胍丁胺对急性小鼠腹膜炎炎性损伤的保护效应及其机制研究

发布时间：2018-01-10 04:08

本文关键词：胍丁胺对急性小鼠腹膜炎炎性损伤的保护效应及其机制研究　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探索胍丁胺(AGM)对酵母多糖(ZYM)诱导急性小鼠腹膜炎炎性损伤的保护效应及其相关机制。方法:1、将72只成年雄性C57BL/6小鼠随机分为假手术组(18只,腹腔注入磷酸盐缓冲液)、ZYM模型组(18只,腹腔注入1mg/ml的酵母多糖溶液0.5ml)、AGM干预组(18只,腹腔注入1mg/ml的酵母多糖溶液0.5ml和胍丁胺溶液400mg/kg)和AGM对照组(18只,腹腔注入胍丁胺溶液400mg/kg),各组分别于建模后2h、6h、24h处死6只小鼠,采集外周血、腹腔灌洗液、肝组织。在处死小鼠前定时察看小鼠精神活动状态。采用酶联免疫吸附实验(ELISA)检测血清、腹腔灌洗液及肝匀浆中的肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、角化细胞来源趋化因子(KC)、巨噬细胞炎性蛋白-2(MIP-2)含量;血细胞计数板计数腹腔灌洗液中浸润炎性细胞总数;流式细胞术分析腹腔浸润的PMN比例;全自动生物化学分析仪检测血清丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)、肌酐(Crea)、尿素氮(Urea)浓度。2、分离小鼠骨髓PMN和腹腔原代巨噬细胞,分别培养于Transwell小室的上下室,ZYM刺激下室的腹腔巨噬细胞,在加或不加胍丁胺干预的情况下,在40分钟后用dapi染色,倒置荧光显微镜下察看各组pmn趋化数目。3、分离并体外培养小鼠腹腔巨噬细胞,采用100μg/ml的酵母多糖溶液刺激,在agm治疗的情况下,于不同时间点收集细胞蛋白及细胞培养上清。elisa检测细胞上清中tnf-α、il-6、kc、mip-2蛋白的浓度,逆转录pcr(qpcr)检测细胞中tnf-α、il-6、kc、mip-2mrna的表达量,免疫印迹法(westernblot)检测胞浆胞核中inos、p65、p-p65的表达水平。此外,分别应用n-甲基d-天冬氨酸受体(nmda-r)拮抗剂mk-801、α2肾上腺素能受体拮抗剂育亨宾(yhb)、咪唑啉1受体(i1-r)高亲和力配基依法克生(efa)及咪唑啉2受体(i2-r)高亲和力配基咪唑克生(ida)预作用巨噬细胞,有或无胍丁胺治疗的条件下,检测zym刺激12h后培养上清中il-6、kc的浓度。结果:1、在酵母多糖攻击2h、6h、24h后zym模型组和agm治疗组小鼠均表现出行动缓慢,反应迟钝,蜷缩成团,腹泻,饮食减少,且随着时间推移症状愈发明显,但agm干预组小鼠精神和活动状态明显好于zym模型组。与对照组比较,agm治疗可显著降低zym刺激2h后小鼠血清中趋化因子kc(pg/ml:1578.8±107.3比2077.4±196.3,p0.05)、mip-2(pg/ml:743.5±77.9比937.6±89.6,p0.05)的浓度和腹腔灌洗液中趋化因子kc(pg/ml:6064.3±577.4比9864.7±851.8,p0.05)、mip-2(pg/ml:1763.4±125.7比2369.7±304.5,p0.05)的浓度;降低zym攻击6h后小鼠血清中tnf-α(pg/ml:513.7±38.5比822.1±47.8,p0.05)、il-6(pg/ml:945.4±107.9比1326.4±178.5,p0.05)的浓度和腹腔灌洗液中炎症因子tnf-α(pg/ml:1661.7±185.4比2812.5±216.4,p0.05)、il-6(pg/ml:11694.1±1503.2比21170.7±3872.4,p0.05)的含量以及腹腔灌洗液中白细胞总数(×106/ml:10.1±1.2比14.7±1.1,p0.05)和中性粒细胞比例(百分比%:77.83%比90.07%,p0.05);削弱zym攻击24h后肝组织匀浆中tnf-α(ng/g:281.6±20.8比358.5±25.3,p0.05)和il-6(ng/g:197.4±22.7比273.5±26.7,p0.05)、趋化因子kc(ng/g:47.2±11.9比77.4±20.3,p0.05)和mip-2(ng/g:67.4±14.6比103.7±19.2,p0.05)的升高(均p0.05);降低zym刺激24h后血清中alt(u/l:392.6±41.4比712.3±55.5,p0.05)、ast(u/l:494.7±34.3比681.7±38.6,p0.05)、urea(mmol/l:31.3±1.8比46.8±3.9,p0.05)、crea(μmol/l:32.5±1.9比38.2±2.7,p0.05)的浓度。2、在体外transwell趋化实验中我们发现,zym组巨噬细胞上清诱导的pmn趋化数目与对照组相比显著增多,但zym+agm组巨噬细胞培养上清诱导的pmn迁移趋化数目与zym组相比极大减少。3、在体外小鼠腹腔原代巨噬细胞培养实验中,采用zym刺激原代巨噬细胞并经agm治疗发现,细胞tnf-α、il-6、kc、mip-2的蛋白释放量和mrna合成量均大大降低,且细胞中inos表达量减少,p65磷酸化和入核受到抑制。此外,zym作用小鼠原代腹腔巨噬细胞12h后,agm、mk-801、ida处理均能抑制巨噬细胞培养上清中il-6、kc的上升,其中mk-801抗炎效果弱于agm,agm与mk-801联用有一定程度的联合抗炎效果,而IDA的抗炎效果与AGM类似,且AGM与IDA联用时联合抗炎效果不明显;YHB、EFA无抑制炎症效果,并对AGM的抗炎效应亦无影响。结论:1、AGM能降低ZYM引起的腹膜炎小鼠血清和腹腔灌洗液中TNF-α、IL-6及趋化因子KC、MIP-2的生成,降低腹腔炎性细胞总数及抑制腹腔PMN的浸润,表明AGM有着良好的全身与局部抗炎作用。2、AGM能降低ZYM诱导腹膜炎小鼠血清中ALT、AST、Urea、Crea的升高,减轻肝脏中炎症因子和趋化因子表达水平,表明AGM对腹膜炎小鼠脏器功能损害有一定的保护作用。3、AGM能减少ZYM体外刺激下小鼠腹腔巨噬细胞TNF-α、IL-6、KC、MIP-2的蛋白释放量和mRNA生成量,抑制iNOS的表达,减弱p65的磷酸化与入核,且AGM的抗炎效应与IDA相似,与MK-801的抗炎效果差异明显,与YHB、EFA无关,表明AGM能通过抑制巨噬细胞iNOS表达,NF-κB信号通路活化,以及激活咪唑啉2受体等途径发挥抗炎作用。
