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1.
J Vis Exp ; (205)2024 Mar 22.
Article En | MEDLINE | ID: mdl-38587373

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.


Cyclic AMP , Respiratory System , Humans , Cyclic AMP/metabolism , Isoproterenol/pharmacology , Colforsin/pharmacology , HEK293 Cells , Respiratory System/metabolism
2.
Front Physiol ; 14: 1149063, 2023.
Article En | MEDLINE | ID: mdl-36926196

Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.

3.
Antioxidants (Basel) ; 12(3)2023 Feb 21.
Article En | MEDLINE | ID: mdl-36978796

Acute and chronic lung injuries are among the leading causes of mortality worldwide. Lung injury can affect several components of the respiratory system, including the airways, parenchyma, and pulmonary vasculature. Although acute and chronic lung injuries represent an enormous economic and clinical burden, currently available therapies primarily focus on alleviating disease symptoms rather than reversing and/or preventing lung pathology. Moreover, some supportive interventions, such as oxygen and mechanical ventilation, can lead to (further) deterioration of lung function and even the development of permanent injuries. Lastly, sepsis, which can originate extrapulmonary or in the respiratory system itself, contributes to many cases of lung-associated deaths. Considering these challenges, we aim to summarize molecular and cellular mechanisms, with a particular focus on airway inflammation and oxidative stress that lead to the characteristic pathophysiology of acute and chronic lung injuries. In addition, we will highlight the limitations of current therapeutic strategies and explore new antioxidant-based drug options that could potentially be effective in managing acute and chronic lung injuries.

4.
SLAS Discov ; 27(3): 185-190, 2022 04.
Article En | MEDLINE | ID: mdl-35227934

Cigarette smoke (CS) and air pollutants (AP) activate pathological processes in bronchial epithelial cells resulting in lung function decline which severely impacts human health. Knowledge about the molecular mechanism(s) by which CS and AP induce pathology is limited. Our previous studies in 2D cultures of human bronchial epithelial (BEAS-2B) cells showed that CS exposure activates transforming growth factor-ß1 (TGF-ß1) release and signaling. Furthermore, CS exposure reduced the expression of E-cadherin, which was prevented by applying a TGF-ß1 neutralizing antibody. Exposure of BEAS-2B cells cultured in 2D to diesel exhaust particles (DEP) increased TGF-ß1 protein expression and reduced the expression of epithelial cell markers, whereas mesenchymal markers are upregulated. Conventional 2D cell culture may, however, not fully reflect the physiology of bronchial epithelial cells in vivo. To simulate the in vivo situation more closely we cultured the bronchial epithelial cells in a 3D environment in the current study. Treatment of epithelial spheroids with TGF-ß resulted in reduced E-cadherin and increased collagen I expression, indicating the activation of epithelial-to-mesenchymal transition (EMT). Similarly, exposure of spheroids to DEP induced and EMT-like phenotype. Collectively, our data indicate AP induces an EMT-like phenotype of BEAS-2B cells in 3D spheroid cultures. This opens new avenues for drug development for the treatment of lung diseases induced by AP. The 3D spheroid cell culture is a novel, innovative and physiologically relevant model for culturing a variety of cells. It is a versatile tool for both high-throughput studies and for identifying molecular mechanisms involved in bronchial epithelial cell (patho)physiology.


Air Pollutants , Transforming Growth Factor beta1 , Air Pollutants/metabolism , Air Pollutants/pharmacology , Bronchi , Cadherins/metabolism , Epithelial-Mesenchymal Transition/physiology , Transforming Growth Factor beta1/metabolism
5.
Pulm Pharmacol Ther ; 69: 102053, 2021 08.
Article En | MEDLINE | ID: mdl-34214692

