RESUMO
Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.
Assuntos
Receptores ErbB/genética , Receptor Muscarínico M3/genética , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Fosforilação , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
ε-Viniferin is a dehydrodimer of resveratrol, a polyphenol synthesized in many plants, including grapevine. The present study investigated the effects of ε-viniferin and resveratrol on epithelial secretory and barrier functions in isolated rat small and large intestinal mucosa. Mucosa-submucosa tissue preparations of various segments of the rat large and small intestines were mounted on Ussing chambers, and short-circuit current (Isc) and tissue conductance (Gt) were continuously measured. The mucosal addition of ε-viniferin (>10(-5) mol/L) and resveratrol (>10(-4) mol/L) to the cecal mucosa, which was the most sensitive region, induced an increase in Isc and a rapid phase decrease (P-1) followed by rapid (P-2) and broad (P-3) peak increases in Gt in concentration-dependent manners. Mucosal ε-viniferin (10(-4) mol/L), but not resveratrol (10(-4) mol/L), increased the permeability of FITC-conjugated dextran (4 kDa). The mucosal ε-viniferin-evoked changes in Isc (Cl(-) secretion), but not in Gt, were attenuated by a selective cyclooxygenase (COX)-1 inhibitor and a selective EP4 prostaglandin receptor. The mucosal ε-viniferin-evoked increase in Isc was partially attenuated, and P-2, but not P-1 or P-3, change in Gt was abolished by a transient receptor potential cation channel, subfamily A, member 1 (TRPA1) inhibitor. Moreover, the mucosal ε-viniferin concentration-dependently attenuated the mucosal propionate (1 mmol/L)-evoked increases in Isc and Gt Immunohistochemical studies revealed COX-1-immunoreactive epithelial cells in the cecal crypt. The present study showed that mucosal ε-viniferin modulated transepithelial ion transport and permeability, possibly by activating sensory epithelial cells expressing COX-1 and TRPA1. Moreover, mucosal ε-viniferin decreased mucosal sensitivity to other luminal molecules such as short-chain fatty acids. In conclusion, these results suggest that ε-viniferin modifies intestinal mucosal transport and barrier functions.
Assuntos
Benzofuranos/farmacologia , Mucosa Intestinal/metabolismo , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Estilbenos/farmacologia , Animais , Benzofuranos/química , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Relação Dose-Resposta a Droga , Mucosa Intestinal/efeitos dos fármacos , Intestino Grosso/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Masculino , Permeabilidade , Ratos , Ratos Sprague-Dawley , Resveratrol , Estilbenos/químicaRESUMO
Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR.
Assuntos
Mucosa Intestinal/citologia , Receptores Muscarínicos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Colo/citologia , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , NF-kappa B/metabolismo , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The present study aimed to design a PEGylated VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), with improved metabolic stability, and develop its respirable powder (RP) formulation for inhalation therapy. IK312532 was chemically conjugated with PEG (5 kDa, P5K), the physicochemical and biochemical properties of which were characterized by CD spectral analysis, binding assays, and metabolic stability. CD spectral analysis demonstrated that PEG conjugation had no impact on the conformational structure of IK312532. Although the receptor-binding activity of IK312532/P5K (IC50: 82 nM) was estimated to be ca. 30-fold less than that of IK312532 (IC50: 2.8 nM), the metabolic stability of IK312532/P5K was highly improved. The IK312532/P5K was jet-milled and blended with lactose carrier particles to provide RP formulation of IK312532/P5K (IK312532/P5K-RP). In vitro inhalation performance and in vivo pharmacological effects of the IK312532/P5K-RP in antigen-sensitized rats were also evaluated. In cascade impactor analyses, fine particle fraction of IK312532/P5K-RP was calculated to be ca. 37%. Insufflation of IK312532/P5K-RP (150 µg of IK312532/P5K) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decreases in inflammatory biomarkers and granulocyte recruitment in pulmonary tissue 24h after the antigen challenge. From these findings, PEGylation of a VIP derivative, as well as its strategic application to the RP formulation, may be a viable approach to improve its therapeutic potential for the treatment of airway inflammatory diseases.
