Mariana Nascimento Machado

P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.

Redox markers and inflammation are differentially affected by atorvastatin, pravastatin or simvastatin administered before endotoxin-induced acute lung injury.

Statins are standard therapy for the treatment of lipid disorders, and the field of redox biology accepts that statins have antioxidant properties. Our aim in this report was to consider the pleiotropic effects of atorvastatin, pravastatin and simvastatin administered prior to endotoxin-induced acute lung injury. Male mice were divided into 5 groups and intraperitoneally injected with LPS (10 mg/kg), LPS plus atorvastatin (10 mg/kg/day; A + LPS group), LPS plus pravastatin (5 mg/kg/day; P + LPS group) or LPS plus simvastatin (20 mg/kg/day; S + LPS group). The control group received saline. All mice were sacrificed one day later. There were fewer leukocytes in the P + LPS and S + LPS groups than in the LPS group. MCP-1 cytokine levels were lower in the P + LPS group, while IL-6 levels were lower in the P + LPS and S + LPS groups. TNF-α was lower in all statin-treated groups. Levels of redox markers (superoxide dismutase and catalase) were lower in the A + LPS group (p < 0.01). The extent of lipid peroxidation (malondialdehyde and hydroperoxides) was reduced in all statin-treated groups (p < 0.05). Myeloperoxidase was lower in the P + LPS group (p < 0.01). Elastance levels were significantly greater in the LPS group compared to the statin groups. Our results suggest that atorvastatin and pravastatin but not simvastatin exhibit anti-inflammatory and antioxidant activity in endotoxin-induced acute lung injury.

LASSBio 596 per os avoids pulmonary and hepatic inflammation induced by microcystin-LR

Cyanobacterial blooms that generate microcystins (MCYSTs) are increasingly recognized as an important health problem in aquatic ecosystems. We have previously reported the impairment of pulmonary structure and function by microcystin-LR (MCYST-LR) exposure as well as the pulmonary improvement by intraperitoneally injected (i.p.) LASSBio 596. In the present study, we aimed to evaluate the usefulness of LASSBio 596 per os on the treatment of pulmonary and hepatic injuries induced by MCYST-LR. Swiss mice received an intraperitoneal injection of 40 μl of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 μg/kg). After 6 h the animals received either saline (TOX and CTRL groups) or LASSBio 596 (50 mg/kg, LASS group) by gavage. Eight hours after the first instillation, lung impedance (static elastance, elastic component of viscoelasticity and resistive, viscoelastic and total pressures) was determined by the end-inflation occlusion method. Left lung and liver were prepared for histology. In lung and hepatic homogenates MCYST-LR, TNF-α, IL-1β and IL-6 were determined by ELISA. LASSBio 596 per os (LASS mice) kept all lung mechanical parameters, polymorphonuclear (PMN) cells, pro-inflammatory mediators, and alveolar collapse similar to control mice (CTRL), whereas in TOX these findings were higher than CTRL. Likewise, liver structural deterioration (hepatocytes inflammation, necrosis and steatosis) and inflammatory process (high levels of pro-inflammatory mediators) were less evident in the LASS than TOX group. LASS and CTRL did not differ in any parameters studied. In conclusion, orally administered LASSBio 596 prevented lung and hepatic inflammation and completely blocked pulmonary functional and morphological changes induced by MCYST-LR.

Redox Imbalance and Pulmonary Function in Bleomycin-Induced Fibrosis in C57BL/6, DBA/2, and BALB/c Mice

The development of bleomycin-induced pulmonary fibrosis (BLEO-PF) has been associated with differences in genetic background and oxidative stress status. The authors’ aim was to investigate the crosstalk between the redox profile, lung histology, and respiratory function in BLEO-PF in C57BL/6, DBA/2, and BALB/c mice. BLEO-PF was induced with a single intratracheal dose of bleomycin (0.1 U/mouse). Twenty-one days after bleomycin administration, the mortality rate was over 50% in C57BL/6 and 20% in DBA/2 mice, and BLEO-PF was not observed in BALB/c. There was an increase in lung static elastance (p < .001), viscoelastic/inhomogeneous pressure (p < .05), total pressure drop after flow interruption (p < .01), and ΔE (p < .05) in C57BL/6 mice. The septa volume increased in C57BL/6 (p < .05) and DBA/2 (p < .001). The levels of IFN-γ were reduced in C57BL/6 mice (p < .01). OH-proline levels were increased in C57BL/6 and DBA/2 mice (p < .05). SOD activity and expression were reduced in C57BL/6 and DBA/2 mice (p < .001 and p < .001, respectively), whereas catalase was reduced in all strains 21 days following bleomycin administration compared with the saline groups (C57BL/6: p < .05; DBA/2: p < .01; BALB/c: p < .01). GPx activity and GPx1/2 expression decreased in C57BL/6 (p < .001). The authors conclude that BLEO-PF resistance may also be related to the activity and expression of SOD in BALB/c mice.

