RESUMEN
The purposes of this study were to determine whether berberine has any effect on phenotype changes and extracellular matrix (ECM) production in nasal polyp-derived fibroblasts (NPDFs) and to investigate the underlying molecular mechanism. NPDFs were pre-treated with berberine prior to induction by transforming growth factor (TGF)-ß1. The expression of α-smooth muscle actin (SMA) and collagen type I mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and collagen type I was determined by western blotting and/or immunofluorescent staining. The total soluble collagen production was analysed by the SirCol collagen assay. The expression of several signaling molecules of the TGF-ß1 pathway was evaluated by western blot analysis. In TGF-ß1-induced NPDFs, berberine significantly inhibited the expression of α-SMA and collagen type I mRNA and reduced α-SMA and collagen protein levels. Berberine only suppressed the expression of pp38 among the evaluated signaling molecules. SB203580 (a specific inhibitor of p38 kinase) markedly suppressed the increased expression of collagen type I and α-SMA in TGF-ß1-induced NPDFs. Berberine exerts suppressive effects on phenotype changes and ECM production in NPDFs via p38 signaling pathway interference. The findings provide new therapeutic options for ECM production in nasal polyps.
Asunto(s)
Berberina/farmacología , Diferenciación Celular/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Pólipos Nasales/patología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Actinas/metabolismo , Adulto , Células Cultivadas , Colágeno Tipo I/metabolismo , Femenino , Humanos , Masculino , Miofibroblastos/citología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Chronic hypoxia is associated with remodeling in various organs. Reactive oxygen species (ROS) derived from NADPH oxidases (Nox), and transforming growth factor-ß(1) (TGF-ß(1)) have been implicated in the pathogenesis of hypoxia-induced remodeling. The aims of this study were to determine in hypoxia-stimulated nasal polyp-derived fibroblasts (NPDF) the effect of hypoxia on the differentiation of myofibroblasts, the role of ROS, the major Nox homolog mediating myofibroblast differentiation, and the role of TGF-ß(1). METHODS: Eight primary cultures of NPDF were established from nasal polyps, which were incubated under hypoxic conditions. Reverse transcription polymerase chain reaction for αSMA, Nox1, Nox3, Nox4, Nox5, and fibronectin mRNA was performed. Western blotting for α-SMA and fibronectin was done. ROS production was detected using a fluorometer. NPDF were pretreated with ROS scavengers and transfected with siNox4. The TGF-ß(1) protein level was measured by ELISA. The effect of treatment with TGF-ß(1) type I tyrosine kinase inhibitor SB431542 on myofibroblast differentiation was observed. RESULTS: Hypoxic stimulation of NPDF significantly increased α-SMA and fibronectin mRNA and protein expression. ROS production was increased by hypoxia, and ROS scavengers inhibited myofibroblast differentiation. Nox4 mRNA was the only Nox homolog increased by hypoxia. Transfection with siNox4 inhibited myofibroblast differentiation. TGF-ß(1) was secreted endogenously by hypoxic NPDF. SB431542 significantly inhibited myofibroblast differentiation. CONCLUSIONS: Hypoxia induces myofibroblast differentiation of NPDF through a signaling pathway involving Nox4-dependent ROS generation and TGF-ß(1). Therapies targeting Nox4 may be effective against remodeling of nasal polyps.
Asunto(s)
Miofibroblastos/enzimología , NADPH Oxidasas/metabolismo , Pólipos Nasales/enzimología , ARN Mensajero/biosíntesis , Actinas/biosíntesis , Adulto , Benzamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/genética , Dioxoles/farmacología , Femenino , Fibronectinas/biosíntesis , Depuradores de Radicales Libres/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Miofibroblastos/efectos de los fármacos , Miofibroblastos/patología , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Pólipos Nasales/patología , Oxígeno/farmacología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Myofibroblasts are detected in nasal polyps and are involved in nasal polyp formation by inducing extracellular matrix accumulation. Reactive oxygen species (ROS) are released during the differentiation of fibroblasts to myofibroblasts. The purpose of this study was to investigate ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in nasal polyp-derived fibroblasts (NPDFs) and to evaluate whether ROS from NOX mediates transforming growth factor (TGF)-ß1-induced production of alpha smooth-muscle actin (α-SMA) and collagen production. METHODS: NPDFs were incubated and treated with TGF-ß1. The mRNA expression of NOXs, α-SMA, and collagen type I and IV was determined by reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the SirCol assay. The ROS generation of cells was investigated using the 2',7'-dichlorfluorescein-diacetate. The fluorescence was captured by fluorescent microscope and measured using a fluorometer. RESULTS: Stimulation with TGF-ß1 increased ROS production by NPDFs compared with NPDFs not treated with TGF-ß1. Stimulation with TGF-ß1 increased the expression of NOX4 mRNA most potently among various Nox enzymes. siNOX4 was able to decrease the level of ROS production. Myofibroblast differentiation and the production of collagen in NPDFs were prevented by inhibition of ROS generation with diphenyliodonium, N-acetylcysteine, ebselen, and siNox4. CONCLUSIONS: This study showed that NOX4 and ROS have a role in myofibroblast differentiation and collagen production of TGF-ß1-induced NPDFs and that these processes are inhibited by the elimination of ROS.
