Efeitos da melatonina na inflamação de vias aéreas em modelos experimentais de asma, DPOC e de sobreposição Asma-DPOC

Background and Objectives: According to previous studies, melatonin, an indolamine, may possibly reduce dyspnea in patients with asthma or COPD. Additionally, it has also been proven, through experimental studies, that the molecule possibly plays an anti-inflammatory role, because it might inhibit transcription of NFk-B in experimental models of asthma and COPD. This study aims to investigate the effects of melatonin in experimental models of asthma, COPD and Asthma-COPD Overlap (OCP). Methods: 64 mice were divided into 8 groups in order to induce COPD (group “ALS”), asthma (“OVA”) or OC (“ACO”). The control group (“SAL”) received saline. The treatment groups (“+MEL”) were submitted to both disease protocols and also received Melatonin (intraperitoneally). After the protocols, the exhaled nitric oxide (Eno) and the total and differential cells of the bronchoalveolar lavage fluid were evaluated. Results: OVA+MEL (11.3±1.65ppb) showed reduction in Eno compared to OVA (24.17±4.30ppb). In the analysis of the bronchoalveolar lavage fluid cells, OVA+MEL (11.3±1.65x104 cells/mL) and ACO+MEL (2.17±0.63x104 cells/mL) showed a reduction in the amount of total cells compared to OVA (25.70±4.59x104 cells/mL) and ACO (14.33±3.11x104 cells/mL), respectively. There was a decrease in eosinophil count in OVA+MEL (5.37±1.41x104 cells/mL) and ACO+MEL (0.87±0.36x104 cells/mL) compared to OVA (18.67±4.01x104 cells/mL) and ACO (1.45±0.41x104 cells/mL), respectively. Additionally, the number of lymphocytes decreased in OVA + MEL (1.00±0.24x104 céls./mL) compared to OVA (3.94±1.15x104 cells/mL). The number of macrophages also decreased in OVA + MEL (3.09±0.39x104 cells/mL) and ACO + MEL (1.10±0.27x104 cells/mL) compared to OVA (7.26±1.93x104 cells/mL) and ACO (6.41±1.54x104 cells/mL), respectively. There was no difference in the comparison of neutrophil counts in the different groups analyzed.Justificativa e Objetivos: De acordo com estudos anteriores, a melatonina, uma indolamina, pode possivelmente reduzir a dispneia em pacientes que apresentam asma ou DPOC. Adicionalmente, foi provado igualmente, através dos estudos experimentais, que a molécula joga possivelmente um papel antiinflamatório, porque pôde inibir a transcrição de NFk-B em modelos experimentais da asma e do COPD. Este estudo tem como objetivo investigar os efeitos da melatonina em modelos experimentais de asma, DPOC e Sobreposição da Asma-DPOC (ACO). Métodos: 64 camundongos foram divididos em 8 grupos, a fim de induzir DPOC (grupo “ELA”), asma (“OVA”) ou ACO (“ACO”). O grupo controle (“SAL”) recebeu solução salina. Os grupos de tratamento (“+MEL”) foram submetidos aos dois protocolos de doença e também receberam Melatonina (intraperitonealmente). Após os protocolos, foram avaliados o óxido nítrico exalado (Eno), bem como as células totais e diferenciais do fluido do lavado broncoalveolar. Resultados: OVA+MEL (11.3±1.65ppb) apresentou redução em eNO em comparação a OVA (24.17±4.30ppb). Além disso, OVA+MEL (11.3±1.65x104 céls./mL) e ACO+MEL (2.17±0.63x104 céls./mL) apresentaram redução na quantidade de células totais em comparação a OVA (25.70±4.59x104 céls./mL) e ACO (14.33±3.11x104 céls./mL), respectivamente. Houve queda na contagem de eosinófilos em OVA+MEL (5.37±1.41x104 céls./mL) e em ACO+MEL (0.87±0.36x104 céls./mL) em comparação a OVA (18.67±4.01x104 céls./mL) e ACO (1.45±0.41x104 céls./mL), respectivamente. Adicionalmente, o número de linfócitos apresentou redução em OVA+MEL (1.00±0.24x104 céls./mL) em comparação a OVA (3.94±1.15x104 céls./mL). O número de macrófagos também apresentou redução em OVA+MEL (3.09±0.39x104 céls./mL) e em ACO+MEL (1.10±0.27x104 céls./mL) em comparação a OVA (7.26±1.93x104 céls./mL) e ACO (6.41±1.54x104 céls./mL), respectivamente (p<0.05 para todas as comparações). Não houve diferença na comparação da contagem de neutrófilos nos diferentes grupos analisados. Conclusão: O tratamento com melatonina mostrou-se, em modelo experimental de asma e ACO, efetivo na redução de parâmetros pró-inflamatórios, podendo representar um papel importante no controle de tais patologias. Além disso, o tratamento com melatonina em modelos experimentais de lesão pulmonar induzida por elastase não se mostrou eficaz no controle de parâmetros pró-inflamatórios no fluido do lavado broncoalveolar, sendo necessários mais estudos para o entendimento dos mecanismos de ação da melatonina no tecido pulmonar e nas vias aéreas

