摘要
一氧化碳(carbon monoxide CO)中毒是一种常见的中毒性疾病，严重危害着人类的健康和生命。部分重度中毒患者常因累及大脑、心脏等多个重要脏器，即使挽救了生命，也常留下迟发性神经精神后遗症，严重影响了生存者的生活质量，给家庭和社会带来了巨大的负担。因此，如何尽早对急性CO中毒脑损伤采取有效治疗手段并防治迟发性中毒后脑病（Delayed neurologic sequelaeDNS），已经成为临床上迫切需要解决的问题。
自由基损伤是急性CO中毒脑损伤的主要发病机制。自由基与人类的健康和疾病密切相关，是当前生命科学研究的热点。过量产生的自由基会导致DNA、脂质和蛋白的氧化损伤，其中羟自由基（.OH）和过氧亚硝酸阴离子（ONOO-）毒性强，目前为止还没有在哺乳动物体内发现内源性的有针对性的清除方式。2007年，有学者在《Nature medicine》报道，动物呼吸2％的氢就可有效清除自由基，显著改善脑缺血再灌注损伤。
本课题采用吸入法建立大鼠急性CO中毒脑损伤模型，该模型为CO中毒的常用模型，大鼠与人类的大脑结构相似，能很好地模拟人脑缺血缺氧的过程。观察氢饱和的生理盐水（简称为富氢水）对急性CO中毒脑损伤大鼠的保护效应，通过症状学、病理学、形态学、行为学等不同指标分别评价富氢水对皮层区以及海马区的保护效应。并从两方面探讨富氢水对急性CO中毒脑损伤保护作用的机制：（1）富氢水治疗对大鼠急性CO中毒后氧自由基及相关抗氧化酶的作用。主要观察急性CO中毒后腹腔注射富氢水，大鼠大脑皮层和海马的氧化应激系统的变化；（2）急性CO中毒后予以富氢水治疗对大鼠神经元细胞凋亡的影响，并测定凋亡酶活性和凋亡相关蛋白变化。通过本课题的实施，旨在为富氢水用于临床治疗急性CO中毒脑损伤提供实验依据。
一、富氢水对大鼠急性CO中毒脑损伤的保护效应
目的：研究腹腔注射富氢水对大鼠急性CO中毒脑损伤是否具有保护效应；同时分别观察皮层和海马对富氢水治疗的反应。
方法：采用吸入法制作急性CO中毒模型，腹腔注射富氢水治疗。动物分为正常对照组（Control）、CO+生理盐水（NS）组、CO+富氢水（Hydrogen）组。造模后24小时观察各组动物的一般精神行为状态、体重变化，14天生存情况，根据Brailowsky描述的神经学评分方法对神经功能缺损进行症状评估，测定脑组织含水量，采用脑组织灌流切片，行HE、NISSL染色，并在镜下对神经细胞进行计数。观察建模后存活14天大鼠的水迷宫实验，评价其空间记忆能力。
结果：富氢水治疗组的动物在建模后24小时精神状态较好，毛色光滑，较为活泼，反应迅速，主动摄入食水，体重下降不明显，其结果明显优于生理盐水组（14.173.76g vs.18.52.43g；P<0.05）；富氢水治疗组的生存率明显增高（80%vs.60%；P<0.05）；神经功能缺失症状减轻明显（13.831.94vs.16.831.72；P<0.05）；脑组织水含量低（0.790.04vs.0.880.04；P<0.01）。HE和NISSL染色均表明富氢水对皮层（NISSL：88.5022.88vs.58.6716.18；P<0.05）和海马(NISSL：102.8326.36vs.51.6716.79，P<0.01)有明显的保护作用。2周后水迷宫测试提示富氢水治疗组逃避潜伏期短于生理盐水组(22.674.03vs.32.334.59，P<0.05)。
结论：1、吸入法模拟急性CO中毒模型制作方法相对简单，重复性好，能比较好地应用于观察动物急性CO中毒后各项指标的变化以及干预措施的疗效比较；2、富氢水注射对大鼠急性CO中毒脑损伤有保护作用。
二、富氢水通过调节氧化应激系统对大鼠急性CO中毒脑损伤的保护作用
目的：研究富氢水的保护效应是否与清除氧自由基、上调抗氧化酶的活性有关。
方法：按照第一部分的方法进行建模和治疗。对大鼠皮层和海马分别取材，制作组织匀浆，根据试剂盒说明用硫代巴比妥酸法（TBA）测定丙二醛（MDA）含量，酶联免疫吸附测试法（ELISA）测定8-羟基鸟嘌呤（8-OHdG）含量，羟胺法测定超氧化物歧化酶（SOD）活力，比色法测定脑组织中谷胱甘肽过氧化物酶（GSH-px）活力。
结果：CO中毒后24小时，与生理盐水组相比，富氢水能降低皮层（1.420.44vs.2.040.44，P<0.05）和海马（1.910.68vs.3.170.69，P<0.01）的MDA含量；降低8-OHdG的含量（皮层54.64±4.93vs.62.98±9.08，P<0.05）；海马60.43±10.49vs.82.66±12.55，P<0.01)；增强抗氧化酶的活性，如SOD（皮层111.35±44.95vs.46.23±28.78，P<0.05；海马173.08±51.43vs.89.11±35.38，P<0.01），但对GSH-px影响不明显（皮层5.41±1.41vs.5.25±0.89，P>0.05；海马6.19±2.67vs.5.87±2.38，P>0.05）。
结论：1、大脑皮层和海马对急性CO中毒均有明显的氧化应激反应；2、富氢水的保护作用可能是通过上调内源性抗氧化酶、清除氧自由基发挥作用。
三、富氢水通过抑制过度凋亡对急性CO中毒脑损伤的保护作用
目的：研究富氢水的保护效应是否与抑制神经细胞过度凋亡有关。
方法：按照第一部分的方法进行建模和治疗。采用TUNEL法检测细胞凋亡；ELISA法检测caspase-3和caspase-9的酶活性；Western Blot检测Bcl-2和Bax蛋白的表达。
结果：CO中毒后24小时，与生理盐水组相比，富氢水能明显减少皮层（57.677.58vs．99.508.73，P<0.01）和海马区（52.3313.17vs．82.1715.79）的凋亡阳性细胞数；降低皮层和海马的caspase-3酶（皮层0.0290.008vs.0.0650.009，P<0.01vs. CO+NS group；海马0.0310.004vs.0.0530.007，P<0.01）和caspase-9酶(皮层1.1960.384vs.1.7970.432，P<0.01；海马1.2080.112vs.1.8970.280，P<0.01)相对活性；也提高了Bcl-2在皮层以及海马（皮层1.363±0.183vs.0.886±0.070，P<0.01；海马1.166±0.139vs.0.807±0.067，P<0.01）的表达，但对Bax水平影响不明显（皮层0.515±0.038vs.0.494±0.029，P>0.05；海马0.437±0.107vs.0.410±0.034，P>0.05）。
