本文關(guān)鍵詞：胍丁胺對(duì)急性小鼠腹膜炎炎性損傷的保護(hù)效應(yīng)及其機(jī)制研究　出處：《重慶醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腹膜炎 胍丁胺 酵母多糖

【摘要】：目的:探索胍丁胺(AGM)對(duì)酵母多糖(ZYM)誘導(dǎo)急性小鼠腹膜炎炎性損傷的保護(hù)效應(yīng)及其相關(guān)機(jī)制。方法:1、將72只成年雄性C57BL/6小鼠隨機(jī)分為假手術(shù)組(18只,腹腔注入磷酸鹽緩沖液)、ZYM模型組(18只,腹腔注入1mg/ml的酵母多糖溶液0.5ml)、AGM干預(yù)組(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對(duì)照組(18只,腹腔注入胍丁胺溶液400mg/kg),各組分別于建模后2h、6h、24h處死6只小鼠,采集外周血、腹腔灌洗液、肝組織。在處死小鼠前定時(shí)察看小鼠精神活動(dòng)狀態(tài)。采用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)檢測(cè)血清、腹腔灌洗液及肝勻漿中的腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素-6(IL-6)、角化細(xì)胞來(lái)源趨化因子(KC)、巨噬細(xì)胞炎性蛋白-2(MIP-2)含量;血細(xì)胞計(jì)數(shù)板計(jì)數(shù)腹腔灌洗液中浸潤(rùn)炎性細(xì)胞總數(shù);流式細(xì)胞術(shù)分析腹腔浸潤(rùn)的PMN比例;全自動(dòng)生物化學(xué)分析儀檢測(cè)血清丙氨酸轉(zhuǎn)氨酶(ALT)、天門冬氨酸轉(zhuǎn)氨酶(AST)、肌酐(Crea)、尿素氮(Urea)濃度。2、分離小鼠骨髓PMN和腹腔原代巨噬細(xì)胞,分別培養(yǎng)于Transwell小室的上下室,ZYM刺激下室的腹腔巨噬細(xì)胞,在加或不加胍丁胺干預(yù)的情況下,在40分鐘后用dapi染色,倒置熒光顯微鏡下察看各組pmn趨化數(shù)目。3、分離并體外培養(yǎng)小鼠腹腔巨噬細(xì)胞,采用100μg/ml的酵母多糖溶液刺激,在agm治療的情況下,于不同時(shí)間點(diǎn)收集細(xì)胞蛋白及細(xì)胞培養(yǎng)上清。elisa檢測(cè)細(xì)胞上清中tnf-α、il-6、kc、mip-2蛋白的濃度,逆轉(zhuǎn)錄pcr(qpcr)檢測(cè)細(xì)胞中tnf-α、il-6、kc、mip-2mrna的表達(dá)量,免疫印跡法(westernblot)檢測(cè)胞漿胞核中inos、p65、p-p65的表達(dá)水平。此外,分別應(yīng)用n-甲基d-天冬氨酸受體(nmda-r)拮抗劑mk-801、α2腎上腺素能受體拮抗劑育亨賓(yhb)、咪唑啉1受體(i1-r)高親和力配基依法克生(efa)及咪唑啉2受體(i2-r)高親和力配基咪唑克生(ida)預(yù)作用巨噬細(xì)胞,有或無(wú)胍丁胺治療的條件下,檢測(cè)zym刺激12h后培養(yǎng)上清中il-6、kc的濃度。結(jié)果:1、在酵母多糖攻擊2h、6h、24h后zym模型組和agm治療組小鼠均表現(xiàn)出行動(dòng)緩慢,反應(yīng)遲鈍,蜷縮成團(tuán),腹瀉,飲食減少,且隨著時(shí)間推移癥狀愈發(fā)明顯,但agm干預(yù)組小鼠精神和活動(dòng)狀態(tài)明顯好于zym模型組。與對(duì)照組比較,agm治療可顯著降低zym刺激2h后小鼠血清中趨化因子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)的濃度和腹腔灌洗液中趨化因子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)的濃度;降低zym攻擊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)的濃度和腹腔灌洗液中炎癥因子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細(xì)胞總數(shù)(×106/ml:10.1±1.2比14.7±1.1,p0.05)和中性粒細(xì)胞比例(百分比%:77.83%比90.07%,p0.05);削弱zym攻擊24h后肝組織勻漿中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)、趨化因子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)的濃度。2、在體外transwell趨化實(shí)驗(yàn)中我們發(fā)現(xiàn),zym組巨噬細(xì)胞上清誘導(dǎo)的pmn趨化數(shù)目與對(duì)照組相比顯著增多,但zym+agm組巨噬細(xì)胞培養(yǎng)上清誘導(dǎo)的pmn遷移趨化數(shù)目與zym組相比極大減少。3、在體外小鼠腹腔原代巨噬細(xì)胞培養(yǎng)實(shí)驗(yàn)中,采用zym刺激原代巨噬細(xì)胞并經(jīng)agm治療發(fā)現(xiàn),細(xì)胞tnf-α、il-6、kc、mip-2的蛋白釋放量和mrna合成量均大大降低,且細(xì)胞中inos表達(dá)量減少,p65磷酸化和入核受到抑制。此外,zym作用小鼠原代腹腔巨噬細(xì)胞12h后,agm、mk-801、ida處理均能抑制巨噬細(xì)胞培養(yǎng)上清中il-6、kc的上升,其中mk-801抗炎效果弱于agm,agm與mk-801聯(lián)用有一定程度的聯(lián)合抗炎效果,而IDA的抗炎效果與AGM類似,且AGM與IDA聯(lián)用時(shí)聯(lián)合抗炎效果不明顯;YHB、EFA無(wú)抑制炎癥效果,并對(duì)AGM的抗炎效應(yīng)亦無(wú)影響。結(jié)論: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及趨化因子KC、MIP-2的生成,降低腹腔炎性細(xì)胞總數(shù)及抑制腹腔PMN的浸潤(rùn),表明AGM有著良好的全身與局部抗炎作用。2、AGM能降低ZYM誘導(dǎo)腹膜炎小鼠血清中ALT、AST、Urea、Crea的升高,減輕肝臟中炎癥因子和趨化因子表達(dá)水平,表明AGM對(duì)腹膜炎小鼠臟器功能損害有一定的保護(hù)作用。3、AGM能減少ZYM體外刺激下小鼠腹腔巨噬細(xì)胞TNF-α、IL-6、KC、MIP-2的蛋白釋放量和mRNA生成量,抑制iNOS的表達(dá),減弱p65的磷酸化與入核,且AGM的抗炎效應(yīng)與IDA相似,與MK-801的抗炎效果差異明顯,與YHB、EFA無(wú)關(guān),表明AGM能通過(guò)抑制巨噬細(xì)胞iNOS表達(dá),NF-κB信號(hào)通路活化,以及激活咪唑啉2受體等途徑發(fā)揮抗炎作用。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R572.2

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