氧醚复合对脓毒症的治疗效应及其机制研究

发布时间：2018-02-03 03:31

本文关键词： 脓毒症氧醚复合 CLP LPS 炎性因子 Nrf2 HO-1 NF-κB mi RNA　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：脓毒症(sepsis)是外科重症监护病房(Intensive Care Unit,ICU)病人死亡的主要原因之一,由于高患病率、高死亡率、高治疗费用的特点,使其成为人类健康的巨大威胁。因此,寻找针对脓毒症的有效救治策略迫在眉睫!前期我们在离体和在体模型上均证实,0.5MAC(minimum alveolar concentration,最低肺泡有效浓度)异氟醚复合60%氧对脓毒症具有保护效应,抑制脓毒症后的异常炎症反应,抑制脂多糖刺激后NF-κB(Nuclear factor Kappa B,核因子-κB)核转位,逆转脓毒症后部分micro RNA(mi RNA,微小RNA)的上调。本研究拟在此基础上,研究一氧化氮(nitric oxide,NO)在氧醚复合治疗脓毒症效应机制中的作用,验证mi RNA是否参与氧醚复合治疗脓毒症效应机制,研究氧醚复合治疗脓毒症效应中对NF-κB信号通路的具体调控方式,研究Nrf2/HO-1(核因子相关因子-2/血红素氧合酶1)抗炎通路在氧醚复合治疗脓毒症效应机制中的作用,并探讨氧醚复合对盲肠结扎穿刺(cecal ligation and puncture,CLP)所致老龄鼠脓毒症模型是否也有治疗效应。相信本研究的实施将为氧醚复合治疗脓毒症提供更多的实验依据,也为研发新的脓毒症治疗策略提供新的可能靶点。研究目的探讨0.5MAC异氟醚复合60%氧对脓毒症治疗效应机制,并在老龄鼠上验证0.5MAC异氟醚复合60%氧及0.5MAC七氟醚复合60%氧对脓毒症的治疗效应。材料与方法实验1.将243只雄性昆明小鼠(25-30g)随机分为7组:Sham+NS+Air组、CLP+NS+Air组、CLP+NS+100%Oxy组、CLP+NS+0.5MAC ISO+60%Oxy组、CLP+L-NAME+100%Oxy组、CLP+L-NAME+0.5MAC ISO+60%Oxy组和CLP+L-NAME+Air组。CLP+NS+Air、CLP+NS+100%Oxy、CLP+NS+0.5MAC ISO+60%Oxy、CLP+L-NAME+100%Oxy、CLP+L-NAME+0.5MAC ISO+60%Oxy和CLP+L-NAME+Air组动物使用盲肠结扎穿刺(CLP)法建立脓毒症模型,Sham+NS+Air组动物只开腹,不实施盲肠结扎穿刺。治疗组动物在CLP或Sham术后1h、6h分别给予0.5MAC异氟醚复合60%氧或单纯100%氧吸入1h;非治疗组直接吸入空气。CLP或Sham前4h,给所有动物腹腔注射20mg/kg L-NAME或等容积的生理盐水。观察动物7天存活情况。检测指标:血清炎症因子水平;动脉血气分析;腹腔灌洗液细菌计数。实验2.在我们以往研究中,0.5MAC异氟醚复合60%氧干预可显著下调CLP后表达升高的4种mi RNAs,即mi R-133a-3p、mi R-141-3p、mi R-3074-2-3p、mi R-542-3p。本研究中,首先构建携带目的基因(mi R-133a-3p、mi R-141-3p、mi R-3074-2-3p、mi R-542-3p)的慢病毒载体,然后用慢病毒感染RAW264.7细胞或小鼠外周血白细胞,使之分别高表达这些mi RNA。治疗组细胞给予0.5MAC异氟醚复合60%氧治疗;非治疗组细胞正常培养。干预完成后收集细胞培养上清液检测炎症因子水平。实验3.培养小鼠巨噬细胞(RAW264.7),随机分为6组:Veh组、Veh+0.5ISO+60%O组、Veh+100%O组、LPS组、LPS+0.5ISO+60%O组和LPS+100%O组。LPS组使用脂多糖刺激细胞建立离体脓毒症模型;Veh组给予正常培养基。治疗组细胞在LPS刺激开始0.5小时后即开始给予0.5MAC异氟醚复合60%氧或单纯100%氧治疗;非治疗组细胞正常培养。LPS刺激2小时后刮取细胞,提取总蛋白,利用Western blot法检测p-IKKα/β、p-IκBα、p-p65蛋白表达。实验4.培养RAW264.7细胞,分组及处理同3。LPS刺激2小时后收集细胞培养上清液检测炎症因子改变;收集细胞沉淀物,利用细胞免疫荧光法检测Nrf2核定位情况,利用实时定量PCR和Western blot法检测细胞Nrf2、HO-1 m RNA或蛋白的表达情况。实验5.将228只雄性老龄SD大鼠(12-14月龄)及120只青年SD大鼠(2-3月龄)随机分为6组:Sham+Air组、Sham+0.5MAC ISO+60%Oxy组、Sham+0.5MAC SEV+60%Oxy组、CLP+Air组、CLP+0.5MACISO+60%Oxy组和CLP+0.5MAC SEV+60%Oxy组。CLP组动物采用盲肠结扎穿刺法建立脓毒症模型,Sham组不进行盲肠结扎穿刺术。治疗组动物于CLP或Sham术后1h、6h分别给予0.5MAC异氟醚复合60%氧气吸入1h,或在CLP或Sham术后6h给予动物0.5MAC七氟醚复合60%氧气吸入2h。非治疗组动物直接吸入空气,作为对照。观察动物7天存活情况。于老龄鼠CLP术后24h取材,包括取一侧肺,做HE染色后观察病理损伤情况;取另一侧肺,计算肺湿干比,评价肺水肿程度;进行肺泡灌洗,收集灌洗液后检测蛋白漏出;从右心室取血,离心后收集血清检测炎症因子、生化指标等;收集腹腔灌洗液,离心后收集上清,检测炎症因子;于股动脉处取血,立即送检进行血气分析。结果实验1.100%氧及0.5MAC异氟醚复合60%氧可显著提高脓毒症小鼠存活率,抑制CLP所致异常的炎症反应,改善氧合情况,并增强细菌清除能力。给予NO合成酶抑制剂后,上述治疗效应均被逆转。这些结果表明,NO参与了100%氧和0.5MAC异氟醚复合60%氧治疗脓毒症效应机制。实验2.用慢病毒感染RAW264.7细胞,高表达mi R-133a-3p、mi R-3074-2-3p、mi R-542-3p后,0.5MAC异氟醚复合60%氧抑制LPS所致异常炎性反应的效应被部分逆转。其次,用慢病毒感染CLP小鼠外周血白细胞,高表达mi R-133a-3p、mi R-3074-2-3p、mi R-542-3p后,0.5MAC异氟醚复合对CLP所致异常炎性反应的抑制效应也被部分逆转。