结论：1、急性CO中毒后，大鼠大脑皮层和海马均呈现不同程度的凋亡；2、抑制凋亡可能是富氢水保护作用机制之一。
Carbon monoxide（CO）poisoning is a common poisoning disease whichseriously endangers human health and life. Critical patients often undergo the injuryof brain, heart and other vital organs. Over half of those with serious poisoningdevelop delayed neurologic sequelae between3days and4weeks afterward, whichseriously affected the survivors’ life quality and brought great burden to familiesand society. It is urgent to take some effective treatments for brain injury of acuteCO poisoning as soon as possible.
Oxidative stress is the main pathogenesis of brain injury resulting from acuteCO poisoning. Free radicals are closely related to human health and disease, whichare also a hot topic of current life science research. The excessive production ofoxygen free radicals can cause oxidative damage to DNA, lipids and proteins.Among which hydroxyl radical (.OH) and peroxynitrite anion (ONOO-) are thepotent members, and so far people have not found any corresponding endogenoustarget to clear them in mammals. In2007, scholars reported in Nature medicine thatanimals breathing2%hydrogen can significantly reduce the cerebral ischemia andreperfusion injury through scavenging free radical effectively.
In this study we copy an animal model of acute CO poisoning by inhalation,which is a commonly used model for CO poisoning. Rats have similar brainstructure as human and can well simulate the human brain ischemia and hypoxiaprocess. We observe the protective effect of hydrogen-saturated saline(hydrogen-rich water) on brain injury of acute CO poisoning rats by the differentindicators of symptoms, pathology and morphology, evaluating the protective effectof hydrogen-rich water on the cortex and hippocampus. Then we explore themechanism of hydrogen-rich water protective function on acute CO poisoning braininjury in two ways:(1) Changes of reactive oxygen species and antioxidantenzymes,(2) Observing neurons apoptosis, determination of apoptosis activity andapoptosis-related protein changes in rats with acute CO poisoning followinghydrogen-rich water treatment. The implementation of this project is to provide theexperimental basis for the clinical treatment of acute CO poisoning brain injurywith hydrogen-rich water.
1. Protective effect of the hydrogen-rich water on rats with brain injuryresulting from acute CO poisoning.
Objective: To study whether hydrogen-rich water have protective effect on ratswith acute CO poisoning brain injury, we take intraperitoneal injection ofhydrogen-rich water to rats at different points, and observe the different responsesof cortex and hippocampus.
