Effect And Mechanism Of Moxifloxacin On Pro-inflammatory Response In Lipopolysaccharide-stimulated Mice Peritoneal Macrophages

Objective: Sepsis is a major infection-induced the host of Systemic inflammatory response syndrome(SIRS)that promotes the multiple organs dysfunction syndrom(MODS).The incidence of sepsis,and death from septic shock,has increased over the past few decades,serious threat to human health.Although sepsis was already known as a severe condition in the times of Hippocrates,the debate on what sepsis represents and how it should be delineated continues today.Many scholars believe that sepsis is a severe condition marked by an overwhelming immune response to a serious infection,and results in the excessive production of various pro-inflammatory cytokines and cellular injury.Macrophages are the key cells that mediate innate and adaptive immunity,and it is the first and main cell that reacts to the inflammation.they are associated with an excessive inflammation response following severe sepsis.Macrophage is the main nucleated cell in the peritoneal fluid.Infection by mycobacteria leads to a signaling response by the host macrophage and subsequent production of pro-inflammatory mediators.Macrophages identify the composition of bacteria,activated by the antimicrobial effector.Previous studies show that early inflammatory response of sepsis associated with lipopolysaccharide(LPS)structure.LPS is The characteristic endotoxin found in the outer membrane of Gram-negative bacteria,can induce the innate immunity system and through the myeloid differentiation protein 2(MD2)and Toll-like receptor 4(TLR4)complex,increase the production of inflammatory mediators.Following infection with Gram-negative bacteria,the host is exposed to microbial LPS through ancillary proteins,such as LPS-binding protein(LBP)and CD14,which transports the microbial LPS to specific target cells and a surface receptor complex composed of Toll-like receptor 4(TLR4)and myeloid differentiation 2(MD2).Subsequent TLR4 activation leads to the recruitment of myeloid differentiation primary-response gene 88(MyD88),activating downstream NF-κB and MAPK pathways.It has also been reported that Sphk-1 is upregulated in stimulated human phagocytes and peritoneal phagocytes of patients with severe sepsis and potentiallyplays a role in the development of sepsis.LPS activates the sphingosine kinase1/sphingosine-1-phosphate(SPHK1/S1P)signaling axis in other cell types including macrophages,leading to translocation of SPHK1 to the plasma membrane where it converts its substrate sphingosine to the bioactive sphingolipid S1 P.SPHK1 is emerging as an important mediator in inflammatory responses activated by various inflammatory stimuli,including lipopolysaccharide(LPS),TNF-α and IL-1β,and involves the toll-like receptor(TLR)signaling pathways.SPHK1 can enhance the AKT activation and TNF-α-induced cyclin D expression,thereby promoting cell survival and proliferation.Moreover,increasing evidence has proved that PI3K/AKT /NF-κB signal pathway can positively regulate the inflammatory responses of various types of cells.and the activation of the NF-κB and MAPK pathways induces the up-regulation and increased expression of pro-inflammatory genes,contributing to multiple organ dysfunction and SIRS.The pro-inflammatory action of LPS is crucial for curbing bacterial infections,but excessive host responses to LPS can lead to systemic inflammatory conditions—sepsis,severe sepsis,and fatal septic shock.Therefore,effective in blocking the LPS signal transduction pathway is of important value for prevention and control of excessive inflammatory response.Despite the advances in the understanding of the pathophysiology and processes involved in sepsis,very little progress has been made towards therapeutic interventions.Recently,a recombinant human activated protein C(drotrecogin alfa)was withdrawn from the market because of a lack of efficacy in septic humans.A large number of immunomodulatory agents have been studied in experimental and clinical settings in an attempt to find an efficacious anti-inflammatory drug that reduces mortality.Even though preclinical results had been promising,the vast majority of these trials actually showed little success in reducing the overwhelmingly high mortality rate of septic shock patients as compared with that of other critically ill intensive care unit patients.It is necessary to use antibiotic to treatment sepsis,so we assume to choose antimicrobials with certain immune regulating effect,so that we can kill two birds with one stone in the treatment,not only for antimicrobial treatment but also can reduce the excessive inflammatory reaction of sepsis,andthen reduce the case fatality rate.Quinolones(QNs)are synthetic,broad-spectrum antimicrobial agents in common use for the activities against both gram-positive and gram-negative bacteria.In recent years,a large number of research results show that a wide range of antimicrobial agents,including quinolones,have been reported to modify immune and inflammatory responses both in vivo and in vitro.Today,anti-infection treatment should not only consider the sensitivity of antibiotics for a particular bacteria,antibacterial strength,also should consider the relationship between the in vivo efficacy and immune regulation of antibiotics and the application of antibiotics are not only limited to the treatment of infection,also can be used to treat the immune system disease.Moxifloxacin(MXF)is a fluoroquinolone with activities against both gram-positive and gram-negative bacteria.It has been suggested that MXF has inhibitory and stimulatory effects on the immune system,primarily by studies that have shown that the production of several cytokines by human and murine leukocytes can be affected by this drug.There is strong evidence that fluoroquinolones additionally exhibit immunomodulatory functions during inflammation and microbial infection besides their bactericidal activities.In vitro studies showed that clinically relevant concentrations