Protective Effects And Mechanism Of Agmatine On Acute Peritonitis Induced By Zymosan In Mice

Objective:To investigate the protective effect of agmatine(AGM)on acute peritoneal inflammatory injury induced by zymosan(ZYM) and related mechanisms.Methods:1.Seventy-two adult male C57BL/6 mice were randomly divided into control group,model group, AGM treatment group and AGM control group(n=18 each). Peritonitis model was reproduced by intra-peritoneal injection 0.5 m L of ZYM(1 mg/ml) in model group and AGM treatment group,while equivalent phosphate buffer saline(PBS) was given in control group,and 400 mg/kg AGM was injected into peritoneal cavity when ZYM challenge in AGM treatment group and AGM control group. Six mice in each group were sacrificed at 2 hours,6 hours and 24 hours, respectively, after modeling. Blood sample,peritoneal lavage fluid(PLF) and liver homogenates were collected. The mental state of mice were observed at regular time before killed. The contents of tumor necrosis factor-α(TNF-α), interleukins-6(IL-6), keratinocyte derived chemokine(KC), macrophage inflammatory protein 2(MIP-2) both in serum,PLF and liver tissue were determined by enzyme linked immunosorbent assay(ELISA). The number of leukocytes and the percentage of neutrophils in PLF were calculated by hemocytometer and flow cytometry, respectively.The levels of ALT,AST,Crea,Urea in serum were analysed by automatic biochemistry analyzer.2.Isolation of mouse bone marrow neutrophils and peritoneal macrophages was cultured in the upper and lower chamber of transwell cells respectively. Peritoneal macrophages were stimulated by ZYM in the lower chamber, with or without agmatine intervention. DAPI staining after 40 min, then fluorescence microscopy was used to observe the chemotaxis of neutrophils.3.Mouse peritoneal macrophages were isolated and cultured in vitro,then stimulated by 100μg/ml ZYM.In the case of agmatine therapy,protein and supernatant were collected at different times.The protein concentration of TNF-α, IL-6, KC and MIP-2 in cell supernatant was measured by ELISA,the expression of TNF-α, IL-6, KC and MIP-2 m RNA in cells was detected by q PCR,the level of i NOS, p65 and p-p65 in cytoplasm and nucleus was determined by western blot. In addition, peritoneal macrophages were pretreatmented with MK-801(non-competitive NMDA receptor antagonist), yohimbine(selecttive α2-adrenoceptor antagonist,YHB),efaroxan(highly seletive imidazoline-1 receptor ligand,EFA) or idazoxan(high-affinity imidazoline-2 receptor ligand,IDA),respectively. With or without agmatine treatment,the concentration of IL-6 and KC in the cell supernatant after 12 hours stimulated by ZYM wasdetected.Results:1.At 2 hours, 6 hours and 24 hours after ZYM attacking,the mice in ZYM model group and AGM treatment group had slow action, diarrhea, diet reduction and a state of curling up into a ball,above which became more and more obvious over time, but the spirit and activity state of AGM treatment group was significantly better than that of ZYM model group. Compared with control group,AGM treatment could significantly reduce the levels of KC[serum KC(pg/ml): 1578.8±107.3 vs 2077.4±196.3;PLF KC(pg/ml): 6064.3±577.4 vs 9864.7±851.8;all P < 0.05], MIP-2[serum MIP-2(pg/ml): 743.5±77.9 vs 937.6±89.6;PLF MIP-2(pg/ml): 1763.4±125.7 vs 2369.7±304.5;all P<0.05] in serum and peritoneal lavage fluid after 2 hours of ZYM stimulation, inhibited the level of TNF-α[serum TNF-α(pg/ml): 513.7±38.5 vs 822.1±47.8;PLF TNF-α(pg/ml): 1661.7±185.4 vs 2812.5±216.4;all P < 0.05],IL-6[serum IL-6(pg/ml): 945.4±107.9 vs 1326.4±178.5;PLF IL-6(pg/ml): 11694.1±1503.2 比21170.7±3872.4;all P<0.05] in serum and peritoneal lavage fluid after 6 hours of ZYM stimulation and the number of total inflammatory cells(×106/ml:10.1±1.2 vs 14.7±1.1,P<0.05) and the percentage of neutrophils(%:77.83% vs 90.07%,P<0.05) in peritoneal lavage fluid after 6 hours of ZYM attack.Meantime,the elevate level of TNF-α(ng/g: 281.6±20.8 vs 358.5±25.3,P<0.05),IL-6(ng/g: 197.4±22.7 vs 273.5±26.7,P<0.05),KC(ng/g: 47.2±11.9 vs 77.4±20.3,P<0.05) and MIP-2(ng/g: 67.4±14.6 vs 103.7±19.2,P<0.05) in tissue homogenates of livers and the contents of ALT(U/L: 392.6±41.4 vs 712.3±55.5,P<0.05),AST(U/L: 494.7±34.3 vs 681.7±38.6,P<0.05),Urea(mmol/L: 31.3±1.8 vs 46.8±3.9,P<0.05) and Crea(μmol/L: 32.5±1.9 vs 38.2±2.7,P<0.05) after 24 hours of ZYM stimulation were attenuated.2.In the vitro transwell chemotaxis test, we found that the chemotaxis number of neutrophil induced by the supernatant of ZYM group was significantly higher than control group. But the chemotaxis number of neutrophil induced by the supernatant of ZYM+AGM group was significantly decreased compared to that of ZYM group.3.In vitro experiment of cell culture, the protein secretion and m RNA expression of TNF-α, IL-6,KC and MIP-2 in peritoneal macrophages after ZYM stimulation and AGM treatment were significantly decreased,also the expression of i NOS in cells was reduced and the phosphorylation and translocation of p65 were inhibited.Moreover,AGM, MK-801 and IDA treatment could attenuate the increase of IL-6 and KC in the cultured supernatant of macrophages after 12 hours of ZYM stimulation,the anti-inflammatory effect of MK-801 was weaker than AGM,but the combination of AGM and MK-801 had a certain degree of joint anti-inflammatory effects,the anti-inflammatory effect of IDA was similar to that of AGM. When AGM combined with IDA, the joint anti-inflammatory effect was not obvious. EFA and YHB had no anti-inflammatory effect, and both of them also had no effect on the anti-inflammatory effect of AGM.Conclusion:1.Agmatine could inhibit the production of TNF-α,IL-6,KC and MIP-2 in serum and peritoneal lavage fluid in peritonitis mice induced by zymosan,and reduce the total number of inflammatory cells and inhibit the infiltration of neutrophils in peritoneal cavity, indicating that AGM has excellent systemic and local anti-inflammatory effects.2.Agmatine could alleviate the increase of ALT,AST,Urea and Crea in serum induced by zymosan peritonitis in mice and reduce the expression of inflammatory cytokines and chemokines in liver,suggesting that AGM has a certain protective effect on organ damagein peritonitis mice.3.Agmatine could depress the protein secretion and m RNA expression of TNF-α, KC, MIP-2 and IL-6 in peritoneal macrophages caused by ZYM,meantime, the expression of i NOS in macrophages decreased and the phosphorylation and translocation of p65 were inhibited. Moreover,agmatine has a similar anti-inflammatory effect to IDA,and the significantly difference of anti-inflammatory effect between MK-801 and agmatine, but YHB and EFA has nothing to do withthat of agmatine.It is indicated that agmatinemay exert anti-inflammatory effect through inhibiting i NOS expression, NF-κB pathway and activating imidazoline 2 receptor inmacrophages.