Simvastatin Posttreatment Controls Inflammation and Improves Bacterial Clearance in Experimental Sepsis.

Sepsis is characterized by a life-threatening organ dysfunction caused by an unbalanced host response to microbe infection that can lead to death. Besides being currently the leading cause of death in intensive care units worldwide, sepsis can also induce long-term consequences among survivors, such as cognitive impairment. Statins (lipid-lowering drugs widely used to treat dyslipidemia) have been shown to possess pleiotropic anti-inflammatory and antimicrobial effects. These drugs act inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, the limiting step in cholesterol biosynthesis. In this work, we evaluated the therapeutic effects of simvastatin in an animal model of sepsis. In previous study from our group, statin pretreatment avoided cognitive damage and neuroinflammation in sepsis survivors. Herein, we focused on acute inflammation where sepsis was induced by cecal ligation and puncture (CLP), and the animals were treated with simvastatin (2 mg/kg) 6 h after surgery. We measured plasma biochemical markers of organ dysfunction, cell migration, cell activation, bacterial elimination, production of nitric oxide 24 h after CLP, survival rate for 7 days, and cognitive impairment 15 days after CLP. One single administration of simvastatin 6 h after CLP was able to prevent both liver and kidney dysfunction. In addition, this drug decreased cell accumulation in the peritoneum as well as the levels of TNF-α, MIF, IL-6, and IL-1ß. Simvastatin diminished the number of bacterial colony forming units (CFU) and increased the production of nitric oxide production in the peritoneum. Simvastatin treatment increased survival for the first 24 h, but it did not alter survival rate at the end of 7 days. Our results showed that posttreatment with simvastatin hampered organ dysfunction, increased local production of nitric oxide, improved bacterial clearance, and modulated inflammation in a relevant model of sepsis.

Na/K-ATPase assay in the intact mice lung subjected to perfusion.

BACKGROUND: Among the characteristics of acute respiratory distress syndrome (ARDS) is edema formation and its resolution depends on pneumocyte Na/K-ATPase activity. Increased concentration of oleic acid (OA) in plasma induces lung injury by targeting Na/K-ATPase and, thus, interfering in sodium transport. FINDINGS: Presently, we adapted a radioactivity-free assay to detect Na/K-ATPase activity in perfused lung mice, comparing the inhibitory effect of ouabain and OA. We managed to perfuse only the lung, avoiding the systemic loss of rubidium. Rb+ incorporation into lung was measured by inductively coupled plasma optical emission spectrometry (ICP OES) technique, after lung tissue digestion. Na/K-ATPase activity was the difference between Rb+ incorporation with or without ouabain. Lung Na/K-ATPase was completely inhibited by perfusion with ouabain. However, OA caused a partial inhibition. CONCLUSIONS: In the present work the amount of incorporated Rb+ was greater than seen in our previous report, showing that the present technique is trustworthy. This new proposed assay may allow researchers to study the importance of Na/K-ATPase activity in lung pathophysiology.

Murine lung injury caused by Leptospira interrogans glycolipoprotein, a specific Na/K-ATPase inhibitor.

BACKGROUND: Leptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation. METHODS: We injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE2 and LTB4). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells. RESULTS: Leptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor. CONCLUSIONS: GLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.

Oleic acid inhibits lung Na/K-ATPase in mice and induces injury with lipid body formation in leukocytes and eicosanoid production.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can emerge from certain pathologies, such as sepsis, fat embolism and leptospirosis, in which the levels of unesterified fatty acids are increased in the patient's plasma. ARDS is characterized by edema formation, and edema resolution occurs mainly due to the pneumocyte Na/K-ATPase activity. As previously described, increased oleic acid (OA) plasma concentrations induce lung injury by interfering with sodium transport. The first aim of this study was to develop a radioactivity-free assay to detect Na,K-ATPase activity ex vivo using a model of OA-induced lung injury in mice. We also investigated the relationship between Na/K-ATPase inhibition and OA-induced lung injury using ouabain-induced lung injury as a comparison, because of the well-described effect of ouabain as a Na/K-ATPase inhibitor. METHODS: We developed a Na/K-ATPase assay based on the capture of non-radioactive Rb+ ions by mice lung tissue in the absence or presence of ouabain, a specific Na/K-ATPase inhibitor. Rb+ incorporation into the lung was measured by inductively coupled plasma-optical emission spectrometry (ICP-OES) after lung tissue mineralization. Na/K-ATPase activity was considered as the difference between Rb+ incorporation in the absence and in the presence of ouabain. Bronchoalveolar lavage fluid was collected for lung injury assessment. For this assessment, cell counting, lipid body enumeration and lipid mediator concentrations were measured. Histological analyses were used to determinate lung pathology. Whole body plethysmographic analysis was performed to assay lung function. RESULTS: The lung Na/K-ATPase activity of mice was completely inhibited by an OA dose of 10 µmol, an effect also obtained with 10-3 µmol of ouabain, as demonstrated by the decreased Rb+ incorporation in the lungs. The same OA dose induced lung edema and inflammation with cell influx, lipid body formation, and leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) production. Ouabain also induced lung inflammation, as detected by histological examinations. As far as we know, this is the first time that ouabain-induced lung injury was shown. Both OA and ouabain induced functional lung pathology in mice simultaneously with inhibition of the lung Na/K-ATPase activity. CONCLUSIONS: We developed a new non-radioactive assay to quantified Na/K-ATPase in vivo. OA and ouabain inhibited in vivo Na/K-ATPase activity in the lungs and induced lung injury. Our data reinforce the idea that Na/K-ATPase inhibitors may worsen lung injury in specific pathological conditions.

Oleic acid induces lung injury in mice through activation of the ERK pathway.

Oleic acid (OA) can induce acute lung injury in experimental models. In the present work, we used intratracheal OA injection to show augmented oedema formation, cell migration and activation, lipid mediator, and cytokine productions in the bronchoalveolar fluids of Swiss Webster mice. We also demonstrated that OA-induced pulmonary injury is dependent on ERK1/2 activation, since U0126, an inhibitor of ERK1/2 phosphorylation, blocked neutrophil migration, oedema, and lipid body formation as well as IL-6, but not IL-1ß production. Using a mice strain carrying a null mutation for the TLR4 receptor, we proved that increased inflammatory parameters after OA challenges were not due to the activation of the TLR4 receptor. With OA being a Na/K-ATPase inhibitor, we suggest the possible involvement of this enzyme as an OA target triggering lung inflammation.