Acute respiratory distress syndrome (ARDS) is the most severe lung inflammatory manifestation and has no effective therapy nowadays. Sepsis is one of the main illnesses among ARDS causes. The use of fluid resuscitation is an important treatment for sepsis, but positive fluid balance may induce pulmonary injury. As an alternative, fluid resuscitation with hypertonic saline ((HS) NaCl 7.5%) has been described as a promising therapeutical agent in sepsis-induced ARDS by the diminished amount of fluid necessary. Thus, we evaluated the effect of hypertonic saline in the treatment of LPS-induced ARDS. We found that hypertonic saline (NaCl 7.5%) treatment in rat model of LPS-induced ARDS avoided pulmonary function worsening and inhibited type I collagen deposition. In addition, hypertonic saline prevented pulmonary injury by decreasing metalloproteinase 9 (MMP-9) activity in tissue. Focal adhesion kinase (FAK) activation was reduced in HS group as well as neutrophil infiltration, NOS2 expression and NO content. Our study shows that fluid resuscitation with hypertonic saline decreases the progression of LPS-induced ARDS due to inhibition of pulmonary remodeling that is observed when regular saline is used.

OBJECTIVE:Volume replacement in septic patients improves hemodynamic stability. This effect can reduce the inflammatory response. The objective of this study was to evaluate the effect of 7.5% hypertonic saline solution versus 0.9% normal saline solution for volume replacement during an inflammatory response in endotoxemic rats.METHODS:We measured cytokines (serum and gut), nitrite, and lipid peroxidation (TBARS) as indicators of oxidative stress in the gut. Rats were divided into four groups: control group (C) that did not receive lipopolysaccharide; lipopolysaccharide injection without treatment (LPS); lipopolysaccharide injection with saline treatment (LPS +S); and lipopolysaccharide injection with hypertonic saline treatment (LPS +H). Serum and intestine were collected. Measurements were taken at 1.5, 8, and 24 h after lipopolysaccharide administration.RESULTS:Of the four groups, the LPS +H group had the highest survival rate. Hypertonic saline solution treatment led to lower levels of IL-6, IL-10, nitric oxide, and thiobarbituric acid reactive substances compared to 0.9% normal saline. In addition, hypertonic saline treatment resulted in a lower mortality compared to 0.9% normal saline treatment in endotoxemic rats. Volume replacement reduced levels of inflammatory mediators in the plasma and gut.CONCLUSION:Hypertonic saline treatment reduced mortality and lowered levels of inflammatory mediators in endotoxemic rats. Hypertonic saline also has the advantage of requiring less volume replacement.

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.

The effects of hypertonic saline solution (HSS) have been shown in several animal models of ischemia and shock. Literature has shown potential benefits of HSS modulating inflammatory response after sepsis in an animal model. We studied the HSS effects in sepsis through cecal ligation and puncture (CLP) in Balb-C mice. Groups studied: 1- CLP without treatment (CLP-C); 2- CLP treated with normal saline solution NaCl 0.9% – 34 ml/Kg (CLP-S); 3- CLP treated with HSS NaCl 7.5% – 4 ml/Kg (CLP-H); and 4- group (Basal) without no CLP or treatment. Volume infusion was always applied 30 min after CLP. Lung and peritoneal lavage were harvested after 6h and 24h of CLP to analyze cytokines amount, oxide nitric, lipid peroxidation and neutrophil infiltration. Neutrophil infiltration, ICAM-1, CXCR-2, and CXCL-1 in lung were reduced by HSS (CLP-H) compared to CLP-C or CLP-S. Neutrophil in peritoneal lavage was increased in 24h with HSS (CLP-H) compared to CLP and CLP-S. Peritoneal CXCR-2 was increased in CLP-C and CLP-S but presented a lower increase with HSS (CLP-H) after 6 hours. GRK-2 presented difference among the groups at 24 h, showing a profile similar to neutrophil infiltration. Pro-inflammatory cytokines (TNF-α and IL-6) were reduced by HSS treatment; CLP-S increased TNF-α. IL-10 was increased in lung tissue by the HSS treatment. The oxidative stress (TBARS and nitric oxide biochemistry markers) was reduced with HSS. Animal survival was 33.3% in CLP-C group, 46.6% in CLP-S group and 60% in the CLP-H group after the sixth day. The HSS protects the animal against sepsis. Our results suggest that the volume replacement modulate pro and anti-inflammatory mediators of an inflammatory response, but HSS presented a more effective and potent effect.