INTRODUCTION: Cigarette smoke (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema. The use of antioxidants has emerged as a potential therapeutic strategy to treat airway inflammation and lung diseases. In the current study, we investigated the potential therapeutic impact of diallyl disulfide (Dads) treatment in a murine model of CS-induced emphysema. METHODS: C57BL/6 mice were exposed to CS for 60 consecutive days and treated with vehicle or Dads (30, 60 or 90 mg/kg) by oral gavage for the last 30 days, three times/week. The control group was sham-smoked and received vehicle treatment. All mice were euthanized 24 h after day 60; bronchoalveolar lavage (BAL) was performed and lungs were processed for further experimentation. Histological (HE stained sections, assessment of mean linear intercept (Lm)), biochemical (nitrite, superoxide dismutase (SOD), glutathione transferase (GST), and malondialdehyde (MDA) equivalents), and molecular biology (metalloproteinase (MMP) 12, SOD2, carbonyl reductase 1 (CBR1), nitrotyrosine (PNK), 4-hydroxynonenal (4-HNE), and CYP2E1) analyses were performed. RESULTS: Treatment with Dads dose-dependently reduced CS-induced leukocyte infiltration into the airways (based on BAL fluid counts) and improved lung histology (indicated by a reduction of Lm). Furthermore, CS exposure dramatically reduced the activity of the antioxidant enzymes SOD and GST in lung tissue and increased nitrite and MDA levels in BAL; these effects were all effectively counteracted by Dads treatment. Western blot analysis further confirmed the antioxidant potential of Dads, showing that treatment prevented the CS-induced decrease in SOD2 expression and increase in lung damage markers, such as CBR1, PNK, and 4-HNE. Furthermore, increased MMP12 (an important hallmark of CS-induced emphysema) and CYP2E1 lung protein levels were significantly reduced in mice receiving Dads treatment. CONCLUSION: Our findings demonstrate that treatment with Dads is effective in preventing multiple pathological features of CS-induced emphysema in an in vivo mouse model. In addition, we have identified several proteins/enzymes, including 4-HNE, CBR1, and CYP2E1, that are modifiable by Dads and could represent specific therapeutic targets for the treatment of COPD and emphysema.


Emphysema , Pulmonary Emphysema , Allyl Compounds , Animals , Bronchoalveolar Lavage Fluid , Disulfides , Lung , Mice , Mice, Inbred C57BL , Pulmonary Emphysema/drug therapy , Pulmonary Emphysema/etiology , Pulmonary Emphysema/prevention & control , Smoke/adverse effects , Smoking
6.
Int J Mol Sci ; 21(24)2020 Dec 18.
Article En | MEDLINE | ID: mdl-33352854

Air pollution is mainly caused by burning of fossil fuels, such as diesel, and is associated with increased morbidity and mortality due to adverse health effects induced by inflammation and oxidative stress. Dimethyl fumarate (DMF) is a fumaric acid ester and acts as an antioxidant and anti-inflammatory agent. We investigated the potential therapeutic effects of DMF on pulmonary damage caused by chronic exposure to diesel exhaust particles (DEPs). Mice were challenged with DEPs (30 µg per mice) by intranasal instillation for 60 consecutive days. After the first 30 days, the animals were treated daily with 30 mg/kg of DMF by gavage for the remainder of the experimental period. We demonstrated a reduction in total inflammatory cell number in the bronchoalveolar lavage (BAL) of mice subjected to DEP + DMF as compared to those exposed to DEPs alone. Importantly, DMF treatment was able to reduce lung injury caused by DEP exposure. Intracellular total reactive oxygen species (ROS), peroxynitrite (OONO), and nitric oxide (NO) levels were significantly lower in the DEP + DMF than in the DEP group. In addition, DMF treatment reduced the protein expression of kelch-like ECH-associated protein 1 (Keap-1) in lung lysates from DEP-exposed mice, whereas total nuclear factor κB (NF-κB) p65 expression was decreased below baseline in the DEP + DMF group compared to both the control and DEP groups. Lastly, DMF markedly reduced DEP-induced expression of nitrotyrosine, glutathione peroxidase-1/2 (Gpx-1/2), and catalase in mouse lungs. In summary, DMF treatment effectively reduced lung injury, inflammation, and oxidative and nitrosative stress induced by chronic DEP exposure. Consequently, it may lead to new therapies to diminish lung injury caused by air pollutants.


