ABSTRACT
Objective To observe the effect of agmatine (AGM) on inflammatory factor in Kupffer cells of liver,and to investigate the protective effects of AGM on severe trauma-induced liver injury in mice and its possible mechanism.Methods Forty-two adult male BALB/c mice were randomly divided into sham group,model group,and AGM treatment group,with 14 mice in each group.The mice model of trauma-hemorrhage was reproduced by hindlimbs fracture combined with 35％ of orbital bleeding.The mice in the sham group were only anesthetized without other treatments.The mice in AGM treatment group were given intraperitoneal injection of 200 mg/kg AGM when limited recovery was performed,and the mice in model group were given the equal amount of normal saline.Seven mice in each group were sacrificed at 12 hours and 24 hours,respectively,after modeling,and blood samples and liver tissue were harvested,and liver Kupffer cells were isolated.Serum alanine aminotransferase (ALT),aspartate transaminase (AST)and lactic dehydrogenase (LDH) were determined with automatic biochemistry analyzer.Hepatic pathological changes were observed with light microscope using hematoxylin and eosin (HE) staining.The levels of tumor necrosis factor-α(TNF-o) and interleukin-6 (IL-6) in serum,hepatic homogenate and Kupffer cell supernatant were determined with enzyme linked immunosorbent assay (ELISA).The mRNA expressions of pro-inflammatory cytokines TNF-α and IL-6 in the Kupffer cell were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR).Results ① The normal liver tissue structure was found in sham group.At 24 hours after modeling in the model group,the changes in pathobiology were found as following:neutrophil infiltration,hepatocytes swelling,hyperemia,and necrosis,as well as the abnormality of parameters reflecting liver function.AGM could significantly improve the pathological changes in liver tissue caused by severe trauma,and ameliorate the liver function.② There were no significant differences in the levels of TNF-α and IL-6 in serum and hepatic tissue at 12 hours after modeling,and the parameters at 24 hours in model group were higher than those at 12 hours,which were significantly higher than those of the sham group [serum TNF-α (ng/L):80.8±4.7 vs.34.7±4.7,IL-6 (ng/L):104.0±9.0 vs.55.4±3.3;liver TNF-α (ng/mg):405.2± 19.6 vs.57.2±10.0,IL-6 (ng/mg):58.4±7.7 vs.14.3±2.1,all P ＜ 0.01].AGM could effectively reduce the levels of TNF-o and IL-6 in serum and hepatic tissue [serum TNF-α (ng/L):58.2 ± 3.1 vs.80.8 ± 4.7,IL-6 (ng/L):74.1 ± 6.6 vs.104.0± 9.0;liver TNF-α (ng/mg):248.7 ± 22.5 vs.405.2 ± 19.6,IL-6 (ng/mg):22.5 ± 3.1 vs.58.4 ± 7.7,all P ＜ 0.01].③ The levels of TNF-o and IL-6 in Kupffer cells supernatant were significantly higher than those of the sham group,and they were further increased after lipopolysaccharide (LPS) stimulation for 24 hours.AGM could effectively reduce the levels of TNF-α and IL-6 in Kupffer cells [TNF-α (ng/L):256.6 ± 5.6 vs.465.5 ± 5.2,IL-6 (ng/L):1 185.5 ± 64.4 vs.2 018.8 ± 53.2,both P ＜ 0.01],and also decreased the mRNA expressions of TNF-α and IL-6 [TNF-α mRNA (2-△△Ct):7.2±0.4 vs.13.5±0.4,IL-6 mRNA (2-△△Ct):13.2±0.7 vs.21.3 ± 1.6,both P ＜ 0.01].Conclusion Agmatine can reduce trauma-induced acute hepatic injury via suppression of cytokines release in Kupffer cells,and can ameliorate the liver function.
ABSTRACT
Objective To investigate the protective effect of agmatine (AGM) against peritoneal inflammatory response and neutrophil (PMN) infiltration induced by zymosan (ZYM) in mice. Methods Thirty-six adult male C57BL/6 mice were randomly divided into sham group, model group, and AGM treatment group. Peritonitis model was reproduced by intra-peritoneal injection of 1 mg/mL ZYM (0.5 mL), while equivalent phosphate buffer saline (PBS) was given to sham group. 200 mg/kg AGM was injected into peritoneal cavity after ZYM challenge in AGM treatment group. Six mice in each group were sacrificed at 2 hours and 6 hours, respectively, after reproduction of the model. Blood sample and peritoneal lavage fluid (PLF) were collected. The levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukins-6 (IL-6) in serum and PLF were determined by enzyme linked immunosorbent assay (ELISA). The number of leukocytes and PMN in PLF were determined by hemocytometer and flow cytometry, respectively. Results Compared with sham group, all serum and PLF levels of KC, MIP-2, TNF-α and IL-6 were greatly elevated at 2 hours after ZYM injection in model group, while AGM treatment could dramatically reduce the levels of the above-mentioned cytokines in serum and PLF as compared with those of the model group [serum KC (ng/L): 990.7±137.9 vs. 2 053.2±262.7, MIP-2 (ng/L): 642.2±124.4 vs. 1 369.7±146.5, TNF-α (ng/L): 608.6±38.1 vs. 1 044.7±101.0, IL-6 (ng/L): 1 058.2±129.1 vs. 1 443.3±190.1; PLF KC (ng/L): 7 462.3±839.6 vs. 12 723.5±1 515.7, MIP-2 (ng/L): 1 570.8±193.4 vs. 3 471.4±384.7, TNF-α (ng/L): 1 115.8±156.7 vs. 1 499.2±231.2, IL-6 (ng/L): 2 646.5±223.2 vs. 3 126.7±291.4; all P < 0.05]. The expressions of KC, MIP-2 and TNF-α at 6 hours were significantly lower than those at 2 hours in model group and AGM treatment group, but IL-6 levels were further increased. The levels of KC and MIP-2 in serum and PLF at 6 hours were decreased to the levels of sham group. At 6 hours after the reproduction of the model, the number of total inflammatory cells and PMN of PLF in the model group was significantly higher than those of the sham group. In contrast, AGM notably lowered the number of inflammatory cells and PMN in peritoneal fluid after ZYM attack [total inflammatory cells (×109/L): 14.7±1.1 vs. 2.0±0.4, 10.1±1.2 vs. 14.7±1.1; PMN (×109/L): 11.37±1.22 vs. 0.18±0.05, 7.69±0.57 vs. 11.37±1.22, all P < 0.05]. Conclusion AGM can effectively alleviate acute peritoneal inflammatory injury induced by ZYM, mainly through reducing the secretion of inflammatory mediators and chemokines, and inhibiting the infiltration of leukocytes and neutrophils.