这些结果提示,mi RNAs参与氧醚复合治疗脓毒症效应机制。实验3.LPS刺激可诱导细胞内p-IKKα/β、p-IκBα、p-p65蛋白表达水平增高,给予100%氧和0.5MAC异氟醚复合60%氧干预后,细胞p-IKKα/β、p-IκBα、p-p65蛋白表达水平明显下调。这些结果提示,100%氧和0.5MAC异氟醚复合60%氧是通过抑制IKKα/β、IκBα、p65蛋白磷酸化,从而抑制NF-κB激活,发挥其抗炎效应。实验4.LPS刺激RAW264.7细胞2h后,细胞培养上清液中炎性因子水平明显增高,细胞Nrf2及其靶基因HO-1的m RNA和蛋白表达水平降低。给予100%氧或0.5MAC异氟醚复合60%氧干预后,可抑制LPS刺激后炎性因子水平的增高,细胞Nrf2及HO-1的m RNA表达水平升高,细胞浆中Nrf2蛋白表达有降低趋势,而细胞核中Nrf2蛋白表达增高,入核明显增多;细胞总蛋白中HO-1的蛋白表达升高。这些结果表明,100%氧和0.5MAC异氟醚复合60%氧干预治疗离体脓毒症效应与Nrf2/HO-1抗炎通路密切相关。实验5.对于老龄SD大鼠及青年SD大鼠,CLP组后7天生存率均明显低于Sham组,而给予0.5MAC异氟醚或七氟醚复合60%氧干预后,动物7天存活率均显著提高。在老龄SD大鼠CLP后24h进行取材并发现,CLP组老龄鼠的肺组织出现明显病理性损伤,血清及腹腔灌洗液中炎症因子和生化指标水平均明显高于Sham组;给予0.5MAC异氟醚或七氟醚复合60%氧干预均可显著改善肺损伤,并减轻CLP后炎症因子、生化指标、动脉血气等各项指标的异常改变。这些结果说明,氧醚复合对老龄鼠脓毒症模型具有治疗效应。结论我们的研究结果表明:NO参与了氧醚复合治疗脓毒症效应机制;mi RNAs参与氧醚复合治疗脓毒症效应机制;氧醚复合是通过抑制IKKα/β、IκBα、p65蛋白磷酸化,抑制NF-κB激活,从而发挥其抗炎效应的;氧醚复合对离体脓毒症的治疗效应与Nrf2/HO-1通路关系密切;氧醚复合对老龄鼠脓毒症模型具有治疗效应。
[Abstract]:Sepsis (sepsis) is a surgical ICU (Intensive Care Unit, ICU) is one of the main causes of death of patients, due to the high prevalence rate, high mortality, high treatment costs, make it become a huge threat to human health. Therefore, in order to find the effective treatment of sepsis early we strategy imminent! In vitro and in vivo models have confirmed that 0.5MAC (minimum alveolar concentration, the minimum alveolar concentration of isoflurane and 60% oxygen) has a protective effect on sepsis, inhibit the abnormal inflammatory response after sepsis, inhibition of lipid polysaccharide stimulated NF- kappa B (Nuclear factor Kappa B, nuclear factor kappa B part RNA) nuclear translocation, micro reversed after sepsis (MI RNA, micro RNA) increase. This study based on the research of nitric oxide (nitric oxide, NO) in the treatment of sepsis oxygen ether compound effect mechanism plays a role in the verification of MI RNA is involved in Oxygen ether compound in the treatment of sepsis effect mechanism, the specific regulation of NF- B pathway of oxygen ether complex in the treatment of sepsis effect, Nrf2/HO-1 (nuclear factor related factor -2/ heme oxygenase 1) the effect of anti-inflammatory pathway in the treatment of sepsis oxygen ether compound effect mechanism, and explore the puncture oxygen ether complex (cecal ligation and of cecal ligation and puncture, CLP) is caused by the aging rat model of sepsis also have therapeutic effect. I believe that the implementation of this study will provide more experimental basis for oxygen ether compound in the treatment of sepsis, may also provide a new target for the development of a new strategy for the treatment of sepsis. Objective to research 0.5MAC isoflurane 60% oxygen for the treatment of sepsis effect mechanism, and verify the therapeutic effect of 0.5MAC isoflurane 60% oxygen and 0.5MAC sevoflurane 60% oxygen for sepsis in aged rats. The experimental materials and methods 1. 