Dimethyl Fumarate/pharmacology , Oxidative Stress , Pneumonia/etiology , Pneumonia/metabolism , Vehicle Emissions , Air Pollutants/adverse effects , Animals , Biomarkers , Bronchoalveolar Lavage Fluid/immunology , Cytokines/metabolism , Disease Models, Animal , Inflammation Mediators/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , Mice , NF-kappa B/metabolism , Oxidation-Reduction , Pneumonia/drug therapy , Pneumonia/pathology , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Vehicle Emissions/toxicity
7.
Curr Opin Pharmacol ; 51: 34-42, 2020 04.
Article En | MEDLINE | ID: mdl-32622335

The second messenger molecule 3'5'-cyclic adenosine monophosphate (cAMP) imparts several beneficial effects in lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). While cAMP is bronchodilatory in asthma and COPD, it also displays anti-fibrotic properties that limit fibrosis. Phosphodiesterases (PDEs) metabolize cAMP and thus regulate cAMP signaling. While some existing therapies inhibit PDEs, there are only broad family specific inhibitors. The understanding of cAMP signaling compartments, some centered around lipid rafts/caveolae, has led to interest in defining how specific PDE isoforms maintain these signaling microdomains. The possible altered expression of PDEs, and thus abnormal cAMP signaling, in obstructive lung diseases has been poorly explored. We propose that inhibition of specific PDE isoforms can improve therapy of obstructive lung diseases by amplifying specific cAMP signals in discreet microdomains.


Cyclic AMP/metabolism , Drug Development/trends , Lung Diseases, Obstructive/drug therapy , Lung Diseases, Obstructive/metabolism , Phosphodiesterase Inhibitors/administration & dosage , Phosphoric Diester Hydrolases/metabolism , Animals , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism
8.
Biochem Soc Trans ; 48(3): 799-811, 2020 06 30.
Article En | MEDLINE | ID: mdl-32597478

Air pollution is a major environmental threat and each year about 7 million people reported to die as a result of air pollution. Consequently, exposure to air pollution is linked to increased morbidity and mortality world-wide. Diesel automotive engines are a major source of urban air pollution in the western societies encompassing particulate matter and diesel exhaust particles (DEP). Air pollution is envisioned as primary cause for cardiovascular dysfunction, such as ischemic heart disease, cardiac dysrhythmias, heart failure, cerebrovascular disease and stroke. Air pollution also causes lung dysfunction, such as chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis (IPF), and specifically exacerbations of these diseases. DEP induces inflammation and reactive oxygen species production ultimately leading to mitochondrial dysfunction. DEP impair structural cell function and initiate the epithelial-to-mesenchymal transition, a process leading to dysfunction in endothelial as well as epithelial barrier, hamper tissue repair and eventually leading to fibrosis. Targeting cyclic adenosine monophosphate (cAMP) has been implicated to alleviate cardiopulmonary dysfunction, even more intriguingly cAMP seems to emerge as a potent regulator of mitochondrial metabolism. We propose that targeting of the mitochondrial cAMP nanodomain bear the therapeutic potential to diminish air pollutant - particularly DEP - induced decline in cardiopulmonary function.


Air Pollutants/toxicity , Heart Diseases/chemically induced , Lung Diseases/chemically induced , Nanotechnology , Humans , Mitochondria/drug effects
9.
Br J Pharmacol ; 176(14): 2402-2415, 2019 07.
Article En | MEDLINE | ID: mdl-30714124

Over the past decades, research has defined cAMP as one of the central cellular nodes in sensing and integrating multiple pathways and as a pivotal role player in lung pathophysiology. Obstructive lung disorders, such as chronic obstructive pulmonary disease (COPD), are characterized by a persistent and progressive airflow limitation and by oxidative stress from endogenous and exogenous insults. The extent of airflow obstruction depends on the relative deposition of different constituents of the extracellular matrix, a process related to epithelial-to-mesenchymal transition, and which subsequently results in airway fibrosis. Oxidative stress from endogenous and also from exogenous sources causes a profound worsening of COPD. Here we describe how cAMP scaffolds and their different signalosomes in different subcellular compartments may contribute to COPD. Future research will require translational studies to alleviate disease symptoms by pharmacologically targeting the cAMP scaffolds. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.


