甲烷对大鼠一氧化碳中毒脑损伤保护作用及机制研究

发布时间：2018-05-15 23:07

本文选题：甲烷 + 一氧化碳中毒　；参考：《第二军医大学》2016年博士论文

【摘要】：一氧化碳(carbon monoxide,CO)是一种无色、无味、无刺激性的有毒气体,分子量为28,主要来源于含碳化合物的不完全燃烧。在大气中,正常的CO浓度不超过0.001%,而0.1%浓度的CO足以致人死亡。目前,CO是世界上最为常见的人类中毒原因。CO中毒症状具有非特异性,轻度CO中毒可表现为头痛、肌痛、头晕、恶心、或神经心理障碍,而严重的CO中毒则可导致意识模糊,昏迷甚至死亡。在CO暴露后3天-4周,超过一半的严重中毒患者会出现迟发性神经精神症状,轻微的症状包括人格的改变,轻度认知功能损害,严重的症状包括严重痴呆,精神错乱,帕金森症,尿失禁或其他异常症状,这些损害被称作迟发性神经精神后遗症(delayed neuropsychological sequelae,DNS)。约有50-70%的DNS患者在一年后部分或完全恢复正常。CO中毒后脑损伤,特别是DNS,给社会和家庭带来严重的经济负担。CO与血红蛋白具有强大的结合能力,是氧气的200倍。CO通过呼吸进入肺泡,扩散至循环系统,与血红蛋白迅速结合,形成碳氧血红蛋白(carboxyhaemoglobin,COHb)。COHb的形成,导致血红蛋白携氧能力的下降,引起低氧血症。CO导致的低氧是脑损伤的关键因素之一,COHb水平高低是判断CO中毒严重程度的重要指标之一。然而,数十年来,临床研究发现,COHb的水平高低与患者症状/体征或最终的预后并非完全一致。研究表明,CO中毒部分病理生理效应与缺血再灌注损伤相似,即在缺氧状态后通常伴随再氧合过程。CO导致的脑组织损伤中,黄嘌呤脱氢酶转换为黄嘌呤氧化酶,催化黄嘌呤氧化产生的活性氧(reactive oxygen species,ROS)导致神经细胞膜的脂质过氧化作用。在CO暴露脑组织缺氧及再氧合状态过程中,都存在着大量的羟基自由基。同时,CO中毒大鼠脑组织中,硝基酪氨酸水平增加10倍,进一步证实在CO中毒过程中,一氧化氮(nitric oxide,NO)和超氧化物大量产生,生成过氧硝酸盐。综上所述,CO暴露导致的脑损伤是自由基的级联反应的结果,即抗氧化系统和氧化应激损伤之间平衡的破坏。应用抗氧化剂是治疗CO中毒后脑损伤的重要策略之一。一些研究小组已经通过应用ROS清除剂作为治疗手段,如氢气,硫化氢,依达拉奉等,得到满意治疗效果。甲烷是最简单的有机化合物和主要的天然气能源。过去认为人类机体不能利用甲烷。近年来,研究表明,甲烷释放可能与机体氧化还原反应调控以及低氧导致的线粒体损伤有关。在肠系膜、肝脏、心肌等缺血再灌注损伤模型以及糖尿病视网膜病变模型研究中发现,甲烷具有显著的保护作用,其机制可能通过抗氧化、抗炎症反应及抗凋亡等作用。甲烷对于氧化应激损伤具有潜在的保护作用。在本研究中,我们推测,甲烷能够有效保护CO中毒后脑损伤,改善脑损伤预后;同时,对于甲烷的保护机制进行了初步研究。第一部分,甲烷对于CO中毒后脑损伤保护效应的研究。我们通过静态吸入CO制备CO中毒大鼠模型,应用甲烷溶液进行处理,通过水迷宫等行为学方法观察大鼠学习记忆能力的变化;通过组织病理学方法,观察模型大鼠大脑海马和皮层神经元损伤情况。研究结果显示,从行为学观察,甲烷能够有效提高CO暴露大鼠的学习和记忆能力;从病理学观察,在CO暴露后,甲烷处理组较单纯CO暴露后大鼠神经元形态相对完整,Nissl小体保存相对完整。第二部分至第四部分,我们分别研究了CO中毒后24小时和第9天,甲烷的抗氧化、抗炎及抗凋亡保护机制。应用试剂盒分别检测CO中毒后24小时和第9天大鼠海马和皮层超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的含量变化,应用酶联免疫法分别检测CO中毒后24小时和第9天大鼠海马和皮层3-硝基酪氨酸(3-nitrotyrosine,3-NT)和8-羟基-2'-脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHd G)的含量变化。应用酶联免疫法检测CO中毒后24小时和第9天大鼠海马和皮层炎症因子水平的变化,肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α),白介素-1β(interleukin-1β,IL-1β)和白介素-6(IL-6)。凋亡指标采用TUNEL(Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling)和蛋白质印迹法(Western Blotting)方法。结果显示,甲烷能够显著提高CO中毒后24小时和第9天大鼠海马和皮层SOD活性,降低海马和皮层MDA水平和海马的3-NT和8-OHd G水平。同时,甲烷能够显著抑制CO中毒后24小时和第9天模型大鼠海马和皮层TNF-α和IL-1β等炎症指标水平。在CO暴露后第9天,甲烷显著能够减少大鼠海马和皮层凋亡细胞数量以及Caspase-3表达水平。第五部分,我们分别采用Western Blotting和实时定量聚合酶链式反应(real time-polymerase chain reaction,RT-PCR)检测了CO暴露后第9天,大鼠脑组织海马和皮层核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf-2)及其下游蛋白过氧化氢酶(catalase,CAT)的蛋白和m RNA表达。结果显示,在CO中毒后第9天,Nrf-2和下游蛋白CAT的表达受到抑制,而甲烷能够提高其表达水平,增强机体抗氧化能力。上述结果可以得出以下结论:1.甲烷能够有效保护CO暴露导致的脑损伤,改善中毒预后。2.与大脑皮层组织相比,CO中毒对于海马组织损伤更为显著。3.甲烷可能通过抗氧化作用,抗炎症作用及抗凋亡作用等机制产生保护作用。4.CO中毒导致的持续存在的氧化应激能够抑制Nrf-2和下游蛋白CAT的表达,甲烷能够解除其抑制作用。
[Abstract]:Carbon monoxide (carbon monoxide, CO) is a colorless, odorless, irritant toxic gas with a molecular weight of 28, mainly from incomplete combustion of carbon containing compounds. In the atmosphere, the normal CO concentration is not more than 0.001%, and the 0.1% concentration of CO is sufficient to cause death. At present, CO is the most common cause of human poisoning in the world, the symptoms of.CO poisoning. Nonspecific, mild CO poisoning may be characterized by headache, myalgia, dizziness, nausea, or neuropsychological disorder, while severe CO poisoning can lead to blurred consciousness, coma and even death. 