Regulatory Effect And Molecular Mechanism Of Exogenous Carbon Monoxide Releasing Molecule(CORM-2) On Neutrophil Function After Lipopolysaccharide Stimulation

Background and objective:Sepsis is caused by infection of the systemic inflammatory response syndrome,severe cases can lead to multiple organ failure, septic shock and even death. The pathogenesis of sepsis is very complex, has not yet been clarified. In the early stage of sepsis, the infection sites recruit a large number of neutrophil and by phagocytosis of pathogenic bacteria, degranulation releasing proteases and other substances to achieve the bactericidal effect. Lipopolysaccharide(LPS) is an important component of the cell wall of Gram-negative bacteria, Gram-negative bacteria is the most important time of sepsis caused by pathogenic ingredients. As an exogenous stimulation factor LPS stimulated neutrophil can promote the release of large amounts of IL-6,leucocyte leukotriene B4, reactive oxygen species(ROS) of inflammatory mediators,and LPS stimulated neutrophil can be in distant organs large number of infiltrating,and this is sepsis SIRS to distant organ injury. Carbon monoxide(CO) is a colorless,odorless, poisonous gas, previous research results show that CORM-2 release of CO can significantly ameliorated cecal ligation and perforation(CLP) mice and LPS stimulated mouse inflammatory reaction, the effective protection of vital organ damage and improve the survival rate. While, the stimulation of LPS on neutrophil,CORM-2 regulation of its function has not been reported.Methods:Set the venous blood from normal adult volunteers and by Ficoll Hypaque centrifugation of neutrophil in the blood of healthy adults, using the random number table method were randomly divided into control group(control), lipopolysaccharide(LPS) group, LPS+CORM-2(10μM) group, LPS+CORM-2(50μM) group,LPS+iCORM-2 group. Flow cytometry was used to detect the neutral granulocyte apoptosis and reactive oxygen species(ROS) release and phagocytic function; flow cytometry technique and ELISA detection of neutrophil exocytosis; agarose cell migration model for the detection of neutrophil migration ability; taken count of the number of neutrophil extracellular traps(NETs) under fluorescence microscope;Western blot(WB) was used to detect the key signaling molecule ERK1 / 2, p38 and Akt expression of neutrophil.Results and Conclusions:1. The early apoptosis of neutrophil can be obviously promoted, and the migration of neutrophil can be obviously inhibited after LPS stimulated, and the use of CORM-2 intervention does not change and LPS on neutrophil early apoptosis and migration ability. The results showed that CORM-2 had no significant effect on the early apoptosis and migration ability of neutrophil.2. The release of neutrophil granules and phagocytic function can be significantly promoted after LPS stimulated, and the use of CORM-2 can significantly inhibit LPS stimulated PMN ROS production and granules release, and further increase the phagocytic function of neutrophil.Experimental results show that CORM-2 can obviously protect LPS stimulated neutrophil and make it function very well.3. The neutrophil extracellular trap production were not been promoted after LPS stimulated, while CORM-2 interventions can effectively promote LPS-stimulated neutrophil extracellular trap generation. Experiments show that CORM-2 can significantly promote neutrophil bactericidal function.4. After the stimulation of LPS expression of neutrophil ERK1/2, P38, AKT key protein were increased in some degree, the expression and the use of CORM-2intervention ERK1/2, P38, AKT has been greatly increased. Experiments show that CORM-2 improved LPS-stimulated neutrophil function may be associated with ERK1/ 2, P38, AKT related.