Cyclic AMP/metabolism , Epithelial-Mesenchymal Transition , Oxidative Stress , Pulmonary Disease, Chronic Obstructive/metabolism , Animals , Humans
10.
Pharmacol Ther ; 197: 225-242, 2019 05.
Article En | MEDLINE | ID: mdl-30759374

Chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma, affect millions of people all over the world. Cyclic adenosine monophosphate (cAMP) which is one of the most important second messengers, plays a vital role in relaxing airway smooth muscles and suppressing inflammation. Given its vast role in regulating intracellular responses, cAMP provides an attractive pharmaceutical target in the treatment of chronic respiratory diseases. Phosphodiesterases (PDEs) are enzymes that hydrolyze cyclic nucleotides and help control cyclic nucleotide signals in a compartmentalized manner. Currently, the selective PDE4 inhibitor, roflumilast, is used as an add-on treatment for patients with severe COPD associated with bronchitis and a history of frequent exacerbations. In addition, other novel PDE inhibitors are in different phases of clinical trials. The current review provides an overview of the regulation of various PDEs and the potential application of selective PDE inhibitors in the treatment of COPD and asthma. The possibility to combine various PDE inhibitors as a way to increase their therapeutic effectiveness is also emphasized.


Lung Diseases/drug therapy , Phosphodiesterase Inhibitors/therapeutic use , Phosphoric Diester Hydrolases/metabolism , Animals , Humans , Lung Diseases/enzymology
11.
Phytomedicine ; 55: 70-79, 2019 Mar 01.
Article En | MEDLINE | ID: mdl-30668445

BACKGROUND: Eucalyptol is a monoterpenoid oil present in many plants, principally the Eucalyptus species, and has been reported to have anti-inflammatory and antioxidative effects. HYPOTHESIS/PURPOSE: Since the potential effect of eucalyptol on mouse lung repair has not yet been studied, and considering that chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide, the aim of this study was to investigate eucalyptol treatment in emphysematous mice. STUDY DESIGN: Male mice (C57BL/6) were divided into the following groups: control (sham-exposed), cigarette smoke (CS) (mice exposed to 12 cigarettes a day for 60 days), CS + 1 mg/ml (CS mice treated with 1 mg/ml eucalyptol for 60 days), and CS + 10 mg/ml (CS mice treated with 10 mg/ml eucalyptol for 60 days). Mice in the CS and control groups received vehicle for 60 days. Eucalyptol (or the vehicle) was administered via inhalation (15 min/daily). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure. METHODS: Histology and additional lung morphometric analyses, including analysis of mean linear intercept (Lm) and volume density of alveolar septa (Vv[alveolar septa]) were performed. Biochemical analyses were also performed using colorimetric assays for myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) activity, in addition to using ELISA kits for the determination of inflammatory marker levels (tumor necrosis factor alpha [TNF-α], interleukin-1 beta [IL-1ß], interleukin 6 [IL-6], keratinocyte chemoattractant [KC], and tumor growth factor beta 1 [TGF-ß1]). Finally, we investigated protein levels by western blotting (nuclear factor (erythroid-derived 2)-like 2 [Nrf2], nuclear factor kappa B [NF-κB], matrix metalloproteinase 12 [MMP-12], tissue inhibitor of matrix metalloproteinase 1 [TIMP-1], neutrophil elastase [NE], and elastin). RESULTS: Eucalyptol promoted lung repair at the higher dose (10 mg/ml), with de novo formation of alveoli, when compared to the CS group. This result was confirmed with Lm and Vv[alveolar septa] morphometric analyses. Moreover, collagen deposit around the peribronchiolar area was reduced with eucalyptol treatment when compared to the CS group. Eucalyptol also reduced all inflammatory (MPO, TNF-α, IL-1ß, IL-6, KC, and TGF-ß1) and redox marker levels (MDA) when compared to the CS group (at least p < 0.05). In general, 10 mg/ml eucalyptol was more effective than 1 mg/ml and, at both doses, we observed an upregulation of SOD activity when compared to the CS group (p < 0.001). Eucalyptol upregulated elastin and TIMP-1 levels, and reduced neutrophil elastase (NE) levels, when compared to the CS group. CONCLUSION: In summary, eucalyptol promoted lung repair in emphysematous mice and represents a potential therapeutic phytomedicine in the treatment of COPD.


