The Protective Effect And Mechanism Of Hydrogen-rich Saline On Brain Injury Of Acute Carbon Monoxide Poisoning In Rat

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.