243 Male Kunming mice (25-30g) were randomly divided into 7 groups: Sham+NS+Air group, CLP+NS+Air group, CLP+NS+100%Oxy group, CLP+NS+0.5MAC ISO+60%Oxy group, CLP+L-NAME+100%Oxy group, CLP+L-NAME+0.5MAC group and ISO+60%Oxy group CLP+L-NAME+Air.CLP+NS+Air, CLP+NS+100%Oxy CLP+NS+0.5MAC, ISO +60%Oxy, CLP+L-NAME+100%Oxy CLP+L-NAME+0.5MAC, ISO+60%Oxy and CLP+L-NAME+Air group animal using cecal ligation and puncture (CLP) method to establish the model of sepsis. Sham+NS+Air group of animal only open, not the implementation of cecal ligation and puncture. The treatment group in the animal CLP or after Sham 1H and 6h 0.5MAC were given isoflurane 60% oxygen or pure oxygen inhalation 100% 1H non treatment group; direct inhalation of air.CLP or Sham before 4h, saline to all animal intraperitoneal injection of 20mg/kg L-NAME or equal volume 7 days. To observe animal survival. Measure the levels of serum inflammatory factor analysis; arterial blood gas; peritoneal lavage fluid Bacteria count. Experiment 2. in our previous study, 60% 0.5MAC isoflurane oxygen intervention expression of 4 mi RNAs increased significantly after MI R-133a-3p downregulation of CLP, MI, R-141-3p, MI, R-3074-2-3p, MI and R-542-3p. in this study, firstly, carrying the target gene (MI R-133a-3p, MI R-141-3p, MI R-3074-2-3p, MI R-542-3p) the lentiviral vector, and then use the peripheral white blood cells RAW264.7 cells or mice infected with lentivirus, which were high expression of these mi RNA. cells treated with 0.5MAC treatment group isoflurane 60% oxygen therapy; treatment of non normal cultured group cells. After intervention collected cell culture supernatant was detected. The level of inflammatory factors in experiment 3. cultured mouse macrophages (RAW264.7), were randomly divided into 6 groups: Veh group, Veh+0.5ISO+60%O group, Veh+100%O group, LPS group, established in vitro sepsis model in LPS+0.5ISO+60%O group and LPS+100%O group group.LPS cells stimulated by lipopolysaccharide Type; Veh group was given normal medium. The treatment group in LPS stimulated cells began to start giving 0.5MAC isoflurane 60% oxygen or pure oxygen treatment is 100% after 0.5 hours; the non treatment group were cultured for 2 hours after.LPS stimulation and scrape cells, the total protein extracted by Western blot method for detecting p-IKK alpha / beta, p-I kappa B alpha, p-p65 protein expression in cultured RAW264.7 cells. In experiment 4., grouping and treatment with 3.LPS stimulation after 2 hours cell culture supernatant was determined by collecting inflammatory