Emphysema/drug therapy , Eucalyptol/pharmacology , Smoking/adverse effects , Animals , Collagen/metabolism , Cytokines/metabolism , Emphysema/chemically induced , Emphysema/pathology , Lung/drug effects , Lung/metabolism , Lung/pathology , Male , Matrix Metalloproteinase 12/metabolism , Mice, Inbred C57BL , NF-kappa B/metabolism , Superoxide Dismutase/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism
12.
Biomed Pharmacother ; 109: 1547-1555, 2019 Jan.
Article En | MEDLINE | ID: mdl-30551407

High-fat diet (HFD)-induced obesity is a worldwide health problem and can cause lipid accumulation in the liver. We evaluated the hepatoprotective effect of mate tea treatment in mice submitted to an HFD. C57BL/6 mice were fed an HFD for 13 weeks with and without mate tea. A separate group of mice was treated with fenofibrate as a positive control (a regular drug for lipid disorders). Histological analyses, glucose tolerance tests (GTT), and quantification of mediators related to lipid peroxidation, oxidative stress and blood biomarkers for lipid profile were performed. The weight of animals and major organs related to hepatic steatosis was determined, and proinflammatory cytokines and the participation of the Nrf2 pathway and adiponectin were evaluated. Mate tea prevented the accumulation of lipid droplets in hepatocytes as well as weight gain in animals submitted to the HFD. Mate tea treatment also prevented increases in the liver weight, heart weight and amount of visceral and subcutaneous white adipose tissue. Mate tea was able to prevent the deregulation of glucose uptake, as evaluated by GTT, and improved the indicators of oxidative stress, such as nitrite levels, catalase activity, and oxidative damage, as evaluated by protein carbonylation and the MDA levels. Mate tea had an anti-inflammatory effect, preventing the increase of IL-1ß and KC and upregulating the expression of Nrf2. Mate tea prevented insulin increase and HDL cholesterol decrease but did not affect total cholesterol or triglycerides levels. Treatment also prevented adiponectin increase. Mate tea may be a good resource to reduce hepatic steatosis in the future since it has anti-diabetic, anti-inflammatory and antioxidant effects, which prevent the accumulation of fat in the liver.


Diet, High-Fat/adverse effects , Fatty Liver/drug therapy , Liver/drug effects , Metabolic Diseases/drug therapy , Plant Extracts/pharmacology , Tea/chemistry , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Fatty Liver/metabolism , Glucose Tolerance Test/methods , Hepatocytes/drug effects , Hepatocytes/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Insulin/metabolism , Insulin Resistance/physiology , Lipid Metabolism/drug effects , Lipid Peroxidation/drug effects , Liver/metabolism , Male , Metabolic Diseases/metabolism , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/metabolism , Obesity/drug therapy , Obesity/metabolism , Oxidative Stress/drug effects
13.
Inflammation ; 42(2): 526-537, 2019 Apr.
Article En | MEDLINE | ID: mdl-30411213

Air pollution caused by fuel burning contributes to respiratory impairments that may lead to death. We aimed to investigate the effects of biodiesel (DB) burning in mouse lungs. DB particulate matter was collected from the exhaust pipes of a bus engine. Mice were treated with 250 µg or 1000 µg of DB particulate matter by intranasal instillation over 5 consecutive days. We demonstrated that DB particulate matter penetrated the lung in the 250-µg and 1000-µg groups. In addition, the DB particulate matter number in pulmonary parenchyma was 175-fold higher in the 250-µg group and 300-fold higher in the 1000-µg group compared to control mice. The instillation of DB particulate matter increased the macrophage number and protein levels of TNF-alpha in murine lungs. DB particulate matter enhanced ROS production in both exposed groups and the malondialdehyde levels compared to the control group. The protein expression levels of Nrf2, p-NF-kB, and HO-1 were higher in the 250-µg group and lower in the 1000-µg group than in control mice and the 250-µg group. In conclusion, DB particulate matter instillation promotes oxidative stress by activating the Nrf2/HO-1 and inflammation by p-NF-kB/TNF-alpha pathways.


