The Effect Of Lipopolysaccharide Binding Protein On Fractalkine Expression And Signal Transduction Mechanism In Acute Respiratory Distress Syndrome

Background: Acute respiratory distress syndrome(Acute respiratory distress syndrome,ARDS)is a common and high mortality critical disease.Inflammatory reactions mediated by endotoxemia is one of the common causes of ARDS.Lipopolysaccharide(LPS)is the main component of endotoxin.Lipopolysaccharide binding protein(lipopolysaccharide binding,protein,LBP)is a glycoprotein in the serum of normal people and animal,which elevated during acute inflammation and involved in regulations of LPS on inflammatory factors.Combined with the formation of LPS-LBP conjugates activated monocytes,macrophages and other cells of the CD14/TLR4 receptor.Activation of TLR4 may be mediated by LPS signal transduction pathway into two directions: one is mitogen activated protein kinase(MAPK)pathway and the other one is nuclear factor kappa B(nuclear factor-κB,NF-κB)pathway.Activation of these two signaling pathway could induce some inflamatoy factor expression.Fractalkine(FKN)is the only member of CX3 C chemokine family,plays an important role on regulating downstream inflammatory factoris,.On the one hand,FKN damage on inflammatory cells in the vascular wall and recruitment of endothelial cells,and the other side through the inhibition of cells apoptosis has anti-inflammatory effects.The effect of LBP on LPS,activation of MAPK and NF-kappa B,influenced the regulation and expression of FKN is unclear.Purpose: Through the study on the LBP,FKN expression in peripheral blood,bronchoalveolar lavage fluid ARDS patients,in order to clear the relationship among LBP,FKN,serum endotoxin level,disease severity,prognosis.Further research the influence of LBP on the expression of FKN and its signal transduction mechanism in LPS stimulated A549 cells and in LPS induced ARDS rat animal model,attempts to clarify the role of LBP and FKN in ARDS,clear the signaling pathway of LBP regulating FKN,find a new target for ARDS therapy.Method: The first part:RT-PCR was used to detect the expression of LBP and FKN m RNA in peripheral blood of healthy controls and ARDS patients;ELISA was used to detect the expression of LBP,FKN protein in blood and bronchoalveolar lavage fluid(BALF).The relationship between the above indexes and the severity and prognosis of ARDS was studied.The second part: LBP plasmid DNA,LBP sh RNA expressiong plasmid DNA,SB203580,SC-514(inhibitor of p38 MAPK,p65NF-κB signaling pathway)were used to pretreatment on A549 cells stimulated with LPS.RT-PCR were used to detect the expression of LBP,FKN m RNA,ELISA was used to determined the LBP,FKN protein in supernatant.Western blotting was used to detection the expression of phosphorylation of p38MAPK(phospho-p38MAPK),,phosphorylated p65 protein(phospho-p65),LBP and FKN protein of cells.Confocal laser scanning microscopy(CLSM)were used to observation phospho-p38 MAPK,phospho-p65 transfer into nuclear.Co-immunoprecipitation assay used to detect the interaction of LBP and phospho-p38 MAPK,phospho-p65.The third part: An acute respiratory distress syndrome(ARDS)rat model was established by tail intravenous injection of LPS(5mg/kg)with or without LBPK95A(5mg/kg),SB203580(5mg/kg)or SC-514(5mg/kg)treatment.LBP and FKN m RNA and protein of lung tissue homogenates was determined by RT-PCR and ELISA respectively and the activation of p38 MAPK and NF-κB was detected by western blotting and immunofluorescence analysis with a confocal laser scanning microscope.Immunohistochemical staining was used to identify the expression of FKN in lung tissue.Result: The first part: Compared with healthy group,the expression of LBP m RNA in blood and LBP protein in serum and bronchoalveolar lavage fluid were increased,expression of FKN m RNA and protein were increased.Compared with mild group,moderate group increased the expression of LBP,severe group increased further;the expression of FKN was decreased,severe group decreased further.the expression of LBP in the death group in higher than the survival group,the expression of FKN decreased as compared with the survival group(all p﹤0.05).The levels of serum LBP protein and serum endotoxin,APACHEⅡscore,the severity of ARDS was positively correlated respectively(all p < 0.05,r were 0.8878,0.8365,0.862).The levels of serum FKN protein and,was negatively related to levels of serum endotoxin,APACHEⅡscore and the severity of ARDS(all p < 0.05,r were-0.7341,-0.7726,-0.695).LBP and FKN m RNA,LBP and FKN protein in BALF and serum were negatively correlated respectively。(all p< 0.05,r were-0.8378,-0.7908,-0.8572。)LBP evaluation of diagnosis index of death in patients withARDS,the sensitivity is as same as APACHE Ⅱ score,but the specificity is superior to the APACHE Ⅱ score.The second part: LPS increased LBP m RNA and protein expression and reduced FKN m RNA and protein expression;LBP overexpression further decreased LPS-induced down-regulation FKN m RNA and protein expression and p38 MAPK and p65 NF-κB activation;LBP gene silencing,SB203580 and SC-514 suppressed LPS-induced down-regulation of FKN m RNA and protein expression and p38 MAPK and p65 NF-κB activation respectively in A549 cells(all p﹤0.05,n=6).LBP gene overexpression promoted phospho-p38 MAPK and phospho-p65 translocation whereas silencing of this gene inhibited this;SB203580 and SC-514 suppressed LPS-induced phospho-p38 MAPK and phospho-NF-κB p65 expression and nuclear translocation respectively in A549 cells.COIP shows that LBP had interacted with phospho-p38 MAPK and phospho-p65.The third part: The expression of LBP and FKN m RNA and protein in lung tissue was significantly increased and decreased respectively following LPS injection(all p﹤0.05,n=10).LPS increased phospho-p38 MAPK and phospho-NF-κB p65 expression in an ARDS rat model.LBPK95 A,SB203580 and SC-514 ameliorated LPS-induced rat lung injury and suppressed LPS-induced down-regulation of FKN m RNA and protein expression by both or respectively decreasing phospho-p38 MAPK and phospho-NF-κB p65(all p﹤0.05,n=10).Conclusion 1.The level of LBP m RNA in peripheral blood,LBP protein in bronchoalveolar lavage fluid and serum of ARDS patients were increased,and the level of FKN m RNA and protein were decreased.LBP,FKN is associated with serum endotoxin levels and severity of illness,and LBP may serve as a new indicator of mortality in ARDS patients.2.In ARDS cells and animal models,LBP may be involved in the process of LPS activing the p38 MAPK,p65NF-κB signaling pathway,down regulated the expression of FKN.3.LBP inhibitory peptides--LBPK95 A intervention may be a new target for ARDS therapy.