change; collecting cells precipitate, detection of nuclear localization of Nrf2 using cell immunofluorescence, cell Nrf2 detection by real-time quantitative PCR and Western blot method, the expression of HO-1 m RNA or protein. In experiment 5., 228 old male SD rats (12-14 months old) and 120 young SD rats (2-3 months old) were randomly divided into 6 groups: Sham+Air group, Sham+0.5MAC ISO+60%Oxy group, Sham+0.5MAC SEV +60%Oxy group, CLP+Air group, C LP+0.5MACISO+60%Oxy group and CLP+0.5MAC group SEV+60%Oxy group.CLP animal model of sepsis was established by cecal ligation and puncture, cecal ligation and puncture without Sham group. Treatment group animal in CLP or Sham after 1h, 6h were given 0.5MAC 60% 1H isoflurane inhalation of oxygen, or as a control group. In the CLP or Sham after 6h to give the animal 0.5MAC sevoflurane 60% oxygen inhalation 2h. non treatment group animal directly into the air, to observe the animal survival. In 7 days old rats after CLP 24h were taken including lung pathological injury after HE staining; and on the other side of the lung, calculate the lung wet to dry ratio, evaluate the degree of pulmonary edema; alveolar lavage detection of protein leakage, collected lavage fluid; blood from the right ventricle, inflammatory factors in serum collected after centrifugation, biochemical indicator; collect peritoneal lavage fluid, after centrifugation the supernatant was collected for detection of inflammation in femoral artery; Blood inspection immediately for blood gas analysis. The results of experiment 1.100% and 60% oxygen oxygen 0.5MAC isoflurane can significantly improve the survival rate of mice with sepsis, inhibiting the inflammatory reaction caused by abnormal CLP, improve oxygenation, and enhance bacterial clearance ability. NO synthase inhibitor, the treatment effects were reversed. These results indicate that in 100%, NO 0.5MAC 60% isoflurane combined oxygen and oxygen in the treatment of sepsis effect. Experiment 2. using lentiviral infection of RAW264.7 cells with high expression of MI, R-133a-3p, MI, R-3074-2-3p, MI, R-542-3p, 0.5MAC effect of isoflurane 60% oxygen inhibits LPS induced abnormal inflammatory response was partially reversed. Secondly, with slow virus infection CLP mice peripheral white blood cells, the high expression of MI R-133a-3p, MI R-3074-2-3p, MI R-542-3p, the inhibitory effect of 0.5MAC isoflurane on CLP induced abnormal inflammatory response was also partially reversed these results. That MI RNAs is involved in oxygen ether complex in the treatment of sepsis. The effect mechanism of 3.LPS stimulation can induce intracellular p-IKK alpha / Beta Kappa B alpha, p-I, p-p65 protein expression level increased, giving 