胍丁胺对酵母多糖诱导急性腹膜炎炎症损伤及中性粒细胞浸润的影响

The effects of agmatine on acute peritoneal inflammatory injury and neutrophil infdtration induced by zymosan in mice

目的：观察胍丁胺（AGM）治疗对酵母多糖（ZYM）诱导的小鼠腹膜炎炎症反应及中性粒细胞（PMN）浸润的影响。方法将36只成年雄性C57BL/6小鼠按随机数字表法分为假手术（Sham）组、模型组、AGM治疗组。采用腹腔注射1mg/mLZYM溶液（0.5mL）制备急性腹膜炎小鼠模型，Sham组腹腔注射等量磷酸盐缓冲液（PBS）；AGM治疗组同时腹腔注射AGM200mg/kg。各组分别于制模后2h和6h各处死6只小鼠，采集眼眶血及腹腔灌洗液，用酶联免疫吸附试验（ELISA）检测血清和腹腔灌洗液中角化细胞来源趋化因子（KC）、巨噬细胞炎性蛋白-2（MIP-2）及肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）的含量，用血细胞计数板计数腹腔灌洗液中白细胞总数，流式细胞仪分析腹腔灌洗液PMN数量。结果制模后2h，模型组血清和腹腔灌洗液中KC、MIP-2、TNF-α、IL-6含量均明显高于Sham组；AGM治疗后血清和腹腔灌洗液中上述各指标均较模型组显著降低〔血清KC（ng/L）：990.7±137.9比2053.2±262.7，MIP-2（ng/L）：642.2±124.4比1369.7±146.5，TNF-α（ng/L）：608.6±38.1比1044.7±101.0，IL-6（ng/L）：1058.2±129.1比1443.3±190.1；腹腔灌洗液KC（ng/L）：7462.3±839.6比12723.5±1515.7，MIP-2（ng/L）：1570.8±193.4比3471.4±384.7， TNF-α（ng/L）：1115.8±156.7比1499.2±231.2，IL-6（ng/L）：2646.5±223.2比3126.7±291.4；均P＜0.05〕；与2h比较，模型组、AGM治疗组6h时血清和腹腔灌洗液中KC、MIP-2、TNF-α明显下降，IL-6进一步升高，其中模型组和AGM治疗组血清及腹腔灌洗液中KC、MIP-2均已降至Sham组水平。制模后6h，模型组腹腔炎性细胞总数与PMN计数均显著高于Sham组，AGM治疗可显著减少腹腔中炎性细胞总数与PMN计数〔炎性细胞总数（×109/L）：14.7±1.1比2.0±0.4、10.1±1.2比14.7±1.1，PMN计数（×109/L）：11.37±1.22比0.18±0.05、7.69±0.57比11.37±1.22，均P＜0.05〕。结论 AGM可显著抑制ZYM诱导急性腹膜炎小鼠趋化因子与炎性因子的分泌，并减轻腹腔炎性细胞的浸润，进而减轻腹腔炎症损伤。

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.