Nathalia Pinheiro

Flavonone treatment reverses airway inflammation and remodelling in an asthma murine model

Asthma is an inflammatory disease that involves airway hyperresponsiveness and remodelling. Flavonoids have been associated to anti‐inflammatory and antioxidant activities and may represent a potential therapeutic treatment of asthma. Our aim was to evaluate the effects of the sakuranetin treatment in several aspects of experimental asthma model in mice.

Pulmonary Inflammation Is Regulated by the Levels of the Vesicular Acetylcholine Transporter

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.

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

BackgroundThe 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.MethodsGuinea 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.ResultsOvalbumin-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).ConclusionsIn 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.

Structurally Related Monoterpenes p-Cymene, Carvacrol and Thymol Isolated from Essential Oil from Leaves of Lippia sidoides Cham. (Verbenaceae) Protect Mice against Elastase-Induced Emphysema

Background: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow obstruction and inflammation. Natural products, such as monoterpenes, displayed anti-inflammatory and anti-oxidant activities and can be used as a source of new compounds to COPD treatment. Our aim was to evaluate, in an elastase-induced pulmonary emphysema in mice, the effects of and underlying mechanisms of three related natural monoterpenes (p-cymene, carvacrol and thymol) isolated from essential oil from leaves Lippia sidoides Cham. (Verbenaceae). Methods: Mices received porcine pancreatic elastase (PPE) and were treated with p-cymene, carvacrol, thymol or vehicle 30 min later and again on 7th, 14th and 28th days. Lung inflammatory profile and histological sections were evaluated. Results: In the elastase-instilled animals, the tested monoterpenes reduced alveolar enlargement, macrophages and the levels of IL-1β, IL-6, IL-8 and IL-17 in bronchoalveolar lavage fluid (BALF), and collagen fibers, MMP-9 and p-65-NF-κB-positive cells in lung parenchyma (p < 0.05). All treatments attenuated levels of 8-iso-PGF2α but only thymol was able to reduced exhaled nitric oxide (p < 0.05). Conclusion: Monoterpenes p-cymene, carvacrol and thymol reduced lung emphysema and inflammation in mice. No significant differences among the three monoterpenes treatments were found, suggesting that the presence of hydroxyl group in the molecular structure of thymol and carvacrol do not play a central role in the anti-inflammatory effects.

Evidences of Herbal Medicine-Derived Natural Products Effects in Inflammatory Lung Diseases.

Pulmonary inflammation is a hallmark of many respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory syndrome distress (ARDS). Most of these diseases are treated with anti-inflammatory therapy in order to prevent or to reduce the pulmonary inflammation. Herbal medicine-derived natural products have been used in folk medicine and scientific studies to evaluate the value of these compounds have grown in recent years. Many substances derived from plants have the biological effects in vitro and in vivo, such as flavonoids, alkaloids, and terpenoids. Among the biological activities of natural products derived from plants can be pointed out the anti-inflammatory, antiviral, antiplatelet, antitumor anti-allergic activities, and antioxidant. Although many reports have evaluated the effects of these compounds in experimental models, studies evaluating clinical trials are scarce in the literature. This review aims to emphasize the effects of these different natural products in pulmonary diseases in experimental models and in humans and pointing out some possible mechanisms of action.

New Pharmacological Targets for Asthma Drug Development

Asthma is an inflammatory disorder characterized by airway hyperresponsiveness, followed by inflammation, remodeling and oxidative stress in the respiratory system and lung tissue. While glucocorticosteroids remain the gold-standard of asthma therapy, they have limitations because of their potentially severe adverse effects and the presence of corticosteroid resistance in some patients. In the present review we will focus in four main groups of experimental pharmacological approaches for future asthma and hyperresponsiveness treatment: proteinase inhibitors and flavonoids, arginase and iNOS inhibition, Rho-kinase inhibitors, cholinergic anti-inflammatory system and nicotinic receptors.

Prophylactic and therapeutic treatment with the flavonone sakuranetin ameliorates LPS-induced acute lung injury

Sakuranetin is the main isolate flavonoid from Baccharis retusa (Asteraceae) leaves and exhibits anti-inflammatory and antioxidative activities. Acute respiratory distress syndrome is an acute failure of the respiratory system for which effective treatment is urgently necessary. This study investigated the preventive and therapeutic effects of sakuranetin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Animals were treated with intranasal sakuranetin 30 min before or 6 h after instillation of LPS. Twenty-four hours after ALI was induced, lung function, inflammation, macrophages population markers, collagen fiber deposition, the extent of oxidative stress, and the expression of matrix metalloprotease-9 (MMP-9), tissue inhibitor of MMP-9 (TIMP-1) and NF-κB were evaluated. The animals began to show lung alterations 6 h after LPS instillation, and these changes persisted until 24 h after LPS administration. Preventive and therapeutic treatment with sakuranetin reduced the neutrophils in the peripheral blood and in the bronchial alveolar lavage. Sakuranetin treatment also reduced macrophage populations, particularly that of M1-like macrophages. In addition, sakurnaetin treatment reduced keratinocyte-derived chemokines (IL-8 homolog) and NF-κB levels, collagen fiber formation, MMM-9 and TIMP-1-positive cells, and oxidative stress in lung tissues compared with LPS animals treated with vehicle. Finally, sakuranetin treatment also reduced total protein, and the levels of TNF-α and IL-1β in the lung. This study shows that sakuranetin prevented and reduced pulmonary inflammation induced by LPS. Because sakuranetin modulates oxidative stress, the NF-κB pathway, and lung function, it may constitute a novel therapeutic candidate to prevent and treat ALI.

Sakuranetin reverses vascular peribronchial and lung parenchyma remodeling in a murine model of chronic allergic pulmonary inflammation.

BACKGROUND AND PURPOSE Asthma is a disease of high prevalence and morbidity that generates high costs in hospitalization and treatment. Although the airway is involved in the physiopathology of asthma, there is also evidence of the importance of vascular and lung parenchyma inflammation and remodeling, which can contribute to the functional pulmonary alterations observed in asthmatic patients. Our aim was to evaluate treatment using sakuranetin, a flavone isolated from the twigs of Baccharis retusa (Asteraceae), on vascular and lung parenchyma alterations in an experimental murine model of asthma. METHODS Male BALB/c mice were subjected to a sensitization protocol with ovalbumin for 30days and were treated with or without sakuranetin (20mg/kg/mice) or dexamethasone (5mg/kg/mice); then, the lungs were collected for histopathological analysis. We evaluated extracellular matrix remodeling (collagen and elastic fibers), inflammation (eosinophils and NF-kB) and oxidative stress (8-isoprostane) in the pulmonary vessels and lung parenchyma. The thickness of the vascular wall was quantified, as well as the vascular endothelial growth factor (VEGF) levels. RESULTS We demonstrated that sakuranetin reduced the number of eosinophils and elastic fibers in both the pulmonary vessels and the lung parenchyma, probably due to a reduction of oxidative stress and of the transcription factor NF-kB and VEGF levels in the lung. In addition, it reduced the thickness of the pulmonary vascular wall. The treatment had no effect on the collagen fibers. In most of the parameters, the effect of sakuranetin was similar to the dexamethasone effect. CONCLUSIONS AND IMPLICATIONS Sakuranetin had anti-inflammatory and antioxidant effects, preventing vascular and distal parenchyma changes in this experimental model of asthma.

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-α (TNF-α), 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.

Vesicular acetylcholine transport deficiency potentiates some inflammatory responses induced by diesel exhaust particles

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-α, and NF-κB in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-α were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.