3 days after exposure to CO, more than half of the severe poisoning patients may have delayed psychosis symptoms, mild symptoms including personality changes, and mild recognition. Cognitive impairment, severe symptoms including severe dementia, mental disorder, Parkinson's, urinary incontinence, or other abnormal symptoms, which are called delayed neuropsychological sequelae (DNS). About 50-70% of DNS patients are divided or fully restored to normal.CO poisoning after one year of DNS, especially DNS, A serious economic burden for society and family.CO has a strong binding capacity with hemoglobin. It is 200 times as much oxygen as.CO to enter the alveoli through breathing, spread to the circulatory system, and quickly combine with hemoglobin to form the formation of carboxyhaemoglobin, COHb.COHb, leading to a decline in the oxygen carrying capacity of hemoglobin. Hypoxemia.CO induced hypoxia is one of the key factors for brain damage, and the level of COHb is one of the important indicators to judge the severity of CO poisoning. However, clinical studies have found that the level of COHb is not completely consistent with the symptoms / signs or final prognosis of the patients. The study shows that the partial pathophysiological effects and deficiency of CO poisoning The blood reperfusion injury is similar, that is, in the brain tissue damage that is usually associated with the reoxygenation process after hypoxia, xanthine dehydrogenase is converted to xanthine oxidase, and the active oxygen (reactive oxygen species, ROS) produced by xanthine oxidation (ROS) leads to lipid peroxidation in the nerve cell membrane. Hypoxia and reoxygenation in the brain tissue are exposed in CO. There are a large number of hydroxyl radicals in the process of occlusion. At the same time, the level of nitro tyrosine in the brain tissue of CO poisoned rats increases by 10 times. It is further confirmed that in the process of CO poisoning, the production of nitric oxide (nitric oxide, NO) and superoxide produced peroxynitrite. To sum up, the brain damage caused by CO exposure is a cascade of free radicals. The result of the reaction is the balance between the antioxidant system and oxidative stress damage. The application of antioxidants is one of the most important strategies for the treatment of brain damage after CO poisoning. Some research teams have obtained satisfactory therapeutic effects by using ROS scavengers as treatments, such as hydrogen, hydrogen sulfide, and edaravone. Methane is the simplest Organic compounds and major natural gas energy sources. In the past, methane was not used by the human body. In recent years, studies have shown that methane release may be related to the regulation of redox reaction and mitochondrial damage caused by hypoxia. Models of ischemia-reperfusion injury in mesentery, liver, myocardium and diabetic retinopathy model study It has been found that methane has a significant protective effect, and its mechanisms may be mediated by antioxidant, anti inflammatory and anti apoptosis. Methane has a potential protective effect on oxidative stress damage. In this study, we speculate that methane can effectively protect the brain injury after CO poisoning and improve the prognosis of brain damage; at the same time, methane protection machine is used. The first part is the preliminary study. Part 1, the study of the protective effect of methane on brain injury after CO poisoning. We use the static inhalation