[Abstract]:Objective: To explore the effects of agmatine (AGM) on yeast polysaccharide (ZYM) protective effect induced by acute peritonitis of mice inflammatory injury and its mechanism. Methods: 1, 72 adult male C57BL/6 mice were randomly divided into sham operation group (18 rats, intraperitoneal injection of phosphate buffer), ZYM model group (18 rats intraperitoneal injection of 1mg/ml, the yeast polysaccharide solution 0.5ml), AGM group (18 rats, intraperitoneal injection of 1mg/ml yeast polysaccharide solution 0.5ml and agmatine solution 400mg/kg) and AGM control group (18 rats, intraperitoneal injection of agmatine solution, 400mg/kg) were determined at 2h after modeling, 6h 24h, killed 6 mice collection of peripheral blood, liver tissue, peritoneal lavage fluid, the mice were killed. Before the timing examine the state of mental activity in mice. Using enzyme-linked immunosorbent assay (ELISA) detection of serum tumor necrosis factor - peritoneal lavage fluid and liver homogenate in alpha (TNF- alpha), interleukin -6 (IL-6), keratosis cell chemotaxis Factor (KC), macrophage inflammatory protein -2 (MIP-2) content; the total number of inflammatory cells infiltrated blood cell count in peritoneal lavage fluid; peritoneal infiltration ratio of PMN analysis by flow cytometry; serum ALT detection automatic chemical analyzer (ALT days), aspartate aminotransferase (AST), creatinine (Crea), urea nitrogen (Urea) concentration of.2, the primary isolation of bone marrow PMN and peritoneal macrophages were cultured in Transwell cells on the lower chamber, the lower chamber of the ZYM stimulated peritoneal macrophages, with or without agmatine in the intervention condition, stained by DAPI in 40 minutes, the inverted fluorescence microscope look over the group of PMN chemokine number.3, isolated and mouse peritoneal macrophages in vitro, polysaccharide solution stimulation by 100 g/ml yeast, in the case of AGM treatment at different time points, cells were collected and protein in cell culture supernatant of.Elisa were detected in the supernatant IL-6, KC, tnf- alpha, MIP-2 protein concentration, reverse transcription PCR (qPCR) were detected by tnf- alpha, IL-6, KC, the expression of mip-2mrna, immunoblotting (Westernblot) detection of intracellular iNOS in the nuclei of p65, and the expression level of p-p65. In addition, respectively using n- methyl d- aspartate receptor (NMDA-R) antagonist MK-801, alpha 2 adrenoceptor antagonist with Henbin (YHB), imidazoline 1 receptor (i1-r) with high affinity ligand efaroxan (EFA) and imidazoline 2 receptor (i2-r) high affinity ligand idazoxan (IDA) pretreatment of macrophages, with or without agmatine treatment conditions the culture supernatant of IL-6, detection of zym after 12h stimulation, the concentration of KC. Results: 1, the yeast polysaccharide attack 2h, 6h, 24h, zym model group and AGM treated mice showed a slow, slow reaction, curls up, diarrhea, eating less, and with time the symptoms become more obvious. But the AGM intervention group and spirit The active state is significantly better than the zym model group. Compared with the control group, AGM treatment can significantly reduce the chemokine KC zym after 2H stimulation in mouse serum (pg/ml:1578.8 + 107.3 to 2077.4 + 196.3, P0.05), MIP-2 (pg/ml:743.5 + 77.9 to 937.6 + 89.6, P0.05) chemokine KC and the concentration of peritoneal lavage fluid in (pg/ml:6064.3 + 577.4 to 9864.7 + 851.8, P0.05), MIP-2 (pg/ml:1763.4 + 125.7 to 2369.7 + 304.5, P0.05) concentration; reduce the zym attack after 6h tnf- in mice serum alpha (pg/ml:513.7 + 38.5 to 822.1 + 47.8, P0.05), IL-6 (pg/ml:945.4 + 107.9 to 1326.4 + 178.5, P0.05) inflammatory factors tnf- alpha