Methods: We copy an animal model of acute CO poisoning by inhalation andtake intraperitoneal injection of hydrogen-rich water as the treatment. The animalswere divided into normal control group (Control), CO+saline (NS) group, CO+hydrogen-rich water (Hydrogen) group. We observe the behavior of animals, weightchange, survival rate of14d, the neurological score which described by Brailowskyas neurological deficit symptoms assessment, brain water content, HE staining andNISSL staining at24h after poisoning. Using Morris water maze test to evaluate thespatial memory ability of animals at14d.
Results: Animals of hydrogen-rich water group were lively and responsive, alsohad a good mental state, smooth coat and were willing to take in food and water at24h after poisoning. They had less weight loss (14.17±3.76g vs.18.5±2.43g, P<0.05), higher survival rate (80%vs.60%, P<0.05), lower score of symptoms ofneurological deficit (13.83±1.94vs.16.83±1.72, P<0.05), and lower water contentof brain tissue (0.790.04vs.0.880.04, P<0.01) than those of the saline group.NISSL staining showed apparent protective effect of hydrogen-rich water on thecortex (88.5022.88vs.58.6716.18, P<0.05NISSL) and hippocampus (102.8326.36vs.51.6716.79, P<0.01NISSL).
Conclusions:1、 To stimulate CO poisoning model by static inhalationsimulating is relatively simple, reproducible, and better used in comparing the effectof hydrogen-rich water effect.2、Hydrogen-rich water injection has a protectiveeffect on animals with acute CO poisoning.
2. The protective effect of hydrogen-rich water on acute CO poisoning throughadjusting the oxidative stress system.
Objective: To study whether the mechanism of protective effect of thehydrogen-rich water by scavenging oxygen free radicals, increasing the activity ofantioxidant enzymes or not.
Methods: According to the method of modeling and treatment in the first part,we took the rats’ cortex and hippocampus of each group and produced tissuehomogenates. We measured malondialdehyde (MDA) by thiobarbituric acid (TBA)method,8-hydroxyguanine (8-OHdG) content by enzyme-linked immunosorbent assay (ELISA), superoxide dismutase (SOD) change by hydroxylamine method andactivity of glutathione peroxidase (GSH-px) at24h after acute CO poisoning.
Results: Hydrogen-rich water can reduce the MDA content of cortex (1.420.44vs.2.040.44, P<0.05) and hippocampus (1.910.68vs.3.170.69,P<0.01), decrease the8-OHdG content of cortex (54.64±4.93vs.62.98±9.08，P<0.05) and hippocampus (60.43±10.49vs.82.66±12.55，P<0.01), enhance theactivity of antioxidant enzymes such as SOD (cortex,111.35±44.95vs.46.23±28.78，P<0.05; hippocampus,173.08±51.43vs.89.11±35.38，P<0.01), but noeffect on GSH-px(cortex,5.41±1.41vs.5.25±0.89， P>0.05, hippocampus,6.19±2.67vs.5.87±2.38，P>0.05).
Conclusions:1、Cerebral cortex and hippocampus suffer significant oxidativestress response from CO poisoning.2、The protective effect of the hydrogen-richwater may be through upregulation of endogenous antioxidant enzymes, scavengingoxygen free radicals.
3、The protective effect of hydrogen-rich water on acute CO poisoning throughinhibiting excessive apoptosis.
Objective: To study whether the mechanism of protective effect of thehydrogen-rich water by inhibiting excessive apoptosis or not.
Methods: We detected apoptosis by TUNEL method, measured activity ofcaspase-3and caspase-9enzyme by enzyme-linked immunosorbent assay (ELISA)and Bcl-2, Bax protein expression by Western blot.
Results: Hydrogen-rich water can significantly reduce the positive apoptoticcells number of cortex (57.677.58vs．99.508.73，P<0.01) and hippocampus(52.3313.17vs．82.1715.79), reduce relative activity of caspase-3(cortex0.0290.008vs.0.0650.009，P<0.01vs. CO+NS group；hippocampus0.0310.004vs.0.0530.007，P<0.01)and caspase-9(cortex1.1960.384vs.1.7970.432，P<0.01vs. CO+NS group；hippocampus1.2080.112vs.1.8970.280，P<0.01vs. CO+NS group)enzyme of cortex and hippocampus, also increase theexpression of Bcl-2in the cortex(1.363±0.183vs.0.886±0.070，P<0.01) andhippocampus(1.166±0.139vs.0.807±0.067，P<0.01), without significant effect onBax levels (cortex0.515±0.038vs.0.494±0.029， P>0.05, hippocampus0.437±0.107vs.0.410±0.034，P>0.05).
Conclusions:1、Cerebral cortex and hippocampus showed varying degree of apoptosis after CO poisoning.2、The protective effect of the hydrogen-rich watermay be through inhibiting apoptosis.
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