Environmental Exposure/adverse effects , Lung/metabolism , Oxidative Stress/drug effects , Particulate Matter/adverse effects , Vehicle Emissions/toxicity , Animals , Heme Oxygenase-1/metabolism , Inflammation/chemically induced , Inflammation/metabolism , Membrane Proteins/metabolism , Metabolic Networks and Pathways , Mice , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Particulate Matter/toxicity , Tumor Necrosis Factor-alpha/metabolism
14.
Biomed Pharmacother ; 102: 160-168, 2018 Jun.
Article En | MEDLINE | ID: mdl-29554594

Emphysema results in a proteinase - antiproteinase imbalance, inflammation and oxidative stress. Our objective was to investigate whether atorvastatin could repair mouse lungs after elastase-induced emphysema. Vehicle (50 µL) or porcine pancreatic elastase (PPE) was administered on day 1, 3, 5 and 7 at 0.6 U intranasally. Male mice were divided into a control group (sham), PPE 32d (sacrificed 24 h after 32 days), PPE 64d (sacrificed 24 h after 64 days), and atorvastatin 1, 5 and 20 mg treated from day 33 until day 64 and sacrificed 24 h later (A1 mg, A5 mg and A20 mg, respectively). Treatment with atorvastatin was performed via inhalation for 10 min once a day. We observed that emphysema at day 32 was similar to emphysema at day 64. The mean airspace chord length (Lm) indicated a recovery of pulmonary morphology in groups A5 mg and A20 mg, as well as recovery of collagen and elastic fibers in comparison to the PPE group. Bronchoalveolar lavage fluid (BALF) leukocytes were reduced in all atorvastatin-treated groups. However, tissue macrophages were reduced only in the A20 mg group compared with the PPE group, while tissue neutrophils were reduced in the A5 mg and A20 mg groups. The redox balance was restored mainly in the A20 mg group compared with the PPE group. Finally, atorvastatin at doses of 5 and 20 mg reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and matrix metalloproteinase-12 (MMP-12) compared with the PPE group. In conclusion, atorvastatin was able to induce lung tissue repair in emphysematous mice.


Atorvastatin/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Inflammation/drug therapy , Pulmonary Emphysema/drug therapy , Animals , Atorvastatin/administration & dosage , Bronchoalveolar Lavage Fluid , Disease Models, Animal , Dose-Response Relationship, Drug , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Inflammation/pathology , Leukocytes/drug effects , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Neutrophils/drug effects , Neutrophils/metabolism , Oxidative Stress/drug effects , Pancreatic Elastase/toxicity , Pulmonary Emphysema/physiopathology , Swine , Time Factors
15.
Bioorg Med Chem ; 25(20): 5557-5568, 2017 10 15.
Article En | MEDLINE | ID: mdl-28886998

Chronic obstructive pulmonary disease (COPD) is an incurable and progressive disease. Emphysema is the principal manifestation of COPD, and the main cause of this condition is cigarette smoke (CS). Natural products have shown antioxidant and anti-inflammatory properties that can prevent acute lung inflammation and emphysema, but there are few reports in the literature regarding therapeutic approaches to emphysema. We hypothesized that supplementation with natural extracts would repair lung damage in emphysema caused by CS exposure. Mice were exposed to 60days of CS and then treated or not with three different natural extracts (mate tea, grape and propolis) orally for additional 60days. Histological analysis revealed significant improvements in lung histoarchitecture, with recovery of alveolar spaces in all groups treated with natural extracts. Propolis was also able to recovery alveolar septa and elastic fibers. Propolis also increased MMP-2 and decreased MMP-12 expression, favoring the process of tissue repair. Additionally, propolis recruited leukocytes, including macrophages, without ROS release. These findings led us to investigate the profile of these macrophages, and we showed that propolis could promote macrophage alternative activation, thus increasing the number of arginase-positive cells and IL-10 levels and favoring an anti-inflammatory microenvironment. We further investigated the participation of Nrf2 in lung repair, but no Nrf2 translocation to the nucleus was observed in lung cells. Proteins and enzymes related to Nrf2 were not altered, other than NQO1, which seemed to be activated by propolis in a Nrf2-independent manner. Finally, propolis downregulated IGF1 expression. In conclusion, propolis promoted lung repair in a mouse emphysema model via macrophage polarization from M1 to M2 in parallel to the downregulation of IGF1 expression in a Nrf2-independent manner.


