Protective Effects And Mechanism Of Agmatine On Lung Injury In Septic Mice

Objective:Establishing cecal ligation puncture(CLP)modle,to explore the effects of agmatine(AGM)on lung injury and polymorphonuclear leukocyte(PMN)function in septic mice.Methods:1.6 to 8-week-old male C57BL/6 mice were randomly divided into three groups: SHAM group(laparotomy but not cecal ligation and puncture,and intraperitoneally injected with saline),CLP group(cecal ligation and puncture,and intraperitoneally injected with saline)and CLP+AGM group(cecal ligation and puncture,and intraperitoneally injected with AGM at200mg/kg).Observe the survival of mice in each group at any time and record death time.2.6 to 8-week-old male C57BL/6 mice were randomly divided into three groups: SHAM group,CLP group and CLP+AGM group.Mice in each group were sacrificed at 24 hours after modeling.Peritoneal lavage fliud,blood and lung tissue in all groups were collected for detecting the bacterial levels.The concentration of interleukin-6(IL-6),tumornecrosis factor-α(TNF-α)and myeloperoxidase(MPO)in lung tissue were detected by enzyme linked immunosorbent assay(ELISA).Pathological changes were observed by Hematoxylin and Eosin stain.Moreover,Terminal deoxynucleotidyl transferase-mediated d UTP Nick-End labeling(TUNEL)assay detects cell apoptosis.At last,tissue W/D ratio was measured.3.6 to 8-week-old male C57BL/6 mice were randomly divided into three groups: SHAM group,CLP group and CLP+AGM group.Mice in each group were executed at 6,12,24 hours after modeling.Peritoneal lavage fliud in all groups were collected.The peritoneal lavage fliud cells were calculated by Neubauer chamber,and the percentage of neutrophils and the level of reactive Oxygen species(ROS)were tested by flow cytomettry.The safety concentration of AGM to neutrophil was detected by methyl thiazolyl tetrazolium assay(MTT).At 24 hours after modeling,the bone marrow neutrophils in all groups were collected for detecting the chemotactic function by Transwell assay.Mice abdominal neutrophils were isolated and cultured in vitro,and the effect of AGM on neutrophils’ phagocytotic function were tested.Results:1.Mice in SHAM group were in good condition with a 100% survival rate.Mice in CLP group were gradually died at 6 hours after modeling and the 72 h survival rate was only 16.67%,significantly difference compared toSHAM group(p<0.001).However,the group treated with AGM after modeling has an obviously improved survival rate(40%),significantly difference compared to CLP group(p=0.027).2.Comparing CLP+AGM group with CLP group,we found AGM could reduce bacterial levels obviously in peritoneal lavage fliud,blood and lung tissue caused by CLP [peritoneal lavage fliud(logCFU/ml): 5.28±0.98 vs.6.85±0.58,p=0.033;blood(logCFU/ml): 3.63±0.45 vs.5.73±0.83,p=0.017;lung tissue(logCFU/ml): 2.31±0.25 vs.3.90±0.71,p=0.018].24 hours after CLP,the levels of IL-6,TNF-α and MPO in lung tissue of CLP group were increased significantly compared to those in SHAM group[IL-6(pg/mg): 1789.67±263.41 vs.169.00±39.04;TNF-α(pg/mg):998.67±172.14 vs.255.33±30.55;MPO(pg/mg): 94.33±8.62 vs.22.00±6.56,all p<0.001].Moreover,clearly pathological changes and pulmonary edema in lung tissue were founded(W/D ratio: 4.95±0.20 vs.3.60±1.45,p<0.001).Comparing CLP+AGM group with CLP group,we found AGM obvious attenuated the levels of IL-6,TNF-α and MPO in lung tissue [IL-6(pg/mg): 1226.27±123.31 vs.1789.67±263.41,p=0.007;TNF-α(pg/mg): 668.00±90.60 vs.998.67±172.14,p=0.012;MPO(pg/mg):68.00±14.42 vs.94.33±8.62,p=0.021],and lung injury and edema caused by CLP was relieved obviously(tissue W/D ratio: 4.39±0.10 vs.4.95±0.20,p=0.001).In addition,confocal laser scanning microscope showed that mice lung tissue in CLP group had the appearance of evident apoptosis,andtissue apoptosis was apparently reduced following with AGM treatment.3.Compared with CLP group,mice in CLP+AGM group after modeling 24 hours showed that AGM treatment could significantiy increased the number and percentage of neutrophils in peritoneal lavage fliud,meanwhile the production of ROS was increased obviously[neutrophils number(×107/ml): 1.92±0.13 vs.0.76±0.70,p<0.01;percentage(%): 92.50±1.41 vs.78.27±4.45,p=0.02;ROS(mean fluorescence intensity×104): 11.81 ± 2.93 vs.7.01 ± 1.5,p=0.02].In the Transwell test,we found that AGM could increase chemotactic function of mice bone marrow neutrophil significantiy.Besides,AGM enhanced the phagocytotic function of mice abdominal neutrophils.Conclusion:1.CLP can induce sepsis in mice and significantly increase death rate.However,treatment with AGM can enhance the survival of mice after this modeling.Those results showed that AGM has protective effect on sepsis mice.2.AGM can reduce bacterial levels,inflammatory cytokine and MPO,and decrease pathological changes and edema in mice lung tissue.Meanwhile,AGM can inhibite apoptosis induced by sepsis in mice lung tissue.Those results showed that AGM has obvious protective effect on lung tissue in sepsis mice.3.AGM can increase the number and percentage of neutrophils inperitoneal lavage fliud and enhance the chemotaxis and phagocytosis functions of neutrophils.