Protective Effect And Molecule Mechanism Of Edaravone On Lipopolysaccharide-induced Acute Lung Injury In Rats

Acute lung injury (ALI) is regarded as a clinical common syndrome with characteristic diffuse inflammation infiltration, disturbance of lung circulation, capillary permeability increasing and hyoxemia associated with fierce state of an illness and unfavorable outcomes. ALI may progress to acute respiratory distress syndrome(ARDS), resulting in multiple organ failure and 30%～70% of mortality rate,therefore. It is vital importance of effective prophylaxis and treatment .While, mass experiments and clinical research have been carried out, pathological process and pathogenesy were not illuminated ,meanwhile, ALI is short of therapeutic tools, so far ,protective MV with lower tidal volume in order to decrease mortality rate is the solo treatment among critical illness.It is initial curative including vasodilator (nitrogen monoxidum inhaling, outside alveolar surfactant, prostacyclin inhaling), glucocorticosteroid, non-steroid anti-inflammatory drug and ketoconazole etc. however, problems of unfavorable therapeutic effect and severity adverse reaction appeared,hitherto, safe and effective curative was not been developed.Elevated levels of proinflammatory mediators combined with a decreased expression of anti-inflammatory molecules are critical components of lung inflammation and play a main role in ALI. The signals that lead to increased gene expression and biosynthesis of proinflammatory mediators by inflammatory and epithelial cells are thus of considerable interest. It is manifested that oxygen free radicals injury is the main mechanism with regard to neutrophil aggregation,activation resulting capillary and pulmonary parenchyma injury. Antioxidant is included by our research. During ALI, active oxygen including superoxide anion, hydroxy radical and hydrogen Dioxide etc. produced by neutrophi, macrophage and alveolar epithelial cell may cause further neutrophil aggregation.Edaravone (MCI-186,3- methylium-1- phenyl- pyrazoline-5- ketone)was produced by Mitsubishi Pharmaceuticals company in Japan, and also went on the market in Jun,2001. So far,edaravone is the only clinical tri-stages using drug regarding newest antioxidant, moreover, effective treatment of acute cerebral infarction has been manifested.Recently, studies have confirmed edaravone can improve the function of microcirculation,decrease capillary wall permeability, act cytoprotection and decrease lysosome releasing etc.. thus, edaravone is potentially beneficial for treatment of ALI.Nowadays,we do not retrieval studies with regard to edaravone protective action in ALI animal models ,thus, we choose ALI rat models in order to further research of ALI protective action related to newest antioxidant through the whole and molecular level,and also supply theory for therapeutics.In first and second chapters of this study, we will validate that if newest antioxidant—edaravone posses protective function to ALI ,through the observation of gas exchange, pathomorphology of inflammatory reaction, pulmonary permeability, neutrophil function, index of oxidation and antioxidation and ultrastructure of pulmonary vascular endothelial cell.Based on the theory about signal passageway of MAPK and NF-κB, our study in sequel will manifest if protective action is associated with activation of MAPKs protein kinase(p38,JNK,ERK) and key protein(NF-κB). Rat models of lipopolysaccharide-induced acute lung injury made and dividen :Based on references and preliminary experiment, male Sprague-Dawley is selected and injected by lipopolysaccharide(5mg/kg) through vena caudalis(P/F ratio < 300),further operation was made after peritoneal injection of 10 % Chloral Hydrate 6h later.Adult male S-D rats were identified according to random digits table: (1)Sodium Chloride group; (2) LPS-induced ALI group; (3) LPS-induced+edaravone (3.0mg/kg);(4)LPS-induced+edaravone(1.0mg/kg);(5)LPS-induced+edaravone(0.3mg/k g); (6) positive control group(lipopolysaccharide+DEX).Objective:To analyze protective effect and molecule mechanism of edaravone on rats with lipopolysaccharide-induced acute lung injury, in order to supply base theory for treatment of acute lung