concentration and peritoneal lavage fluid (pg/ml:1661.7 + 185.4 to 2812.5 + 216.4, P0.05), IL-6 (pg/ml:11694.1 + 1503.2 to 21170.7 + 3872.4, P0.05) and the content of the total number of white blood cells in peritoneal lavage fluids (106/ml:10.1 * + 1.2 14.7 + 1.1, P0.05) and neutrophil percentage (%: 77.83% vs 90.07%, P0.05); zym 24h tnf- after the attack weakened in liver homogenate alpha (ng/g:281.6 + 20.8 to 358.5 + 25.3, P0.05) and IL-6 (ng/g:197.4 + 22.7 to 273.5 + 26.7, P0.05), chemokine KC (ng/g:47.2 + 11.9 to 77.4 + 20.3, P0.05 + 14.6 and MIP-2 (ng/g:67.4) 103.7 + 19.2, P0.05) increased (P0.05); serum zym decreased after 24h stimulation in ALT (u/l:392.6 + 41.4 to 712.3 + 55.5, P0.05), AST (u/l:494.7 + 34.3 to 681.7 + 38.6, P0.05), urea (mmol/l:31.3 + 1.8 to 46.8 + 3.9, P0.05, crea (mol/l:32.5) 38.2 + 1.9 + 2.7 P0.05), the concentration of.2, Transwell in vitro chemotaxis experiment we found that zym group macrophage supernatant induced PMN chemotaxis number increased significantly compared with the control group, but zym+agm group of macrophage supernatant induced PMN migration number compared with the zym group greatly reduced.3 in vitro primary mouse peritoneal macrophage culture experiment Primary macrophages stimulated by zym, and found that tnf- cells treated by AGM, IL-6, KC, alpha, MIP-2 protein release and mRNA synthesis were greatly reduced, and the cells in the iNOS expression decreased, p65 phosphorylation and nuclear translocation was inhibited. In addition, the role of zym in mouse primary peritoneal macrophages after 12h AGM, MK-801, IDA treatment can inhibit the macrophage in culture supernatant of IL-6, the increase of KC, the anti-inflammatory effect of MK-801 was weaker than that of AGM, AGM and MK-801 combined with combined anti-inflammatory effect to a certain extent, while IDA's anti-inflammatory effect similar to AGM, AGM and IDA with combined anti-inflammatory effect is not obvious; YHB no, inhibit the inflammatory effect of AGM and EFA, the anti-inflammatory effects have no effects. Conclusion: 1. AGM can reduce TNF- alpha ZYM induced peritonitis in mice serum and peritoneal lavage fluid, IL-6 and chemokine KC, MIP-2 generation, reduce the number of inflammatory cells and peritoneal invasion inhibition of abdominal PMN, showed that A GM has a good local and systemic anti-inflammatory effects of.2, AGM can reduce ZYM induced peritonitis in mice serum ALT, AST, Urea, Crea increased, reduce the inflammatory factor in liver and chemokine expression levels, the protective effect of.3 AGM on mice showed that the peritonitis of organ damage, AGM can reduce ZYM in vitro stimulation mouse peritoneal macrophage of TNF- alpha, IL-6, KC, and mRNA generation release of MIP-2 protein, inhibiting the expression of iNOS decreased the phosphorylation of p65 and nuclear, anti inflammation and the effect of AGM is similar to IDA, the difference and the anti-inflammatory effect of MK-801 obviously, and YHB, not EFA, show that AGM can inhibit macrophages the expression of iNOS, NF- and B signaling pathway activation and activation of imidazoline receptors play 2 way anti-inflammatory effect.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R572.2

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