Asunto(s)
Fibroblastos/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , Pólipos Nasales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Acetilcisteína/farmacología , Actinas/antagonistas & inhibidores , Actinas/biosíntesis , Adulto , Azoles/farmacología , Compuestos de Bifenilo/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/antagonistas & inhibidores , Colágeno Tipo I/biosíntesis , Colágeno Tipo IV/antagonistas & inhibidores , Colágeno Tipo IV/biosíntesis , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fluoresceínas , Colorantes Fluorescentes , Expresión Génica/efectos de los fármacos , Humanos , Isoindoles , Masculino , Microscopía Fluorescente , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Pólipos Nasales/patología , Compuestos Onio/farmacología , Compuestos de Organoselenio/farmacología , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Fibroblast migration is crucial for normal wound repair after sinonasal surgery. Prostaglandin E2 (PGE2) is a potent inhibitor of fibroblast functions including chemotaxis, proliferation, and matrix production. The purpose of this study was to determine whether PGE2 affects the migration of nasal fibroblasts and to investigate the mechanism of action of PGE2 on nasal fibroblasts. METHODS: Primary cultures of nasal fibroblasts were established from inferior turbinate samples. Fibroblast migration was evaluated with scratch assays. Reverse-transcription polymerase chain reaction was performed for E prostanoid (EP) 1, EP2, EP3, and EP4 receptors. EP receptor-selective agonists and antagonists were used to evaluate receptor functions. Stimulatory G (Gs) proteins were activated to evaluate mechanisms. Intracellular cyclic adenosine monophosphate (cAMP) levels were measured by ELISA, and fibroblast cytoskeletal structures were visualized with immunocytochemistry. RESULTS: PGE2 significantly reduced the migration of nasal fibroblasts. Agonists selective for the EP2 and EP4 receptors significantly reduced the nasal fibroblast migration. Antagonists of the EP2 and EP4 receptors inhibited the effect of PGE2 on nasal fibroblast migration. Activation of Gs protein and adenyl cyclase reduced nasal fibroblast migration. CONCLUSION: PGE2 inhibited the migration of nasal fibroblasts via the EP2 and EP4 receptors, and this inhibition was mediated by cAMP elevation. Targeting specific EP receptors could offer therapeutic opportunities for conditions such as delayed wound healing after nasal surgery.
Asunto(s)
Alprostadil/análogos & derivados , Compuestos de Bifenilo/farmacología , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Endoscopía , Fibroblastos/efectos de los fármacos , Senos Paranasales/efectos de los fármacos , Pirrolidinonas/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Xantonas/farmacología , Alprostadil/farmacología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/farmacología , Fibroblastos/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Terapia Molecular Dirigida , Senos Paranasales/patología , Senos Paranasales/cirugía , Subtipo EP1 de Receptores de Prostaglandina E/agonistas , Subtipo EP1 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP3 de Receptores de Prostaglandina E/agonistas , Subtipo EP3 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Cicatrización de HeridasRESUMEN
PURPOSE: Angiogenesis is involved in the pathogenesis of chronic rhinosinusitis with nasal polyps. We aimed to investigate the effects of prostaglandin E2 (PGE2) on vascular endothelial growth factor (VEGF) production, the role of E-prostanoid (EP) 4 receptors, and the signal transduction pathway mediating VEGF production in nasal polyp-derived fibroblasts (NPDFs). METHODS: Eight primary NPDF cultures were established from nasal polyps, which were incubated with or without PGE2. Reverse transcription-polymerase chain reaction amplification of EP receptors (EP1, EP2, EP3, and EP4) and immunofluorescence staining for VEGF production were performed. VEGF production via the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways was evaluated by enzyme-linked immunosorbent assay. RESULTS: All EP receptors were expressed in NPDFs. PGE2 significantly increased VEGF production concentration- and time dependently, and VEGF production was regulated by an EP4 receptor. Activation of intracellular cAMP regulated VEGF production. VEGF production was decreased by PKA and PI3K inhibitors via intracellular cAMP. CONCLUSIONS: PGE2 stimulates VEGF production via the EP4 receptor in NPDFs. These results indicate that PGE2-induced VEGF production is mediated, at least partially, through cAMP-dependent signaling pathways. Therapies targeting the EP4 receptor may be effective in inhibiting the development of nasal polyps.