injury.Method:Chapter one Protective effect and pathophysiological mechanism of edaravone on LPS-induced acute lung injury in rats: After 6 hours of intravenous injection of LPS or Sodium Chloride, arterial blood gas analysis, lung permeability index (LPI),wet to dry weight ratio (W/D) and protein content in bronchoalveolar lavage fluid (BALF) were measured. Pathological changes of lung tissue were measured by light and electron microscopy.Chapter two To manifest whether pretreatment of edaravone is beneficial to LPS-induced ALI in rats through inhibition of the pulmonary neutrophil sequestration and oxygen free radicals injury: The ratio of neutrophiles in bronchoalveolar lavage fluid (BALF), the activities of superoxide dismutase (SOD),myeloperoxidase (MPO),lactate dehydrogenase (LDH) and malondialdehyde (MDA) content in blood serum and lung were measured. Chapter three To investigate whether the protective effect of edaravone in the treatment of ALI could result from its direct interference with the LPS-induced activation of NF-kappaB: After 6 hrs of intravenous injection of NS or LPS, Lowry and western blotting were manipulated to identify activation of NF-kappaB in pulmonary tissue.Chapter four To investigate whether the therapeutic effect of edaravone in ALI could result from its direct interference with the LPS-induced activation of p38 mitogen-activated protein kinase,c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation.: After 6 hrs of intravenous injection of NS or LPS, Lowry and western blotting were manipulated to identify activation of p38 mitogen-activated protein kinase,c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation in pulmonary tissue.Results:Chapter one Compared to LPS-challenged group, significantly decreased arterial partial pressure of carbon dioxide,W/D ratio,LPI,protein content in BALF ,and increased partial pressure of oxygen of arterial blood and PH appeared in edravone pretreatment groups. Moreover, relief of pulmonary inflammation reaction , injury of endothelial cells were observed under light microscope and transmission electron microscope in LPS-challenged rats, dose-effect relationship correlating edaravone also appeared.Chapter two Edaravone pretreatment significantly decreased MDA level,MPO and LDH activity,the ratio of neutrophiles in BALF, and dramatically enhanced SOD activity (P<0.01, or P<0.05) . dose-effect relationship correlating edaravone also appeared.Chapter three Compared to controls, LPS-challenged rats had significantly increased the levels of phosphorylated NF-κB in lung tissues. High- and moderate-dose of edaravone pretreatment resulted in a drastic down-regulation of phospho-NF-kappaB. Edaravone significantly attenuated LPS-induced phospho-NF-kappaB expression in dose-dependent.Chapter four Compared to controls, LPS-challenged rats had significantly increased the levels of phosphorylated p38 mitogen-activated protein kinase,c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation in lung tissues. A high-dose and moderate-dose of edaravone pretreatment resulted in a drastic down-regulation of phospho-p38, phosphor -ERK and phospho-JNK(P<0.01 or P< 0.05).Conclutions:These results demonstrate that edaravone can attenuate acute lung injury and exert protective effect in ALI rats induced by LPS. Furthermore, edaravone can acquire inhibition of pulmonary inflammation reaction and increase of permeability in order to improve gas exchange; Pretreatment of edaravone is beneficial to LPS-induced ALI in rats through inhibition of the lung neutrophil sequestration and relief of injury due to lipid peroxidation in order to rectify imbalance of oxidation/antioxidation; Edaravone relieves the inflammation in acute lung injury rats ,possibly through the inhibition of NF-kappaB activation. It may be elucidated that protective effect of edaravone to ALI is associated with the inhibition of NF-kappaB activation,meanwhile, Edaravone also takes part in inhibition of another signal transduction-MAPKs,furthers interrupt of inflammation cascade reaction and secondary lesion; solo three-stage clinical antioxidant-edaravone may be the new choice to treat ALI through antioxidative pathway,therefore,our study supply experimental foundation.