100% oxygen and 60% 0.5MAC isoflurane oxygen intervention, cell p-IKK alpha / beta, p-I kappa B alpha, p-p65 expression protein was down regulated. These results suggest that 100% 0.5MAC oxygen and isoflurane 60% oxygen through inhibition of IKK alpha / beta, I kappa B alpha, p65 protein phosphorylation, thereby inhibiting NF- kappa B activation, exert its anti-inflammatory effect. Experimental 4.LPS stimulation of RAW264.7 cells after 2H cell culture supernatant in inflammatory factor level obviously increased expression of M RNA and protein in Nrf2 cells and its target gene HO-1 decrease. Given 100% oxygen or 0.5MAC isoflurane 60% oxygen intervention can increase the level of inflammatory factors in the inhibition of LPS stimulation, increased the expression level of M Nrf2 and HO-1 RNA cells, the expression of Nrf2 protein in the cytoplasm Decreased, and the expression of Nrf2 protein in the nucleus increased into the nucleus increased significantly; the expression of HO-1 protein in total cell protein increased. These results indicated that 100% 0.5MAC oxygen and isoflurane 60% oxygen treatment in vitro sepsis effect and Nrf2/HO-1 anti-inflammatory pathway are closely related. In experiment 5. aged SD rats and young SD in group CLP, after the 7 day survival rate was significantly lower than that of group Sham, and given 0.5MAC of isoflurane or sevoflurane combined with 60% oxygen intervention, 7 animal survival rate was significantly improved. In aged SD rats after CLP 24h were collected and found that the CLP group in aged rat lung tissues appeared obvious pathological injury, serum and peritoneal lavage fluid in inflammatory cytokines and biochemical indexes were obviously higher than Sham group; 0.5MAC isoflurane or sevoflurane combined 60% oxygen intervention can significantly improve lung injury, and reduce inflammatory factors after CLP, arterial blood gas and other biochemical indicators. The abnormal changes of indexes. These results indicated that oxygen ether composite on the aging rat model of sepsis has a therapeutic effect. Conclusion our results suggest that NO is involved in the oxygen ether compound in the treatment of sepsis effect mechanism; MI RNAs in oxygen ether complex in the treatment of sepsis effect mechanism; oxygen ether composite by inhibiting IKK alpha / beta, I kappa B alpha, p65 phosphorylation and inhibition of NF- K B activation, so as to exert its anti-inflammatory effect; oxygen ether complex on the treatment effect and Nrf2/HO-1 pathway in vitro sepsis closely; oxygen ether compound on aged rat model of sepsis has a therapeutic effect.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R614

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