RESUMEN
BACKGROUND: Nasal polyposis is associated with a chronic inflammatory condition of the sinonasal mucosa and involves myofibroblast differentiation and extracellular matrix (ECM) accumulation. Epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been reported to have inhibitory effects on myofibroblast differentiation in lung and renal fibroblasts. The purpose of this study was to investigate the inhibitory effect of TSA on myofibroblast differentiation and ECM production in nasal polyp organ cultures. METHODS: Nasal polyp tissues from 18 patients were acquired during endoscopic sinus surgery. After organ culture, nasal polyps were stimulated with TGF-beta1 and then treated with TSA. Alpha-smooth muscle actin (α-SMA), fibronectin, and collagen type I expression levels were examined by reverse transcription-polymerase chain reaction (PCR), real-time PCR, Western blot, and immunofluorescent staining. HDAC2, HDAC4, and acetylated H4 expression levels were assayed by Western blot. Cytotoxicity was analyzed by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. RESULTS: The expression levels of α-SMA, fibronectin, and collagen type 1 were increased in nasal polyp after transforming growth factor (TGF) beta1 treatment. TSA-inhibited TGF-beta1 induced these gene and protein expression levels. Furthermore, TSA suppressed protein expression levels of HDAC2 and HDAC4. However, TSA induced hyperacetylation of histones H4. Treatment with TGF-beta1 with or without TSA did not have cytotoxic effect. CONCLUSION: These findings provide novel insights into the epigenetic regulation in myofibroblast differentiation and ECM production of nasal polyp. TSA could be a candidate of a therapeutic agent for reversing the TGF-beta1-induced ECM synthesis that leads to nasal polyp development.
Asunto(s)
Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/enzimología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Miofibroblastos/efectos de los fármacos , Pólipos Nasales/enzimología , Pólipos Nasales/patología , Acetilación/efectos de los fármacos , Actinas/genética , Actinas/metabolismo , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Miofibroblastos/enzimología , Miofibroblastos/patología , Técnicas de Cultivo de Órganos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
Nasal polyps are associated with chronic inflammation of the sinonasal mucosa and are involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. Ginsenoside Rg1, a compound derived from Panax ginseng, shows antifibrotic and anticancer effects. However, the molecular effects of Rg1 on myofibroblast differentiation and ECM production remain unknown. The aims of this study were to investigate the effect of Rg1 on transforming growth factor (TGF)-ß1-induced myofibroblast differentiation and ECM production and to determine the molecular mechanism of Rg1 in nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyps of seven patients who had chronic rhinosinusitis with nasal polyp. NPDFs were exposed to TGF-ß1 with or without Rg1. Expression levels of α-smooth muscle actin (SMA), fibronectin and collagen type Iα1 were determined by reverse transcription polymerase chain reaction, Western blot and immunofluorescent staining. TGF-ß1 signaling molecules, including Smad2/3, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were analyzed by Western blotting. Transcription factors involved with TGF-ß1 signaling, nuclear factor (NF)-κB and activator protein 1 (AP-1) were also assessed by Western blot. The cytotoxic effect of Rg1 was measured by an established viability assay. The mRNA and protein expression levels of α-SMA, fibronectin and collagen type Iα1 were increased in TGF-ß1-induced NPDFs. Rg1 inhibited these effects. The inhibitory molecular mechanism of Rg1 was involved in the ERK pathway. Rg1 inhibited the transcription factor activation of AP-1. Rg1 itself was not cytotoxic. The ginsenoside Rg1 has inhibitory effects on myofibroblast differentiation and ECM production. The inhibitory mechanism of Rg1 is involved with the ERK and AP-1 signaling pathways. Rg1 may be useful as an inhibitor of ECM deposition, and has potential to be used as a novel treatment option for nasal polyps.
Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ginsenósidos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Pólipos Nasales/patología , Transducción de Señal/fisiología , Factor de Transcripción AP-1/metabolismo , Actinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/patología , Fibronectinas/metabolismo , Humanos , FN-kappa B/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Intelectin-1 is a new type of Ca(2+)-dependant soluble lectin in humans that has affinity for galactofuranose in carbohydrate chains of bacterial cell walls, indicating that intelectin-1 may play a role in immune defense against bacteria. The purpose of the current study was to determine the expression of intelectin-1 mRNA and protein and to localize intelectin-1 protein in nasal polyps and tissues from control subjects. METHODS: Normal sphenoid sinus mucosa was obtained from 10 patients undergoing surgery for pituitary tumor. Nasal polyp samples were obtained from 10 patients undergoing endoscopic sinus surgery for chronic polypoid rhinosinusitis. Real-time polymerase chain reaction (PCR) was performed for intelectin-1 mRNA. Immunofluorescent staining was done for localization of intelectin-1 and quantitatively analyzed using computer-based image analysis. Western blot analysis was performed. RESULTS: Real-time PCR and Western blot analysis showed that intelectin-1 expression in nasal polyps was increased compared with normal sinus mucosa. Using immunofluorescent staining, intelectin-1 was strongly stained in epithelium and submucosa of nasal polyps, and faint staining was found in normal sinus mucosa. CONCLUSIONS: Intelectin-1 is expressed in human sinus mucosa and is increased in patients with nasal polyps. These results suggest a possible contribution for intelectin-1 in the pathophysiology of nasal polyps.
Asunto(s)
Citocinas/genética , Lectinas/genética , Pólipos Nasales/metabolismo , Adulto , Western Blotting , Citocinas/análisis , Citocinas/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI/análisis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Regulación de la Expresión Génica , Humanos , Lectinas/análisis , Lectinas/fisiología , Masculino , Persona de Mediana Edad , Mucosa Nasal/química , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Statins are the most commonly prescribed drugs for the treatment of hypercholesterolemia. Statins exert not only lipid-lowering but also other cellular effects, including antifibrotic properties. The purpose of this study was to determine the effect of simvastatin on transforming growth factor (TGF)-beta-1-induced myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts (NPDFs) and to verify the mechanism of the effect of simvastatin in TGF-beta-1-induced myofibroblast differentiation in NPDFs. METHODS: NPDFs were pretreated with simvastatin with or without mevalonate or Y-27643 for 2 hours before induction by TGF-beta-1. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by immunofluorescent cytochemical staining. Total soluble collagen production was analyzed by the SirCol collagen dye-binding assay (Biocolor, Belfast, U.K.). Phosphorylation of Smad 2/3 was evaluated by Western blot analysis. RESULTS: In TGF-beta-1-induced NPDFs, simvastatin significantly inhibited the expressions of α-SMA and collagen type IV mRNA and reduced alpha-SMA and collagen protein levels. Pretreatment with mevalonate reversed the effect of simvastatin. The expression of alpha-SMA mRNA and protein was significantly decreased by pretreatment with Y-27632. The TGF-beta-1-induced expression of pSmad 2/3 protein was notably decreased by pretreatment with simvastatin. CONCLUSION: We showed that simvastatin inhibits TGF-beta-1-induced myofibroblast differentiation (expression of alpha-SMA) and collagen production in NPDFs and Rho/Rock and TGF-ß/Smad signaling is involved as an underlying mechanism. The results of our study suggest that simvastatin is a possible candidate for the suppression of nasal polyp formation.
Asunto(s)
Antifibrinolíticos/farmacología , Fibroblastos/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Pólipos Nasales/tratamiento farmacológico , Simvastatina/farmacología , Actinas/genética , Actinas/metabolismo , Adulto , Amidas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ácido Mevalónico/farmacología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Pólipos Nasales/patología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
BACKGROUND: [6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp-derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. METHODS: NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2â³,7â³-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. RESULTS: TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1-induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. CONCLUSION: These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.