Assuntos
Anti-Inflamatórios/química , Polietilenoglicóis/química , Peptídeo Intestinal Vasoativo/análogos & derivados , Administração por Inalação , Alérgenos , Animais , Anti-Inflamatórios/administração & dosagem , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , L-Lactato Desidrogenase/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Ovalbumina , Tamanho da Partícula , Peroxidase/metabolismo , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Polietilenoglicóis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Peptídeo Intestinal Vasoativo/administração & dosagem , Peptídeo Intestinal Vasoativo/químicaRESUMO
The present study was undertaken to develop a respirable sustained-release powder (RP) formulation of long-acting VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), using PLGA nanospheres (NS) with the aim of improving the duration of action. NS formulation of IK312532 (IK312532/NS) was prepared by an emulsion solvent diffusion method in oil, and a mixture of the IK312532/NS and erythritol was jet-milled and mixed with lactose carrier to obtain the IK312532/NS-RP. Physicochemical properties were characterized focusing on appearance, particle size, and drug release, and in vivo pharmacological effects were assessed in antigen-sensitized rats. The IK312532/NS with a diameter of 140 nm showed a biphasic release pattern in distilled water with ca. 20% initial burst for 30 min and a sustained slow release up to ca. 55% for 24h. Laser diffraction analysis demonstrated that IK312532/NS-RP had fine dispersibility and suitable particle size for inhalation. In antigen-sensitized rats, insufflated IK312532/NS-RP (10 µg of IK312532/rat) could suppress increases of granulocyte recruitment and myeloperoxidase in pulmonary tissue for up to 24h after antigen challenge, although IK312532-RP at the same dose was less effective with limited duration of action. From these findings, newly prepared IK312532/NS-RP might be of clinical importance in improving duration of action and medication compliance for treatment of airway inflammatory diseases.
Assuntos
Asma/tratamento farmacológico , Pneumonia/tratamento farmacológico , Peptídeo Intestinal Vasoativo/análogos & derivados , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/citologia , Preparações de Ação Retardada , Modelos Animais de Doenças , Granulócitos/efeitos dos fármacos , Granulócitos/fisiologia , Pulmão/efeitos dos fármacos , Pneumopatias/tratamento farmacológico , Masculino , Nanosferas , Peroxidase/metabolismo , Ratos , Ratos Sprague-Dawley , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/imunologia , Peptídeo Intestinal Vasoativo/administração & dosagem , Peptídeo Intestinal Vasoativo/farmacologia , Peptídeo Intestinal Vasoativo/uso terapêuticoRESUMO
Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca(2+)-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.
Assuntos
Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Receptores Odorantes/efeitos dos fármacos , Timol/farmacologia , Idoso , Análise de Variância , Animais , Anquirinas/genética , Anquirinas/metabolismo , Bicarbonatos/metabolismo , Transporte Biológico , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cloretos/metabolismo , Colo/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Células Epiteliais/metabolismo , Ácidos Graxos/metabolismo , Feminino , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Masculino , Potenciais da Membrana , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Permeabilidade , Inibidores de Fosfodiesterase/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Antagonistas da Serotonina/farmacologia , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Cigarette smoke (CS) has been identified as a predominant causative factor for chronic obstructive pulmonary disease (COPD), so CS-exposed rodent model of COPD has drawn considerable interest and attention for fundamental study and drug discovery. In the present study, using experimental COPD model rats, the therapeutic potential of a newly prepared respirable powder (RP) formulation of a long-acting VIP derivative, [Arg(15,20,21), Leu(17)]-VIP-GRR (IK312532), was assessed with a focus on pro-inflammatory biomarkers, morphological and histochemical changes, and infiltrated cells in the respiratory system. CS exposure of rats for 11 days led to the marked infiltration of inflammatory cells, except for eosinophils, in bronchiolar epithelium, followed by goblet cell metaplasia and hyperplasia. However, inhalation of IK312532-RP (50µg/rat) in the CS-exposed rats resulted in 74 and 71% reductions of granulocyte recruitment in bronchoalveolar lavage fluids and lung tissues, respectively, with 68% decrease of goblet cells. Biomarker study demonstrated that the inhaled IK312532-RP could suppress the CS-evoked increase of myeloperoxidase in both plasma and lung by 87 and 70%, respectively, possibly leading to potent suppression of neutrophilic inflammatory symptoms. The results from TUNEL staining were indicative of apoptotic damage in respiratory tissues of the CS-exposed rats, and there appeared to be marked decrease of TUNEL-positive cells in the CS-exposed rat with inhaled IK312532-RP. The present findings suggest that an inhalable formulation of IK312532 might be efficacious as a therapy for COPD or other airway inflammatory diseases because of its potent immunomodulating activities.