of CO to prepare the rat model of CO poisoning, use the methane solution and observe the changes of the learning and memory ability of the rats by the water maze, and observe the model rats by histopathological method. The results showed that methane can effectively improve the learning and memory ability of CO exposed rats from behavioral observation. From pathological observation, after exposure to CO, the neuronal morphology of the methane treatment group was relatively complete and the Nissl corpuscle was relatively intact. Second to fourth parts were preserved in the methane treatment group. We studied the antioxidant, anti-inflammatory and anti apoptotic mechanisms of methane at 24 hours and ninth days after CO poisoning. The changes in the content of superoxide dismutase (SOD) and malondialdehyde (malondialdehyde, MDA) in hippocampus and cortex of rats after CO poisoning were detected by the reagent kit, and the enzyme linked immunosorbent assay was applied. The changes of 3- nitrotyrosine (3-nitrotyrosine, 3-NT) and 8- hydroxyl -2'- deoxy guanosine (8-hydroxy-2-deoxyguanosine, 8-OHd G) in the hippocampus and cortex of rats after CO poisoning were detected respectively. The changes in the levels of inflammatory factors in hippocampus and cortex in 24 and ninth days after CO poisoning were detected by ELISA, and the tumor necrosis was detected. Factor alpha (tumour necrosis factor- alpha, TNF- alpha), interleukins -1 beta (interleukin-1 beta, IL-1 beta) and interleukins -6 (IL-6). The apoptotic indexes are TUNEL (Terminal deoxynucleotidyl) and Western blotting. The results show that methane can significantly increase the 24 small amounts after poisoning. The hippocampal and cortical SOD activity of rats in the time and ninth days decreased the level of hippocampal and cortex MDA and the level of 3-NT and 8-OHd G in hippocampus, and methane could significantly inhibit the levels of TNF- A and IL-1 beta in hippocampus and cortex of rat model of CO poisoning after CO poisoning. Ninth days after CO exposure, methane could significantly reduce the hippocampus and cortex of rats. The number of apoptotic cells and the expression level of Caspase-3. Fifth, we used Western Blotting and real-time quantitative polymerase chain reaction (real time-polymerase chain reaction, RT-PCR) to detect the ninth days after CO exposure, and the hippocampus and cortical nuclear factor E2 phase factor 2 (nuclear factor 2). Nrf-2) and its downstream protein catalase (catalase, CAT) protein and m RNA expression. The results showed that ninth days after CO poisoning, the expression of Nrf-2 and downstream protein CAT was inhibited, and methane could improve its expression level and enhance the body's antioxidant capacity. The results can be concluded as follows: 1. methane can effectively protect the CO exposure guide. Induced brain damage, improving the prognosis of.2. and cerebral cortex, CO poisoning is more significant for hippocampal tissue damage,.3. methane may be induced by oxidative stress, anti-inflammatory and anti apoptosis, and the persistent oxidative stress caused by.4.CO poisoning can inhibit the expression of Nrf-2 and downstream protein CAT. Methane can relieve its inhibition.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R595.1;R747.9

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