Both the isoform-nonselective PMCA inhibitor caloxin 2a1 and PMCA4-selective inhibitor caloxin 1b1 decreased the decay rate of Ca2+ transient induced by TpG or BK. PMCA inhibitors except caloxin 2a1 promoted ASMCs proliferation. Annexin-V apoptosis assay detected that caloxin 2a1 increased ASMCs apoptosis, suggesting that inhibition of PMCA with different blockers results in different [Ca2+]i and thus different cellular response. Our results provide evidences to support the hypothesis that PMCA is involved in the regulation of Ca2+ homeostasis and ASMCs proliferation. These data suggest that PMCA may be a new target in the treatment of chronic asthma.


Assuntos
Sinalização do Cálcio , Miócitos de Músculo Liso/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Brônquios/citologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular , Lantânio/farmacologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Peptídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley
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