Assuntos
Inflamação/tratamento farmacológico , Neutrófilos/fisiologia , Nicotiana , Fumaça/efeitos adversos , Peptídeo Intestinal Vasoativo/análogos & derivados , Administração por Inalação , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Biomarcadores , Peroxidase de Eosinófilo/metabolismo , Inflamação/induzido quimicamente , Pneumopatias/induzido quimicamente , Pneumopatias/tratamento farmacológico , Pneumopatias/patologia , Masculino , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Pós , Ratos , Ratos Sprague-Dawley , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Peptídeo Intestinal Vasoativo/administração & dosagem , Peptídeo Intestinal Vasoativo/química , Peptídeo Intestinal Vasoativo/uso terapêuticoRESUMO
Vasoactive intestinal peptide (VIP) exerts immunomodulating and anti-inflammatory activities through its specific receptors, such as VPAC1 and 2 receptors. Previously, a stabilized VIP derivative, [R(15,20,21), L(17)]-VIP-GRR (IK312532), was proposed as a candidate of anti-asthma drug, and a dry powder inhaler system of IK312532 was also developed for inhalation therapy with minimal systemic side-effects. In the present study, the anti-inflammatory properties of IK312532 respirable powder (RP) were characterized in an asthma/COPD-like animal model, with the use of newly developed ovalbumin (OVA)-RP for lung inflammation. Marked inflammatory events in the lung were observed after OVA-RP challenge in rats as evidenced by significant increase of inflammatory biomarkers such as eosinophil peroxidase (EPO), myeloperoxidase (MPO) and lactate dehydrogenase (LDH). However, intratracheal administration of IK312532-RP led to significant attenuation of plasma EPO, MPO and LDH activities, as well as significant reduction of recruited inflammatory cells in BALF, especially macrophages and eosinophils. In the rats pretreated with IK312532-RP, histochemical examinations revealed that the inflammatory cells infiltrating to the lung and the epithelial wall thickness decreased significantly by 85% and 58%, respectively. Thus, inhalable powder formulation of IK312532 exerts its anti-inflammatory activity by suppressing granulocyte recruitment to the lung and epithelial hyperplasia, followed by the reduction of cytotoxic peroxidases.
Assuntos
Anti-Inflamatórios , Asma/tratamento farmacológico , Pós/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Peptídeo Intestinal Vasoativo/análogos & derivados , Administração por Inalação , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Asma/imunologia , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , L-Lactato Desidrogenase/metabolismo , Masculino , Peroxidase/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Ratos , Ratos Sprague-Dawley , Peptídeo Intestinal Vasoativo/administração & dosagem , Peptídeo Intestinal Vasoativo/uso terapêuticoRESUMO
Interaction between substance P (SP) and PGE(2) on Cl(-) secretion in the guinea pig distal colonic epithelia was investigated. A short-circuit current (I(sc)) was measured as an index of ion transport. Mucosa preparations deprived of muscle and submucosa of distal colon were mounted in the Ussing flux chamber and treated with TTX and piroxicam to remove the influences of neuronal activity and endogenous PG synthesis, respectively. Although SP (10(-7) M) itself evoked little increase in I(sc), exogenous PGE(2) concentration dependently enhanced the response of SP. The effect of PGE(2) on the SP-evoked response was mimicked by forskolin and 8-bromoadenosine cAMP. Depletion of Ca2+ from the bathing solution reduced the PGE(2)-dependent response of SP. Effects of PGE(2), SP, and SP in the presence of PGE(2) on intracellular Ca2+ concentration ([Ca2+](i)) in isolated crypt cells were measured by the confocal microscope fluorescence imaging system. SP, but not PGE(2), temporally evoked an increase in [Ca2+](i) but declined to the baseline within 3 min. A return of the SP-evoked increase in [Ca2+](i) was slower in the presence of PGE(2) than SP alone. These results suggest that PGE(2) synergistically enhances SP-evoked Cl(-) secretion via an interaction between the intracellular cAMP and [Ca2+](i) in the epithelial cells. In conclusion, SP and PGE(2) could cooperatively induce massive Cl(-) secretion in guinea pig distal colon at epithelial levels.