A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

Aims: To determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice.Methods: C57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. in both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). the inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target.Laboratorios de Investigacao Medica do Hospital das Clinicas da Faculdade de Medicina da USP (LIM/HC)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Dept Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUNIFESP EPM, Dept Bioquim, São Paulo, BrazilUFABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUNIFESP EPM, Dept Bioquim, São Paulo, BrazilWeb of Scienc

Antileukotriene Reverts the Early Effects of Inflammatory Response of Distal Parenchyma in Experimental Chronic Allergic Inflammation

Aims. Compare the effects of montelukast or dexamethasone in distal lung parenchyma and airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods. GP have inhaled ovalbumin (OVA group-2x/week/4weeks). After the 4th inhalation, GP were treated with montelukast or dexamethasone. After 72 hours of the 7th inhalation, GP were anesthetised, and lungs were removed and submitted to histopathological evaluation. Results. Montelukast and dexamethasone treatments reduced the number of eosinophils in airway wall and distal lung parenchyma compared to OVA group (P < 0.05). On distal parenchyma, both treatments were effective in reducing RANTES, NF-kappa B, and fibronectin positive cells compared to OVA group (P < 0.001). Montelukast was more effective in reducing eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (P < 0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (P < 0.001). On airway walls, montelukast and dexamethasone were effective in reducing IGF-I, RANTES, and fibronectin positive cells compared to OVA group (P < 0.05). Dexamethasone was more effective in reducing the number of eotaxin and NF-kappa B positive cells than Montelukast (P < 0.05). Conclusions. in this animal model, both treatments were effective in modulating allergic inflammation and remodeling distal lung parenchyma and airway wall, contributing to a better control of the inflammatory response.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Sch Med, Dept Med, BR-01246903 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-09972270 Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, BR-09972270 Diadema, SP, BrazilCNPq: LIM-20-HC-FMUSPWeb of Scienc

A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice

Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor- (TNF-), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Sao Paulo, Sch Med, Dept Clin Med, BR-01246903 Sao Paulo, BrazilHosp Sirio Libanes, Phys Therapy Dept, BR-01308050 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biosci, BR-09972270 Diadema, BrazilUniv Fed Sao Paulo, Dept Biochem, BR-09972270 Diadema, BrazilUniv Fed Sao Paulo, Dept Biosci, BR-09972270 Diadema, BrazilUniv Fed Sao Paulo, Dept Biochem, BR-09972270 Diadema, BrazilWeb of Scienc

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. the treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. the lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. in conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Sch Med, Dept Med, BR-01246903 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

Effects of Anti-IL-17 on Inflammation, Remodeling, and Oxidative Stress in an Experimental Model of Asthma Exacerbated by LPS

Inflammation plays a central role in the development of asthma, which is considered an allergic disease with a classic Th2 inflammatory profile. However, cytokine IL-17 has been examined to better understand the pathophysiology of this disease. Severe asthmatic patients experience frequent exacerbations, leading to infection, and subsequently show altered levels of inflammation that are unlikely to be due to the Th2 immune response alone. This study estimates the effects of anti-IL-17 therapy in the pulmonary parenchyma in a murine asthma model exacerbated by LPS. BALB/c mice were sensitized with intraperitoneal ovalbumin and repeatedly exposed to inhalation with ovalbumin, followed by treatment with or without anti-IL-17. Twenty-four hours prior to the end of the 29-day experimental protocol, the two groups received LPS (0.1 mg/ml intratracheal OVA-LPS and OVA-LPS IL-17). We subsequently evaluated bronchoalveolar lavage fluid, performed a lung tissue morphometric analysis, and measured IL-6 gene expression. OVA-LPS-treated animals treated with anti-IL-17 showed decreased pulmonary inflammation, edema, oxidative stress, and extracellular matrix remodeling compared to the non-treated OVA and OVA-LPS groups (p < 0.05). The anti-IL-17 treatment also decreased the numbers of dendritic cells, FOXP3, NF-kappa B, and Rho kinase 1-and 2-positive cells compared to the non-treated OVA and OVA-LPS groups (p < 0.05). In conclusion, these data suggest that inhibition of IL-17 is a promising therapeutic avenue, even in exacerbated asthmatic patients, and significantly contributes to the control of Th1/Th2/Th17 inflammation, chemokine expression, extracellular matrix remodeling, and oxidative stress in a murine experimental asthma model exacerbated by LPS.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)National Council of Scientific and the Technological Development (CNPq)Laboratory of Medical InvestigationsUniv Sao Paulo, Sch Med, Dept Med Sci, Sao Paulo, Brazil|Hosp Sirio Libanes, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Biol Sci, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Sao Paulo, BrazilFAPESP: 2013/17944-1Laboratory of Medical Investigations: LIM-20 FMUSPWeb of Scienc

