| Background and objective:Sepsis is characterized by an uncontrolled and harmful host reaction to microbial infection with life-threatening organ dysfunction,which is one of serious complications of infection,burn/trauma,shock and other critically ill patients.With the advancements in medical technology,well-equipped intensive care units and better practice treatments,the rate of sepsis-related mortality continues to be high.During sepsis,the disorder of the host's immune response could decrease the elimination of microbial infection,which lead to recurrent severe infections and multiple organ dysfunction.Neutrophils,as the first line of defence against pathogens,depend on chemotaxis to eradicate bacteria.During sepsis,neutrophils are stimulated with bacterial toxins and cytokines,which lead to impaired functions,showing severely damaged recruitment and chemotaxis function,decreased phagocytosis,increased release of inflammatory mediators and formation of NETs.These cause the immune dysfunction of patients with sepsis.Therefore,how to regulate immune function in patients with sepsis to effectively exert anti-infection function,could become one of an important measures of treatment of sepsis.LPS is highly acylated saccharolipid located on the outer leaflet of the outer membrane of Gram-negative(G-)bacteria.Infections caused by gram-negative bacilli is the most important pathogenic elementsis the pathogenic elements of endoxemia and sepsis.Early studies in vivo and in vitro showed that LPS can inhibit chemotaxis of neutrophils.Adenosine(ADO)is one hydrolysis product of adenosine triphosphate(ATP)with a very short tissue half-life and potent signaling functions.ADO exert its function through the four kinds of G protein coupled receptors: A1,A2 A,A2B and A3 receptors.All ADO receptors are expressed in the surface of neutrophils.ADO mediates neutrophil functions through its different receptors including neutrophil chemotaxis.However,there is no report on ADO mediating the recovery of LPS-inhibited neutrophil chemotaxis.Methods:1.Elbow venous blood from healthy adult volunteers was separated by Ficoll Hypaque density gradient centrifugation to achieve healthy adult peripheral blood neutrophils.The activity and purity of neutrophils were measured by Wright's staining,trypan blue staining and flow cytometry to detect the CD66 b.2.Under agarose chemotaxis model was set up,which was used to examine neutrophil directional migrating distance to chemoattractant IL-8 or f MLP.Chemotaxis model was identified and the concentrations of chemoattractant IL-8 and f MLP were established.3.Neutrophils were stimulated with LPS,ADO,ADO receptor inhibitor and agonist,c AMP analogue and inhibitor,AC agonist,and p38 MAPK inhibitor intervention and so on.And neutrophil directional migrating distances were measured by under agarose chemotaxis model.4.Flow cytometry and Western blot(WB)were used to test the ADO receptor A1 level on neutrophils.5.The relative phosphorylation levels of MAPK signaling pathway protein were detected by protein microarray.WB was used to detect total phosphorylation level of p38 MAPK.Results: 1.Cells separation from Ficoll Hypaque density gradient centrifugation,were stained by Wright's staining.Neutrophils were characterized that the nucleus are horseshoe or lobulated,which were stained with purple,and cell cytoplasm appeared light red.Trypan blue staining and flow cytometry to detect CD66 b,showed the activity and purity of cell are ?97%,respectively.2.Under agarose chemotaxis experiments showed that the optimum concentration of IL-8 and f MLP-induced neutrophil chemotaxis was 1 ?mol/L and 0.1 ?mol/L,respectively.3.After LPS stimulation,neutrophil directional migration to IL-8 or f MLP was inhibited with a dose dependent way.Administration of ADO could reverse LPS-inhibited chemotaxis of neutrophils in a concentration dependent manner.4.ADO mediated the restoration of LPS-inhibited neutrophil chemotaxis mainly by ADO receptor A1 on the surface of neutrophils.5.After LPS stimulation,the A1 receptor(A1R)expression on neutrophils did not obviously alter.6.A1 R that recovered LPS-inhibited neutrophil chemotaxis might not rely on the classical AC-c AMP-PKA signaling pathway,but inhibit the phosphorylation level of p38 MAPK. |
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