The Effect Of Lps On Neutrophil Phagocytosis And The Molecular Mechanism

Background and objective: Sepsis refers to multiple organ dysfunction when human body affected by exogenous microbes,especially in patients with severe trauma,shock,burns and so on.Even in today with updated medical technologies,the mortality of sepsis is still high.Based on an epidemic survey from North American,there are about 7.5 million suffering from sepsis.In it,severe sepsis account for 9%,septic shock account for 3% and more than 2.1 million of these patients can’t survive.The mortality of patients with sepsis remains high.Sepsis,in some degree,it is a direct consequence of MODS and infection.The relevant study addressed that 90% sepsis patients are subjected to infection and 60% would come from G-bacterial and in it,E.coli was the main pathogen.LPS,a glycolipid highly acylation,mainly consists of polysaccharides,lipids,and polysaccharide antigen.As a main agent of G-bacterials wall,LPS is a striking causes of sepsis.Neutrophils challenged with LPS by Toll4 receptor release a lot of cytokines,suh as il-6,ltb4 and ROS.In the early stage of sepsis,neutrophils infiltrated in the organs(liver,spleen et.al)with rich blood supply.In sepsis,the pro-inflammatory cytokines from bacterial would induce neutrophils dysfunction and infiltrate in organs in organs such as the liver and all of this would contribute the SIRS.In current clinical therapy,with the inappropriate usage of antibiotic,it contributes to the problem of antimicrobial resistance which reflects the shortage of antibiotic.Therefore,a new antimicrobial drug or methods should be created in the clinical therapy.Previous study showed that LPS stimulating neutrophils would increase its phagocytosis,but the detail mechanism is still unknown.Methods:Neutrophils were obtained from venous blood of healthy adult volunteers by Ficoll gradient centrifugation.Then neutrophils were randomly divided into control group,LPS 10 min group,LPS 20 min group,LPS 30 min group and LPS plus antagonist group.Some parameters were assayed in the study.In each experiment different methods would be used.Flow cytometry was used to examine neutrophil phagocytosis and F-actin expression.TEM was applied to detect morphological changes of neutrophils and IFM was also used to observe neutrophils morphology,F-actin level and the capacity of neutrophil phagocytosis to 25922 GFP.In terms of the expression of Pi3k、P-Pi3k、AKT and P-Akt,WB was applied.Results and Conclusions: 1.Neutrophils treated with LPS showed morphological change with irregular shape,reduced granules,folded membrane and lots of pseudopod.2.Neutrophils treated with LPS enhanced its phagocytosis in a time-dependent way.3.Neutrophils treated with LPS increased the level of F-actin and its polarization,which was closely associated with the enhancement of neutrophil phagocytosis.4.LPS enhanced the ability of neutrophils to devour ATCC 25922 GFP by activating the pi3k/Akt signaling pathway in neutrophils.5.Neutrophils treated with LPS would increase the phosphorylation level of PI3 K,Akt.The enhancement of phagocytosis after stimulating neutrophilic granulocytes may be be related with the pi3k/Akt signaling pathway.