| BackgroundSepsis is a common syndrome that is difficult to be cured. It is asevere systemic inflammatory response caused by microbial invasion after infection or trauma, and it is alsoa pathological and physiological processes of clinical syndrome can cause secondary damage in tissue or organ. Sepsis is the leading cause of death in ICUwith a high incidence and high mortality (30-50%), and more than 18 million cases of severe sepsis were happened each yearworldwide.The pathogenesis of sepsis is very complicated, in which the infection and inflammation plays a vital role in the development of sepsis.Infection caused a large number of pro-inflammatory mediators releasing at the early stage of sepsis, which resulted in severe tissue and organ damage, and the lung is the first target organ of sepsis.If the disease can not be controlled in time, the immune state of the body will be changed from accentuation to inhibitory, which induced the pathogenic microorganisms mass rearing and opportunistic infections increasing.Macrophage plays a key role in immune surveillance and defense as an important effector cells of innate immune system.Macrophage can secrete many kinds of bioactive substances induced by pathogen, such as NO. TNF-α、ROS, etc. ROS is an important immune regulatory factor as a second messenger in cell signal transduction.ROS can interfere with DNA transcription, ATP synthesis, enzyme and kill bacteria or damage the cell membrane in an appropriate intracellular level. And remove microorganisms finally.Although the pattern recognition receptors, led by TLRs, and their mediated signaling pathways occupy a very important position in the pathogenesis of sepsis, TLRs targeted treatment has not achieved the desired effect in recent years. S1PRs is a class of G protein coupled receptors, which can initiate transmembrane signal transduction, mediate S1P acting as a first messenger, and regulate cell proliferation, angiogenesis and inflammation. S1PR3 as a key member of the S1PRs family which can coupled of three G proteins (Gi,Gq, G12/G13), and mediate a wide range of biological effects.In recent years, a large number of studies focused on how the S1PR3 effects in pro-inflammatory in regulating central and outer innate immune cells. But the function of S1PR3 in bacterial clearance remains unclear.ObjectiveThe research was carried out in two parts. Part 1:A sepsis model was induced by abdominal cavity bacterial infection was established. KRX-725, a specific agonist was designed and synthesized for interveningS1PR3, so the survival rate of septic mice, and complex bacteria in the lung injury effected by KRX-725 can be analyzed.Part 2:The effect of KRX-725 on ROS productionregulation and the function of bacterial clearance was observed for investigating the molecular mechanism.MethodsSeptic mice model wereestablished by intraperitoneal injection of E.coli, And treated with KRX-725 through a intraperitoneal injection way, which is a specific agonist of S1PR3. The equal volume of solvent was treated in control group.Survival rate of septic mice were observed at 8,16,24,32,40 and 48 hours after KRX-725 therapy. Peripheral blood and peritoneal irrigation fluid in KRX-725 treated group and control were acquired and diluted in proportion, and CFU was also calculated after 24 hours. The lung tissue was acquired, fixed for paraffin section making and HE staining, so the lung injury score can beevaluated after 24 hours.Mice peritoneal macrophages were induced by Fluid Thioglycollate Mediumin vitro. And the ROS level in macrophages was detected after stimulated by E.coli for 0, 10,20,30,40,50,and 60 minutes. The same experiment was carried out again under the protection of gentamycin.Experimental data wasanalyzed by SPSS 17.0 software, and survival rate wasanalyzed by Test Log-rank.The measurement data were expressed in mean ± SD, Data comparison between groups was analyzed by student’T test.The cut-off for statistical significance was set at P<0.05.Result1 Septic mice model wereestablished by intraperitoneal injection of E.colisuccessfully, and 48-hour survival rate of KRX-725 treated group weresignificantly higher than those in control group(n=12, P<0.05).2 CFU value of blood in KRX-725 treated group weresignificantly lower than those in control group(P<0.05), while the CFU value of peritoneal irrigation fluid show moreSignificantly by comparison after 24hours (P<0.01).3 The pathological changes in lung tissues including alveolar congestion, a large number of inflammatory cells infiltration, diffuse alveolar damage, etc, was significantly reduced in KRX-725 treated group as well as acute lung injury scale after 24 hours (P<0.05).4 The ROS level in KRX-725 treatedmacrophage weresignificantly higher than those in control group after 20 minutes (P<0.05) and 30 minutes (P<0.01).5 The gentamycin protection showed that bacteria survival in KRX-725 treated group was significantly lower than those in control group(P<0.05), especially after 6 hours (P<0.01).ConclusionKRX-725, a S1PR3 specific agonist, which can improve the survival rate of septic mice significantly, decrease the abdominal cavity and blood bacterial load ofseptic mice and reduce the lung injury in vivo. S1PR3 also can increase the capability of bacterial clearanceby upregulating the ROS level of macrophage, and improve the prognosis of septic mice in vitro.The study not only provides new insight into the role of S1PR3 in the early prevention and treatment of sepsis, but also provides new potential therapeutic targets for sepsis and other infectious diseases. |
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