ABSTRACT
ObjectiveTo observe protective effects of agmatine (AGM) on inflammatory response and spleen immune function in mice with trauma.Methods Forty-eight adult male C57BL/6 mice were randomly divided into three groups (n= 16 each), including control group, model group (bilateral femoral fracture and removal of 35% of the total blood volume), and AGM group (trauma/hemorrhage & AGM 200 mg/kg). Eight mice in each group were sacrificed at 3 hours and 24 hours, respectively, after modeling, and blood samples and tissue homogenate of spleen and liver were collected. The contents of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β) in serum and liver tissue were determined with enzyme linked immunosorbent assay (ELISA). Serum aspartate transaminase (AST), alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) were determined with automatic biochemistry analyzer. Spleen proliferation response stimulated with concanavalin A (ConA) was evaluated with methyl thiazolyl tetrazolium colourimetry (MTT).γ-interferon (IFN-γ) and IL-2 releases were determined with ELISA.Results Compared with control group, 3 hours after trauma/hemorrhage, the levels of serum TNF-α, IL-6, and IL-1β in model group were significantly elevated [TNF-α (ng/L): 145.38±31.50 vs. 23.06±11.14, IL-6 (ng/L): 496.94±50.76 vs. 47.13±17.47, IL-1β (ng/L): 321.31±43.02 vs. 29.25±16.24,allP< 0.01]. It was found that AGM treatment could alleviate the increase in serum pro-inflammatory mediators induced by trauma/hemorrhage, such as TNF-α (ng/L:111.56±25.47 vs. 145.38±31.50), IL-6 (ng/L: 412.56±44.33 vs. 496.94±50.76), IL-1β (ng/L: 273.38±45.25 vs. 321.31±43.02,P< 0.05 orP< 0.01). Twenty-four hours after trauma/hemorrhage, serum pro-inflammatory mediators were recovered to the levels in control group. There was no significant difference in TNF-α and IL-6 levels at 3 hours after trauma/hemorrhage among groups. Compared with control group, the expressions of liver TNF-α and IL-6 in model group were increased at 24 hours following trauma [TNF-α (ng/mg): 32.93±4.90 vs. 26.58±2.33, IL-6 (ng/mg): 11.20±1.66 vs. 8.38±0.89,bothP< 0.01]. However, AGM 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) in the liver as induced by trauma/hemorrhage (P< 0.05 andP< 0.01). At 24 hours after modeling, model group and AGM group had distinctly higher serum AST, ALT, LDH levels than those of control group [AST (U/L): 405.9±31.2, 245.7±22.1 vs. 128.2±15.9; ALT (U/L): 92.1±6.3, 51.6±5.0 vs. 30.1±3.2; LDH (U/L): 606.7±36.3, 478.7±25.3 vs. 384.0±16.6, allP< 0.01]. Nevertheless,the increase in serum AST, ALT and LDH was alleviated in AGM group (allP< 0.01). Meantime, trauma/hemorrhage produced a noticeable depression of proliferation of splenic cells and IFN-γ and IL-2 release stimulated with ConA compared with control group [proliferation rate: (40.97±4.13)% vs. (89.99±7.76)%, IFN-γ(ng/L): 91.6±12.3 vs. 353.2±21.5,IL-2 (ng/L): 53.4±6.4 vs. 91.0±12.2,allP< 0.01]. In contrast, AGM notably restored the capacity of proliferation response of splenic cells [proliferation rate: (74.86±5.75)% vs. (40.97±4.13)%, P< 0.01],enhanced the release of IFN-γ and IL-2 stimulated with ConA [IFN-γ (ng/L): 327.8±23.6 vs. 91.6±12.3, IL-2 (ng/L): 74.8±10.4 vs. 53.4±6.4, bothP< 0.01].Conclusion AGM can dramatically alleviate spleen immunosuppression, excessive inflammation and organ damage induced by trauma/hemorrhage.