Survival Protective Effects Of Agmatine On Posttraumatic Sepsis In Mice And Its Mechanism

Objective:To observe the survival protective effects of agmatine(AGM) on septic mice following trauma and its mechanism.Methods:① The mortality rate of three murine models, containing trauma/hemorrhage(bilateral femoral fracture and drawing 35% of the total blood, TH), sepsis(lipopolysaccharide, LPS 25mg/kg) and posttraumatic sepsis(TH+LPS), were determinated. The dose and time-dependent effects of agmatine on survival of posttraumatic sepsis in mice were observed and recorded as well.② Adult male C57BL/6 mice were randomly divided into three groups, containing control group,TH group and AGM group(TH & AGM 200mg/kg). Mice in each group were sacrificed at 3 hours and 24 hours, respectively. After modeling, blood samples, tissue homogenates of liver were collected for detection of tumor necrosis factor-α(TNF-α)、interleukin(IL-6、IL-1β) by enzyme linked immunosorbent assay(ELISA). Blood routine examination such as red blood count(RBC), white blood count(WBC), hemoglobin(HGB), platelet(PLT) and hematocrit(HCT) were tested by automated hematology analyzer. Serum aspartate transaminase(AST), alanine aminotransferase(ALT) and lactic dehydrogenase(LDH) were determinated by automatic biochemistry analyzer, spleen proliferation response stimulated with Concanavalin A(Con A) was evaluated by MTT, γ-interferon(IFN-γ) and interleukin-2(IL-2) releases were detected by ELISA.③ Mice induced by different doses of LPS(10mg/kg or 40mg/kg) were pretreated with yohimbine(selective α2-adrenoceptor antagonist, YHB), MK-801(non-competitive NMDA receptor antagonist), efaroxan(highly selective imidazoline-1 receptor ligand, EFA) or idazoxan(high-affinity imidazoline-2 receptor ligand, IDA) before administration of agmatine or saline. A part of mice were sacrificed for serum IL-6 detection, another part of mice were observed for survival analysis. The effects of agmatine on polymicrobial sepsis induced by cecal ligation and puncture were also detected.Results:① The mortality rate induced by TH in mice was 6.67%. Nevertheless, TH dramatically augmented the risk of dying from sepsis in mice(83.33% vs. 13.33%, P<0.05). Campared with TH+LPS group, treatment with AGM(400mg/kg) after LPS noticeably alleviated the increased mortality(31.25% vs. 87.50%, P<0.05). Combined AGM treatment after trauma(100, 200, 400mg/kg) and LPS(400mg/kg) dose-dependently improved the survival of mice after trauma/hemorrhage and the induction of sepsis(40%, 33.33%, 25% vs. 80%, all P<0.05).② Campared with control group, 24 hours following trauma, the RBC, HGB and HCT in model and AGM group were significantly diminished[RBC(×1012/L): 6.16±1.13, 6.56±1.44 vs. 9.60±0.42;HGB(g/L): 88.75±24.68, 96.75±20.50 vs. 144.50±8.54;HCT(%): 29.80±7.74, 32.31±7.11 vs. 45.60±2.34; all P<0.05]. Treatment with agmatine was found to alleviate increased contents of serum inflammatory mediators induced by trauma [ng/L: TNF-α(111.56±25.47 vs. 145.38±31.50), IL-6(412.56±44.33 vs. 496.94±50.76), IL-1β(273.38±45.25 vs. 321.31±43.02); all P<0.05]. Moreover, agmatine inhibited the level of TNF-α(ng/mg: 28.92±3.16 vs. 32.93±4.90)and IL-6(ng/mg: 9.03±1.28 vs. 11.20±1.66) induced by TH in the liver(both P<0.05). Similarly, the level of serum liver enzymes were declined in AGM group following trauma[U/L : AST(245.7±22.1 vs. 405.9±31.2), ALT(51.6±5.0 vs. 92.1±6.3) and LDH(478.7±25.3 vs. 606.7±36.3); all P<0.05]. Campared with TH group, AGM also notably improved the capacity of spleen proliferation response stimulated with Con A[ratio(%):74.86±5.75 vs. 40.97±4.13, P<0.05], increased the release of IFN-γ and IL-2[ng/L:IFN-γ(327.8±23.6 vs. 91.6±12.3) and IL-2(74.8±10.4 vs. 53.4±6.4), both P<0.05].③ AGM reduced mortality induced by LPS(35% vs. 85%, P<0.05). It was found that administration of AGM also could decrease mortality following CLP(40% vs. 80%, P<0.05). MK-801, AGM and IDA could inhibit the level of serum IL-6 induced by LPS[IL-6(μg/L): 51.03±5.92, 39.5±9.24, 36.63±4.19 vs. 67.95 ± 5.95; all P<0.05]. However, IDA did not produce a synergistic anti-inflammatory effect with AGM. YHB, MK-801 or EFA could not cut mortality rate induced by LPS, respectively. In addition, pretreatment with YHB, MK-801 or EFA did not affect the survival protection action of AGM on murine sepsis. Both AGM and IDA could reduce mortality rate induced by LPS(43.75%, 43.75% vs. 81.25%; both P<0.05). Meanwhile, pretreatment with IDA did not produce a synergistic survival protection effect with AGM.Conclusion:Trauma/hemorrhage can significantly increase the risk of dying from sepsis in mice. However, treatment with AGM in the time window between TH and sepsis can dose-dependently enhance the survival of mice after this model. The mechanisms include: ①AGM can inhibit excessive inflammatory reaction, protect liver damage against trauma, and improve depressed immune functions following trauma. ②AGM can reduce mortality induced by LPS or CLP. The survival protective effects of agmatine may be primarily mediated by I2 receptors, independent of α2-adrenoceptors, NMDA receptors or I1 receptors.