Modulation of the oscillatory mechanics of lung tissue and the oxidative stress response induced by arginase inhibition in a chronic allergic inflammation model

Abstract\ud \ud \ud \ud Background\ud The importance of the lung parenchyma in the pathophysiology of asthma has previously been demonstrated. Considering that nitric oxide synthases (NOS) and arginases compete for the same substrate, it is worthwhile to elucidate the effects of complex NOS-arginase dysfunction in the pathophysiology of asthma, particularly, related to distal lung tissue. We evaluated the effects of arginase and iNOS inhibition on distal lung mechanics and oxidative stress pathway activation in a model of chronic pulmonary allergic inflammation in guinea pigs.\ud \ud \ud \ud Methods\ud Guinea pigs were exposed to repeated ovalbumin inhalations (twice a week for 4 weeks). The animals received 1400 W (an iNOS-specific inhibitor) for 4 days beginning at the last inhalation. Afterwards, the animals were anesthetized and exsanguinated; then, a slice of the distal lung was evaluated by oscillatory mechanics, and an arginase inhibitor (nor-NOHA) or vehicle was infused in a Krebs solution bath. Tissue resistance (Rt) and elastance (Et) were assessed before and after ovalbumin challenge (0.1%), and lung strips were submitted to histopathological studies.\ud \ud \ud \ud Results\ud Ovalbumin-exposed animals presented an increase in the maximal Rt and Et responses after antigen challenge (p<0.001), in the number of iNOS positive cells (p<0.001) and in the expression of arginase 2, 8-isoprostane and NF-kB (p<0.001) in distal lung tissue. The 1400 W administration reduced all these responses (p<0.001) in alveolar septa. Ovalbumin-exposed animals that received nor-NOHA had a reduction of Rt, Et after antigen challenge, iNOS positive cells and 8-isoprostane and NF-kB (p<0.001) in lung tissue. The activity of arginase 2 was reduced only in the groups treated with nor-NOHA (p <0.05). There was a reduction of 8-isoprostane expression in OVA-NOR-W compared to OVA-NOR (p<0.001).\ud \ud \ud \ud Conclusions\ud In this experimental model, increased arginase content and iNOS-positive cells were associated with the constriction of distal lung parenchyma. This functional alteration may be due to a high expression of 8-isoprostane, which had a procontractile effect. The mechanism involved in this response is likely related to the modulation of NF-kB expression, which contributed to the activation of the arginase and iNOS pathways. The association of both inhibitors potentiated the reduction of 8-isoprostane expression in this animal model.FAPESP and LIM20HCFMUSP.FAPESP and LIM-20-HC-FMUSP

Y-27632 is associated with corticosteroid-potentiated control of pulmonary remodeling and inflammation in guinea pigs with chronic allergic inflammation

Abstract\ud \ud Background\ud Previously, we showed that treatment with the Rho-kinase inhibitor Y-27632 was able to control airway responsiveness, inflammation, remodeling, and oxidative stress in an animal model of asthma, suggesting that this drug is beneficial in asthma. However, studies evaluating the effects of these inhibitors in conjunction with corticosteroids on chronic pulmonary inflammation have not been conducted. Therefore, we evaluated the effects of treatment with the Rho-kinase inhibitor Y-27632, with or without concurrent dexamethasone treatment, on airway and lung tissue mechanical responses, inflammation, extracellular matrix remodeling, and oxidative stress in guinea pigs with chronic allergic inflammation.\ud \ud \ud Methods\ud The guinea pigs were subjected to seven ovalbumin or saline inhalation exposures. Treatment with Y-27632 (1 mM) and dexamethasone (2 mg/kg) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the pulmonary mechanics were evaluated and exhaled nitric oxide (ENO) levels were determined. The lungs were removed and histological analysis was performed using morphometry.\ud \ud \ud Results\ud The treatment of guinea pigs with the Rho-kinase inhibitor and dexamethasone (ORC group) decreased ENO, the maximal mechanical responses after antigen challenge, inflammation, extracellular matrix remodeling and oxidative stress in the lungs.\ud This therapeutic strategy reduced the levels of collagen and IFN-γ in the airway walls, as well as IL-2, IFN-γ, 8-iso-PGF2α and NF-κB in the distal parenchyma, when compared to isolated treatment with corticosteroid or Rho-kinase inhibitor (P < 0.05) and reduced the number of TIMP-1-positive cells and eosinophils in the alveolar septa compared to corticosteroid-treated animals (P < 0.05). The combined treatment with the Rho-kinase inhibitor and the corticosteroid provided maximal control over the remodeling response and inflammation in the airways and parenchyma.\ud \ud \ud Conclusions\ud Rho-kinase inhibition, alone or in combination with corticosteroids, can be considered a future pharmacological tool for the control of asthma.We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for their financial support