Lipopolysaccharide Binding Protein In The Regulation Of Endotoxic Lung Injury Effects And Mechanisms

Lipopolysaccharide (LPS), a component of the outer membrane of Gram- gegative bacteria, is known to induce in the host a variety of pathophysiologic responses, including systemic inflammatory response syndrome (SIRS), acute lung injury (ALl) and acute respiratory distress syndrome (ARDS). lipopolysaccharide binding protein(LBP) ,a 6OkD serum acute phase glycoprotein,binds to LPS and transfers it to the [PS receptor CD 14. In cytoplasm, LPS-induced cytokine expression has been reported to depend on the phosphorylation of protein tyrosine kinases at different levels and the activationltranslocation of the transcription factor NF-kB .LPS mediated stimulation of macrophages is enhanced when LBP is added to serum-free systems and therefore LBP has been thought to participate in the pathogenesis of endotoximea. Recently, it has also been shown that LBP not only mediate the binding of LPS to CD 14, but also binding of scavenger receptor on macrophage membrane or high density lipoprotein (HDL) leading to phagocytosis and subsepuent clearance of LPS.The mechanisms of the regulatory effects of LBP on LPS?biologic affects has remained unclear. In the present studies, LBP from actue phase rat serum was purified by precipitation of ammonium sulphate, Bio-Rex7O resin and MonoQ column. Rat alveolar macrophages were exposed to LPS (0.0 lmg/L or lmg/L) in the various concentrations of LBP. RT-PCR and Western blotting were used to detect expressions of TNF- a ,IL-6 and IL- 10 mRNA and phospho-p3 8 respectively.Lastly, in order to investigate the potential role of LBP in rat acute Lung injury following 憈wo hit?and the therapeutic effects of anisodamine.The ?III ? ~L animal model of acute lung injury was establised with the oleic acid (OA ,0.2m1/kg) and lipopolysaccharide (LPS , 2mg/kg) . LBP, TNF- a and IL- 10 mRNA expression in pulmonary tissue were determined with RT-PCR Also, the pathological changes of pulmonary tissue and arterial blood gas were observed. The results showed: 0762 i~ g rat LBP were purified and the purification folds reached to 1570. SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and had molecular weight of 60 kD. The binding of lipopolysaccharide to mononuclear cells was enhanced by purified rat LBP. ㏒timulation of rat alveolar macrophages with LPS at concentrations of 0.Olmg/L was LBP dependent. LBP at concentrations up to 1 mg/L was able to increase TNF- a , IL-6 and IL- 10 mRNA expressions.however,when LBP concentrations were further increased to lOmg/L , the TNF- a ,IL-6 and IL-b mRNA levers were lower as compared with that in the presence of lmg/L. Stimulation of rat alveolar macrophages with LPS at concentrations of lmg/L was LBP independent.甌he regulatory effects of LBP on cytokines mRNA expressions were not changed at different time points.OFor the p38 kinase activity, the greatest activity was at 30 mm, but there was detectable kinase activity as early as 15 mm and as late as 60 mm after LPS stimulation. p38 kinase activity following stimulation with LPS at concentration of 0.0 lmg/L was LBP dependent and with LPS at concentration of 1 mg/L was LBP independent.甃BP mRNA exprexxion was up-regulated after injection of OA alone . Both of TNF- a and IL-10 mRNA exprexxion in rat treated with OA and LPS were higher than in rat treated with OA al...