Anti-Inflammatory Agents/pharmacology , Macrophages/drug effects , NF-E2-Related Factor 2/metabolism , Propolis/pharmacology , Pulmonary Emphysema/drug therapy , Smoking/drug therapy , Animals , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Pulmonary Emphysema/metabolism , Pulmonary Emphysema/pathology , Smoking/metabolism
16.
Inflammation ; 40(5): 1487-1496, 2017 Oct.
Article En | MEDLINE | ID: mdl-28534139

Elastase (PPE) is usually used for emphysema models, whereas bleomycin (BLM) is used for fibrosis models. The aim of this study was to investigate the effect of BLM in PPE-induced emphysema, as well as the effect of PPE in BLM-induced fibrosis. C57BL/6 mice were divided into five groups: control, PPE, BLM, PPE + BLM, and BLM + PPE. Mice received saline, PPE (3 U/mouse), or BLM (20 U/kg) by intranasal instillation. Mice from the BLM and BLM + PPE groups received BLM on day 0 and saline or PPE on day 21, respectively. Those in the PPE and PPE + BLM groups received PPE on day 0 and saline or BLM on day 21, respectively. Mice were euthanized on day 42. We performed histology, morphometry in lung sections and ELISA, zymography and western blotting in BAL samples or lung homogenates. In the lungs of PPE + BLM and BLM + PPE groups, we observed inflammation, oxidative stress and expression of MMP-2 and MMP-9. The alveolar enlargement was reduced in the PPE + BLM group, suggesting that the BLM could participate in the alveolar remodeling process. The significance of this result supports future therapeutic approaches targeting extracellular-matrix deposition in patients with emphysema as a way to repair the enlargement of alveoli and airspaces.


Bleomycin/therapeutic use , Pancreatic Elastase/therapeutic use , Pulmonary Emphysema/drug therapy , Pulmonary Fibrosis/drug therapy , Animals , Bleomycin/adverse effects , Inflammation/chemically induced , Lung/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice, Inbred C57BL , Oxidative Stress/drug effects , Pancreatic Elastase/adverse effects , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/pathology , Pulmonary Emphysema/chemically induced , Pulmonary Fibrosis/chemically induced
17.
Inflammation ; 40(3): 965-979, 2017 Jun.
Article En | MEDLINE | ID: mdl-28251446

Cigarette smoke (CS) induces pulmonary emphysema by inflammation, oxidative stress, and metalloproteinase (MMP) activation. Pharmacological research studies have not focused on tissue repair after the establishment of emphysema but have instead focused on inflammatory stimulation. The aim of our study was to analyze the effects of atorvastatin and simvastatin on mouse lung repair after emphysema caused by CS. Male mice (C57BL/6, n = 45) were divided into the following groups: control (sham-exposed), CSr (mice exposed to 12 cigarettes a day for 60 days and then treated for another 60 days with the vehicle), CSr+A (CSr mice treated with atorvastatin for 60 days), and CSr+S (CSr mice treated with simvastatin for 60 days). The treatment with atorvastatin and simvastatin was administered via inhalation (15 min with 1 mg/mL once a day). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure. We performed biochemical, morphological, and physiological analyses. We observed decreased levels of leukocytes and cytokines in statin-treated mice, accompanied by a reduction in oxidative stress markers. We also observed a morphological improvement confirmed by a mean linear intercept counting in statin-treated mice. Finally, statins also ameliorated lung function. We conclude that inhaled atorvastatin and simvastatin improved lung repair after cigarette smoke-induced emphysema in mice.


Atorvastatin/pharmacology , Lung/drug effects , Pulmonary Emphysema/drug therapy , Simvastatin/pharmacology , Animals , Atorvastatin/therapeutic use , Male , Mice , Mice, Inbred C57BL , Pulmonary Emphysema/etiology , Simvastatin/therapeutic use , Smoking/adverse effects
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