Severe acute pancreatitis (AP) induces a systemic inflammatory disease that is responsible for high mortality rates, particularly when it is complicated by infection. Therefore, differentiating sepsis from the systemic inflammation caused by AP is a serious clinical challenge. Considering the high metabolic rates of leukocytes in response to stress induced by infection, we hypothesized that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1α), a master regulator of mitochondrial biogenesis and function, would be distinctly expressed during inflammation or infection and, therefore, could constitute a useful marker to differentiate between these two conditions. Rats were subjected to injection of taurocholate into the main pancreatic duct, which caused a severe AP with high amylase levels and white blood cell counts. In these animals, a marked increase in PGC-1α mRNA levels in circulating leukocytes was observed 48 h after the surgical procedure, a time when bacteremia is present. Antibiotic treatment abolished PGC-1α up-regulation. Moreover, PGC-1α expression was higher in peritoneal macrophages from animals subjected to a bacterial insult (cecal ligation and puncture) than in animals with AP. In isolated macrophages, we also observed that PGC-1α expression is more prominent in the presence of a phagocytic stimulus (zymosan) when compared to lipopolysaccharide-induced aseptic inflammation. Moreover, abolishing PGC-1α expression with antisense oligos impaired zymosan phagocytosis. Together, these findings suggest that PGC-1α is differentially expressed during aseptic inflammation and infection and that it is necessary for adequate phagocytosis. These results could be useful in developing new tests for differentiating infection from inflammation for clinical purposes in patients with AP.

Ao meu orientador, Prof. Dr. Francisco Garcia Soriano, por me aceitar como aluna, pela orientação, apoio, disponibilidade, compreensão, respeito e dedicação, que foram necessários para realizar esta tese. Ao Prof. Dr. Irineu Tadeu Velasco, pela oportunidade de realizar esta tese no LIM 51, pela confiança e apoio.

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Sepsis is a systemic inflammatory response to infection and a major cause of mortality. Hypertonic saline solution (HSS) showed improvement on hemodynamic and inflammatory response. We studied the HSS effects in sepsis through cecal ligation and puncture (CLP) in mice Balb‐C, divided in 4 groups: CLP without treatment (CLP); CLP treated with saline solution 0,9% ‐ 34ml/Kg (SS); CLP treated with HSS 7,5% ‐ 4ml/Kg (SH); and a basal group without CLP and treatment; both treatments were applied 30 min after CLP. Lung and gut were collected after 6, 12 and 24h to analyze neutrophil recruitment using myeloperoxidase as index (MPO), ICAM, VCAM by ELISA and differential cell counter from peritoneal fluid. The SH group showed higher survival rate (60%) than SS (46,6%) and CLP (33,3%). CLP group (3 ± 0,4mU/mg; 29,4 ± 5,8 ng/mg) increased MPO and ICAM levels when compared to SH (1,5 ± 0,2 mU/mg; 15,9 ± 2,3 ng/mg) in lung at 24h. In gut MPO level increased in SH (0,9 ± 0,3mU/mg) when compared to CLP (0,1± 0,03mU/mg) at 6h; at 12h and 24h, SH decreased (0,2 ± 0,08; 0,2±0,05 mU/mg) in relation to CLP (3,5 ± 1,4; 0,6 ±0,05 mU/mg), ICAM levels in SS (1,7 ± 0,1 ng/mg) increased compared to SH (1,13±0,06ng/mg) at 6h, and SH (0,9±0,07ng/mg) decreased in relation to CLP (1,3 ± 0,07ng/mg) and SS (1,3 ± 0,1 ng/mg) at 12h. HSS protects the lung against tissue damage during sepsis, decreasing neutrophil recruitment into tissue through of a reduction in ICAM. CAPES.