Protective Effects Of Agmatine On Sepsis Induced By Zymosan In Mice

Objective：To investigate the effects of agmatine, a biological amine, on the acutephase of the sepsis in mice caused by zymosan.Methods：1. Thirty-two adult male C57BL/6mice were randomly divided intofour groups(n=8in each group): normal control group[SHAM+PBS group,phosphate buffer saline(PBS)0.5ml],AGM control group(SHAM+AGMgroup, AGM200mg/kg), model group(ZYM+PBS group,ZYM500mg/kg+PBS0.5ml),AGM treatment group(ZYM+AGM group,ZYM500mg/kg+AGM200mg/kg). AGM,ZYM and PBS were all intraperitoneally injectioninto mice. Mental status of mice were observed12h after administration ofzymosan and/or agmatine. Blood samples and peritoneal exudates of micewere collected for measuring tumor necrosis factor-α(TNF-α) andinterleukin-6(IL-6) with enzyme linked immunosorbent assay(ELISA),nitric oxide(NO) with Total Nitric Oxide Assay Kit.2. TNF-α,IL-6, myelopcroxidase(MPO) activity and apoptosis in thelung and small intestine were determind at the same time. The pathological changes were observed.The DNA-binding capacity of nuclear factor-Bp65(NF-B p65) in the lung and small intestine were determind by ELISA.3. The marker enzymes of liver,kidney and pancreas were measured inserum in clinical laboratory using standard laboratory techniques.4.Using zymosan(80ug/ml) activated macrophages, differentconcentration of agmatine was added to the medium2h before zymosan.Pro-or anti-inflammatory mediators were detected in supernatant12h afterstimulated by zymosan.Results：1.12hours after ZYM injection, mice became lethargic, with lessactivity and drink, while mice in AGM treatment group, showed muchbetter mental status, activity and water ingest. Treatment of mice withAGM attenuated the increase of TNF-α（ng/L:252.6±32.1vs.421.7±76.7,295.7±78.6vs.592.0±84.3，all P<0.05）, IL-6(ng/L:2198.8±281.8vs.4725.3±615.4,19829.3±3647.0vs.47751.3±5264.8，all P<0.05) and NO(μmol/L:33.2±4.3vs.50.2±5.2,14.0±3.6vs.45.4±5.2，all P<0.05) levels inserum and peritoneal exudates caused by ZYM.2. Treatment of mice with AGM also attenuated the increase ofTNF-α(ng/L:245.7±39.1vs.378.3±67.6, P<0.05)、IL-6(ng/L:810.3±175.6vs.1172.4±203.3, P<0.05)、MPO activity(ng/mg:24.9±4.4vs.37.3±5.8,P<0.05) in the lung and small intestine caused by ZYM. Confocalfluorescence microscopy showed that AGM attenuated apoptosis in lung and small intestine caused by zymosan. ZYM-injected led to severeinflammatory reaction in lung and small intestine tissus, includingvasodilatation,edema and neurophils infiltration. The treatment with AGMresulted in a significant reduction of lung and small intestineinjury.Moreover, compared with zymosan group, treatment of mice withAGM directly inhibited DNA binding ability of NF-B p65（OD:0.272±0.029vs.0.347±0.037, P<0.05）in lung. But, there was no observiousdifference between ZYM+PBS group and ZYM+AGM group in smallintestine.3. Compared with zymosan group,treatment of mice with AGMattenuated the increase of marker enzymes caused by zymosan in the lung,liver and pancreatic.4.In vitro, we demonstrated that AGM inhibited the production ofIL-6,TNF-α and increased slightly the production of IL-10.Conclusion：1.AGM can attenuate the increase of inflammatory factors(IL-6、TNF-α、NO) in local and all over the body in mice induced by zymosan.Theseresults demonstrate that AGM could produce a marked effect ofanti-inflammatory.2.AGM can lighten the alteration of inflammation in lung and smallintestine; decrease the production of inflammatory factors; inhibitsapoptosis; protects the lung tissue and small intestine tissue on sepsis in mice induced by zymosan.Anti-inflammatory effects of AGM may beconcerned with NF-B p65pathway.3. Treatment of mice with AGM attenuated the lung, liver andpancreatic injury and renal dysfunction caused by zymosan.4. AGM can attenuates IL-6、TNF-αsecreted by macrophages,increase the anti-inflammatory factor IL-10slightly. These results showedAGM can produce the effect of anti-inflammatory in vitro.