N-(2-mercaptopropionyl)-glycine but not Allopurinol prevented cigarette smoke-induced alveolar enlargement in mouse.

We investigated the possible protective effects of the Allopurinol (A), N-(2-mercaptopropionyl)-glycine (M) and N-acetylcysteine (N) against lung injury caused by long-term exposure to cigarette smoke (CS) in mouse. C57BL6 mice were exposed to 12 cigarettes a day for 60 days and concomitantly treated with either one of the antioxidant drugs diluted in saline (CS+A-50 mg/kg; CS+M-200 mg/kg/day; CS+N-200 mg/kg/day). Control groups were sham-smoked (AA). Long-term CS exposure results in extensive parenchyma destruction in CS group. Both CS+N and CS+M groups showed preserved alveolar structure and showed preserved lung function when compared to CS group. Macrophage and neutrophil counts were decreased in CS+M, and CS+N groups when compared to CS group (p<0.05). Antioxidant enzyme activities were reduced in all treated groups. CS+A showed the highest reduction in catalase activity (-25%, p<0.01). We conclude that M treatment reduced long-term CS-induced inflammatory lung parenchyma destruction and lung function, comparable to N treatment, however, antioxidant administration did not reverse CS-induced antioxidant enzyme activity reduction.

Papain-induced experimental pulmonary emphysema in male and female mice

In papain-induced models of emphysema, despite the existing extensive description of the cellular and molecular aspects therein involved, sexual hormones may play a complex and still not fully understood role. Hence, we aimed at exploring the putative gender-related differences in lung mechanics, histology and oxidative stress in papain-exposed mice. Thirty adult BALB/c mice received intratracheally either saline (50 μL) or papain (10 U/50 μL saline) once a week for 2 weeks. In males papain increased lung resistive and viscoelastic/inhomogeneous pressures, static elastance, and viscoelastic component of elastance, while females showed higher static elastance and resistive pressure only. Both genders presented similar higher parenchymal cellularity and mean alveolar diameter, and less collagen-elastic fiber content and body weight gain than their respective controls. Increased functional residual capacity was more prominent in males. Female papain-treated mice were more susceptible to oxidative stress. Thus, male and female papain-exposed mice respond differently, which should be carefully considered to avoid confounding results.

The influence of 5-lipoxygenase on cigarette smoke-induced emphysema in mice.

BACKGROUND Pulmonary emphysema is characterized by the loss of lung architecture. Our hypothesis is that the inhibition of 5-lipoxygenase (5-LO) production may be an important strategy to reduce inflammation, oxidative stress, and metalloproteinases in lung tissue resulting from cigarette smoke (CS)-induced emphysema. METHODS 5-LO knockout (129S2-Alox5(tm1Fun)/J) and wild-type (WT) mice (129S2/SvPas) were exposed to CS for 60days. Mice exposed to ambient air were used as Controls. Oxidative, inflammatory, and proteolytic markers were analyzed. RESULTS The alveolar diameter was decreased in CS 5-LO(-/-) mice when compared with the WT CS group. The CS exposure resulted in less pronounced pulmonary inflammation in the CS 5-LO(-/-) group. The CS 5-LO(-/-) group showed leukotriene B4 values comparable to those of the Control group. The expression of MMP-9 was decreased in the CS 5-LO(-/-) group when compared with the CS WT group. The expression of superoxide dismutase, catalase, and glutathione peroxidase were decreased in the CS 5-LO(-/-) group when compared with the Control group. The protein expression of nuclear factor (erythroid-derived 2)-like 2 was reduced in the CS 5-LO(-/-) group when compared to the CS WT group. CONCLUSION In conclusion, we show for the first time that 5-LO deficiency protects 129S2 mice against emphysema caused by CS. We suggest that the main mechanism of pathogenesis in this model involves the imbalance between proteases and antiproteases, particularly the association between MMP-9 and TIMP-1. General significance This study demonstrates the influence of 5-LO mediated oxidative stress, inflammation, and proteolytic markers in CS exposed mice.

