本文選題：甲烷 + 一氧化碳中毒　； 參考：《第二軍醫(yī)大學》2016年博士論文

【摘要】：一氧化碳(carbon monoxide,CO)是一種無色、無味、無刺激性的有毒氣體,分子量為28,主要來源于含碳化合物的不完全燃燒。在大氣中,正常的CO濃度不超過0.001%,而0.1%濃度的CO足以致人死亡。目前,CO是世界上最為常見的人類中毒原因。CO中毒癥狀具有非特異性,輕度CO中毒可表現(xiàn)為頭痛、肌痛、頭暈、惡心、或神經心理障礙,而嚴重的CO中毒則可導致意識模糊,昏迷甚至死亡。在CO暴露后3天-4周,超過一半的嚴重中毒患者會出現(xiàn)遲發(fā)性神經精神癥狀,輕微的癥狀包括人格的改變,輕度認知功能損害,嚴重的癥狀包括嚴重癡呆,精神錯亂,帕金森癥,尿失禁或其他異常癥狀,這些損害被稱作遲發(fā)性神經精神后遺癥(delayed neuropsychological sequelae,DNS)。約有50-70%的DNS患者在一年后部分或完全恢復正常。CO中毒后腦損傷,特別是DNS,給社會和家庭帶來嚴重的經濟負擔。CO與血紅蛋白具有強大的結合能力,是氧氣的200倍。CO通過呼吸進入肺泡,擴散至循環(huán)系統(tǒng),與血紅蛋白迅速結合,形成碳氧血紅蛋白(carboxyhaemoglobin,COHb)。COHb的形成,導致血紅蛋白攜氧能力的下降,引起低氧血癥。CO導致的低氧是腦損傷的關鍵因素之一,COHb水平高低是判斷CO中毒嚴重程度的重要指標之一。然而,數(shù)十年來,臨床研究發(fā)現(xiàn),COHb的水平高低與患者癥狀/體征或最終的預后并非完全一致。研究表明,CO中毒部分病理生理效應與缺血再灌注損傷相似,即在缺氧狀態(tài)后通常伴隨再氧合過程。CO導致的腦組織損傷中,黃嘌呤脫氫酶轉換為黃嘌呤氧化酶,催化黃嘌呤氧化產生的活性氧(reactive oxygen species,ROS)導致神經細胞膜的脂質過氧化作用。在CO暴露腦組織缺氧及再氧合狀態(tài)過程中,都存在著大量的羥基自由基。同時,CO中毒大鼠腦組織中,硝基酪氨酸水平增加10倍,進一步證實在CO中毒過程中,一氧化氮(nitric oxide,NO)和超氧化物大量產生,生成過氧硝酸鹽。綜上所述,CO暴露導致的腦損傷是自由基的級聯(lián)反應的結果,即抗氧化系統(tǒng)和氧化應激損傷之間平衡的破壞。應用抗氧化劑是治療CO中毒后腦損傷的重要策略之一。一些研究小組已經通過應用ROS清除劑作為治療手段,如氫氣,硫化氫,依達拉奉等,得到滿意治療效果。甲烷是最簡單的有機化合物和主要的天然氣能源。過去認為人類機體不能利用甲烷。近年來,研究表明,甲烷釋放可能與機體氧化還原反應調控以及低氧導致的線粒體損傷有關。在腸系膜、肝臟、心肌等缺血再灌注損傷模型以及糖尿病視網膜病變模型研究中發(fā)現(xiàn),甲烷具有顯著的保護作用,其機制可能通過抗氧化、抗炎癥反應及抗凋亡等作用。甲烷對于氧化應激損傷具有潛在的保護作用。在本研究中,我們推測,甲烷能夠有效保護CO中毒后腦損傷,改善腦損傷預后;同時,對于甲烷的保護機制進行了初步研究。第一部分,甲烷對于CO中毒后腦損傷保護效應的研究。我們通過靜態(tài)吸入CO制備CO中毒大鼠模型,應用甲烷溶液進行處理,通過水迷宮等行為學方法觀察大鼠學習記憶能力的變化;通過組織病理學方法,觀察模型大鼠大腦海馬和皮層神經元損傷情況。研究結果顯示,從行為學觀察,甲烷能夠有效提高CO暴露大鼠的學習和記憶能力;從病理學觀察,在CO暴露后,甲烷處理組較單純CO暴露后大鼠神經元形態(tài)相對完整,Nissl小體保存相對完整。第二部分至第四部分,我們分別研究了CO中毒后24小時和第9天,甲烷的抗氧化、抗炎及抗凋亡保護機制。應用試劑盒分別檢測CO中毒后24小時和第9天大鼠海馬和皮層超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的含量變化,應用酶聯(lián)免疫法分別檢測CO中毒后24小時和第9天大鼠海馬和皮層3-硝基酪氨酸(3-nitrotyrosine,3-NT)和8-羥基-2'-脫氧鳥苷(8-hydroxy-2-deoxyguanosine,8-OHd G)的含量變化。應用酶聯(lián)免疫法檢測CO中毒后24小時和第9天大鼠海馬和皮層炎癥因子水平的變化,腫瘤壞死因子-α(tumour necrosis factor-α,TNF-α),白介素-1β(interleukin-1β,IL-1β)和白介素-6(IL-6)。凋亡指標采用TUNEL(Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling)和蛋白質印跡法(Western Blotting)方法。結果顯示,甲烷能夠顯著提高CO中毒后24小時和第9天大鼠海馬和皮層SOD活性,降低海馬和皮層MDA水平和海馬的3-NT和8-OHd G水平。同時,甲烷能夠顯著抑制CO中毒后24小時和第9天模型大鼠海馬和皮層TNF-α和IL-1β等炎癥指標水平。在CO暴露后第9天,甲烷顯著能夠減少大鼠海馬和皮層凋亡細胞數(shù)量以及Caspase-3表達水平。第五部分,我們分別采用Western Blotting和實時定量聚合酶鏈式反應(real time-polymerase chain reaction,RT-PCR)檢測了CO暴露后第9天,大鼠腦組織海馬和皮層核因子E2相關因子2(nuclear factor erythroid-2 related factor 2,Nrf-2)及其下游蛋白過氧化氫酶(catalase,CAT)的蛋白和m RNA表達。結果顯示,在CO中毒后第9天,Nrf-2和下游蛋白CAT的表達受到抑制,而甲烷能夠提高其表達水平,增強機體抗氧化能力。上述結果可以得出以下結論:1.甲烷能夠有效保護CO暴露導致的腦損傷,改善中毒預后。2.與大腦皮層組織相比,CO中毒對于海馬組織損傷更為顯著。3.甲烷可能通過抗氧化作用,抗炎癥作用及抗凋亡作用等機制產生保護作用。4.CO中毒導致的持續(xù)存在的氧化應激能夠抑制Nrf-2和下游蛋白CAT的表達,甲烷能夠解除其抑制作用。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R595.1;R747.9

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