Role Of Caveolin-1in Acute Lung Injury Induced By Hpopolysaccharide

ObjectiveThe aim of this study is firstly to investigate changes of caveolin-1(Cav-1) in the lung tissues of lung injury and rat pulmonary microvascular endothelial cells (RPMVEC) induced by lipopolysaccharide (LPS). And then, we observed the influence of lipopolysaccharide (LPS) on rat pulmonary microvascular permeability; investigating the intervention of protein kinase A (PKA) inhibitor in the Cav-1expression and RPMVEC permeability elicited by LPS. These results may be helpful for providing experiment basis for delineating the function and importance of Cav-1in the pathogenesis of acute respiratory distress syndrome (ARDS).MethodsRPMVEC was isolated and cultured in vitro; A RPMVEC monolayer was constructed to determine changes of transendothelial electrical resistance (TER) and evans blue-labeled albumin flux (Pd) across the monolayer. Western blot was used to determine the Cav-1protein and the phosphorylation-Cav-1expression in RPMVEC induced by lipopolysaccharide (LPS). Rats were challenged with LPS intravenously to establish the lung injure model. Hematoxylin-eosin staining (HE) was used to observe the changes of pathology of lung tissue.And then, the expression of Cav-1mRNA and protein in lung tissues were determined by western-blot, reverse transcription polymerase chain reaction and immunohistochemistry.Results1. RPMVEC was isolated and cultured successfully in vitro, and was confirmed by morphology and fluorescein isotbiocyanate-banderiraea simplicifolia I isolectin B4 (FITC-BSI) integration experiment.2. After RPMVEC monolayers were challenged with lOug/ml LPS for1h,1h,3h,6h,12h,24h, there were significant increase in time-dependent manner in the permeability coefficient as measured by TER and Pd:Pd increased as compared with quiescent levels after RPMVEC monolayers were treated by LPS for1h, peaked at3h and prolonged lag time of24h. Similarly, the progressive decrease in TER produced by LPS was time-dependent when compared with untreated monolayer. PKA inhibitor (PKI) pretreated with RPMVEC monolayer, the increased Pd and decreased TER induced by LPS can be significantly increased.3. After RPMVEC was treated with10μg/ml LPS, Western blot revealed that the expression of Cav-1protein raised at lh, peaked at3h, then decreased gradually, but still higher at24h, When compared with Oh group, there were significant difference.4. LPS increased Cav-1phosphorylation in a time-dependent manner in RPMVEC. This phosphorylation inereased within10min, peaked at30min and then returned to basal levels120min after LPS treatment. And PKA inhibitor (PKI) pretreated with RPMVEC can significantly increase the expression of Cav-1and phosphorylation-Cav-1which induced by LPS.5. The model of acute lung injury was successfully made by LPS injection.6. LPS down-regulated the expression of Cav-1protein and mRNA in lung tissue in a time-dependent manner.7. Compared with the group of LPS stimulation for6h, methylprednisolone significantly inhibited the down-regulated expression of Cav-1protein and mRNA, and lessened the pathologic damage in lung challenged by LPS.Conclusion1. LPS could increase RPMVEC monolayer permeability in time-dependent manner.2. LPS could up-regulate the expression of Cav-1and phosphorylation-Cav-1in time-dependent manner.3. PKI could induce RPMVEC permeability injury alone, but only cooperated with LPS to increase RPMVEC permeability injury seriously, and mechanism may have be associated with the thing that PKI can significally up-regulate the effect of LPS on the expression of Cav-1and phosphorylation-Cav-1in RPMVEC.4. Cav-1mRNA and protein expression decreased in lung tissue of the lung injury model induced by LPS.5. Methylprednisolone can effectively relieve LPS-induced lung injury by partly inhibiting the down-regulated expression of Cav-1.