Intratracheal instillation of coal and coal fly ash particles in mice induces DNA damage and translocation of metals to extrapulmonary tissues

Continuous exposure to coal mining particles can cause a variety of lung diseases. We aimed to evaluate the outcomes of exposure to detailed characterized coal and coal fly ash (CFA) particles on DNA, lung and extrapulmonary tissues. Coal samples (COAL11 and COAL16) and CFA samples (CFA11 and CFA16) were included in this study. Intending to enhance the combustion process COAL16 was co-fired with a mixture of fuel oil and diesel oil, producing CFA16. Male BALB/c mice were intratracheally instilled with coal and CFA particles. Measurements were done 24h later. Results showed significant rigidity and obstruction of the central airways only for animals acutely exposed to coal particles. The COAL16 group also showed obstruction of the peripheral airways. Mononuclear cells were recruited in all treatment groups and expression of cytokines, particularly TNF-α and IL-1β, was observed. Only animals exposed to COAL16 showed a significant expression of IL-6 and recruitment of polymorphonuclear cells. DNA damage was demonstrated by Comet assay for all groups. Cr, Fe and Ni were detected in liver, spleen and brain, showing the efficient translocation of metals from the bloodstream to extrapulmonary organs. These effects were associated with particle composition (oxides, hydroxides, phosphates, sulfides, sulphates, silciates, organic-metalic compounds, and polycyclic aromatic hidrocarbons) rather than their size. This work provides state of knowledge on the effects of acute exposure to coal and CFA particles on respiratory mechanics, DNA damage, translocation of metals to other organs and related inflammatory processes.

Pulmonary functional and morphological damage after exposure to tripoli dust

Tripoli is a microcrystalline siliceous rock used to polish metals and precious stones. Its inhalation has been associated with increased prevalence of breathing complaints and pneumoconiosis. However, its acute human exposure has not been so far studied. We aimed at evaluating the putative mechanical, morphological, biochemical and inflammatory lung damage in mice acutely exposed to Tripoli dust. BALB/c mice were randomly assigned to 2 groups: In control group (CTRL, n=6) animals received intratracheally (i.t.) 0.9% NaCl (50μl), while Tripoli group (TRIP, n=15) received 20mg of Tripoli powder diluted in 50μL of saline i.t. The experiments were done 15 days later. TRIP mice showed higher pulmonary mechanical impedance, polymorphonuclear cells, TNF-α, IL1-β and IL-6 than CTRL. TRIP presented granulomatous nodules containing collagenous fibers that occupied 35% of the lung tissue area. In conclusion, acute exposure to Tripoli dust triggered important lung damage in mice lungs that if found in human workers could trigger severe illness.

The anti-inflammatory and anti-oxidative actions of eugenol improve lipopolysaccharide-induced lung injury

Acute lung injury (ALI) remains a major cause of mortality. In lipopolysaccharide (LPS)-stimulated macrophages, eugenol reduces cyclooxygenase-2 expression, NF-κB activation, and inflammatory mediators. We examined the anti-inflammatory and anti-oxidative action of eugenol in an in vivo model of LPS-induced lung injury. Lung mechanics and histology were analyzed in mice 24 h after LPS exposure, with and without eugenol treatment at different doses. Additional animals, submited to the same protocol, were treated with eugenol at 150 mg/kg to determine its effect on inflammatory cytokines (ELISA) and oxidative markers. LPS-induced lung functional and histological changes were significantly improved by eugenol, in a dose-dependent way. Furthermore, eugenol (150 mg/kg) was able to inhibit the release of inflammatory cytokines (TNF-α, IL-1β and IL-6), NADPH oxidase activity, as well as antioxidant enzymes activity (superoxide dismutase, catalase and glutathione peroxidase). Finally, eugenol reduced LPS-induced protein oxidation. In conclusion, eugenol improved in vivo LPS-induced ALI through both anti-inflammatory and anti-oxidative effects, avoiding damage to lung structure.