of moxifloxacin inhibit the synthesis of inflammatory mediators(e.g.IL-1,TNF-α,IL-6,IL-8)in human peripheral blood mononuclear cells and in the monocytic cell and endothelial Cells stimulated with Lipopolysaccharide(LPS).And the effection of MXF on LPS stimulate macrophage inflammatory response research is very few.We have known that macrophages play an important role in immune defense,and whether the immunoregulatory effects of moxifloxacin can affect LPS induced macrophage secreted pro-inflammatory factor,and its regulating pathway is involved in TLR4 and SPHK1? This study will use moxifloxacin on LPS stimulated mice abdominal cavity macrophage to find out the pro-inflammatory factor secretion and its mechanism.Discussion if moxifloxacin through adjusting the function of macrophage to improve the prognosis of sepsis.This study by LPS stimulated macrophages to establishment of in vitro sepsis inflammatory reaction model,detecting and analyzing the TLR4,SPHK1,proinflammatory factor level and the expression of cytokines and so on.whileobserving the role MXF in LPS stimulated macrophages inflammatory responses.as a means of intervention,explore the MXF effect in signaling transduction mechanisms of LPS stimulated macrophage.And small interfering RNA(siRNA)technology targeted silence TLR4 gene to block specific signaling molecule,Western blot,Real-time PCR and ELISA techniques were used to discusse in cellular level to macrophages TLR4,the influence of SPHK1 may and the mechanism of moxifloxacin inhibiting proinflammatory factor.Method:1.The mice peritoneal macrophages were isolated by lavage of the peritoneal cavity and then cells were cultured.In all experiments,macrophages were incubated with LPS(500 ng/mL)for different time points.The levels of TLR4、SPHK1 and NF-κB mRNA in were determined by;The relative levels of TLR4、SPHK1 and NF-κB in were determined by Western blot.The levels of TNF-α and IL-6 secreted by macrophages were detected by ELISA.In order to obtained the optimal point in time after LPS stimulation each index increased,for the next experiment.2.Objective To study the effects of different concentrations of the MXF in mice peritoneal macrophages induced by LPS.According the last experiment to determine the LPS stimulation to find the optimal point in time(i.e.,the corresponding index changes when the most obvious point in time).The macrophages were divided into 5groups.The levels of TLR4 、 SPHK1 and NF-κB mRNA were determined by realtime-PCR;The relative levels of TLR4、SPHK1 and NF-κB were determined by Western blot.The levels of TNF-αand IL-6 secreted by macrophages were detected by ELISA.The expression level of TLR4,SPHK1 and NF-κB in each group cells was examined by immunofluorescence.According to the experiment,to determine the optimum concentration of the MXF,for γmg/L,for follow-up study.3.Effective TLR4 siRNA was screened through real-time PCR and Western blot.The influence of SPHK1 siRNA on mice peritoneal macrophages was detected.The experiment is set up 5 groups group.The levels of TLR4、SPHK1 and NF-κB mRNA were determined by;The relative levels of TLR4、SPHK1 and NF-κB were determined by Western blot.The levels of TNF-α and IL-6 secreted by macrophages were detected by ELISA.The expression level of TLR4,SPHK1 and NF-κB in each group cells wasexamined by immunofluorescence.Results:1.The levels of TLR4,SPHK1,NF-κB p65 mRNA and protein were significantly higher than control group after LPS stimulated mice peritoneal macrophages,and the difference was statistically significant(P<0.0001).The cell supernatant of TNF-αand IL-6 levels were increased in varying degrees within 24 h,compared with the control group the difference was statistically significant(P<0.0001).2.Certain concentration of the MXF have inhibitory effect on the increasing of TLR4,SPHK1,NF-κB expression level and cell supernatant of TNF-αand IL-6 in LPS stimulation of mice peritoneal macrophages.Especially in the concentration of moxifloxacin for 16 mg/L,have the strongest inhibitory effect.The difference was statistically significant(P < 0.0001).3.Compared with other 2 group of oligo and NC oligo,1567 oligo exhibited the strongest interfering action to TLR4,which as a result is the effective target gene sequence for following experiment.Transfection with TLR4 siRNA significantly reduced the levels of TLR4 expression by 81.2% in mice peritoneal macrophages.The the level of SPHK1,NF-κB expression in SHPK1 siRNA-transfected cells were significantly reduced as compared with that of un-manipulated cells(p<0.001).The MXF have synergies with TLR4 siRNA,make the expression of SPHK1 and NF-κB further reduced,the difference was statistically significant(P < 0.001).The cell supernatant of TNF-α and IL-6 levels also reduce,the difference was statistically significant(P < 0.001).Conclusion:1.LPS stimulated mice peritoneal macrophages at different time points cause TLR4,SPHK1,NF-κB p65 mRNA expression and protein level increased obviously,the TNF alpha and increased secretion of IL-6,increased secretion of TNF-α and IL-6,which indicated that TLR4,SPHK1,NF-κB might involve the macrophage inflammatory response induced by LPS.2.Certain concentration of the MXF can inhibit the inflammatory response in LPS stimulation of mice peritoneal macrophages by decrease the level of TLR4,SPHK1,NF-κB expression and cell supernatant of TNF-αand IL-6 in LPS stimulation of miceperitoneal macrophages,thereby inhibiting the release of the macrophage pro-inflammatory factor.The inhibition may be associated with TLR4,SPHK1 protein levels decrease,suggested the MXF may affect the TLR4 and SPHK1 to modulation macrophage inflammatory response.3.The condition of TLR4 gene silence inhibited the LPS-induced produce of inflammatory response from mice peritoneal macrophage.After TLR4 gene silence,SPHK1,nf-kappa B p65 protein levels decrease,the cell supernatant of TNF-α and IL-6levels also reduced.The MXF have synergies with TLR4 siRNA,can further inhibit inflammatory response of the mice peritoneal macrophages.