【作者】 郑兴锋；

【导师】 夏照帆； 朱世辉； 路卫； 唐洪泰； 贲道锋； 王广庆； 王光毅； 程大胜； 肖仕初；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 富氢水对大鼠肠缺血再灌注损伤以及四氯化碳诱导的肝损伤的保护作用及其机制背景：肠缺血再灌注(intestinal ischemia-reperfusion, IIR)损伤和药物／化学性肝损伤(drug/chemical induced liver injury)是烧伤科以及危重病领域常见的两个临床问题。严重烧伤、急性失血、休克、脓毒症、肠梗阻、肠系膜血栓等因素常常引起肠缺血再灌注损伤,ⅡR可导致肠动力不足,通透性增加,粘膜屏障功能受损,ⅡR不仅引起肠道的局部损伤,尚可引发全身炎症反应和远隔器官的损伤,是引起多器官功能障碍综合征(MODS)的重要原因。药物／化学性肝损伤也是临床常见并且棘手的问题之一,随着药物的不断开发利用,随之而来的副作用也不断增加。另外,肝脏作为体内最大的代谢和解毒器官,常常受到多种不同外源性以及内源性因素的损伤,保肝护肝成为细胞保护策略的首要任务。四氯化碳(carbon tetrachloride, CCl4)是一种亲肝的细胞毒性物质,CCl4诱导的肝损伤模型被广泛用作药物／化学性肝损伤的首选实验动物模型,此外,CCl4还是工业生产中常用的化合物,CCl4中毒的处理也是危重病与急救医学的重要内容之一在肠缺血再灌注损伤以及的四氯化碳诱导的化学性肝损伤的致病过程中,氧化应激起到了非常重要的作用。活性氧自由基的大量产生和激活作用是这两种模型共同的发病机制之一。活性氧自由基是活泼的氧自由基与具有氧自由基反应特性的其他含氧物质的总称,包括超氧阴离子自由基(·O2-)、过氧化氢(H2O2)、羟自由基(·OH)以及单线态氧(102)等。过量产生的氧自由基会导致DNA、脂质和蛋白的氧化损伤,其中·OH的毒性最强,并且到目前为止还没有发现哺乳动物体内内源性针对·OH的清除途径。近来,Ohsawa等发现了氢气分子可选择性清除细胞体系中的·OH,动物呼吸气体中一定浓度的氢气可以有效缓解脑缺血再灌注损伤。因此,本研究开发了更为安全和方便使用的氢饱和的生理盐水,这里称为富氢水,以肠缺血再灌注损伤以及的四氯化碳诱导的肝损伤为研究对象,观察富氢水是否具有保护作用并探讨其机制。方法：(一)将健康雄性SD大鼠随机分为假手术组(sham)、模型组(I/R)、富氢水处理组(I/R+H2)以及富氮水处理组(I/R+N2)。用无创小血管夹夹闭肠系膜上动脉(superior mesenteric artery, SMA) 45 min,松开血管夹再灌注120 min,建立肠缺血再灌注损伤模型。再灌注开始10 min前,于静脉分别注射5 ml/kg生理盐水、氢饱和生理盐水以及氮饱和生理盐水。再灌注结束后,取血和小肠组织。检测如下指标：1)HE染色和病理学检查；2)血清二胺氧化酶(diamine oxidase, DAO),组织丙二醛(malondialdehyde, MDA)、蛋白质羰基(protein carbonyl, PC)和髓过氧化物酶(myeloperoxidase, MPO);3)血清肿瘤坏死因子-a(tumor necrosis factor-α, TNF-α)、白介素-1β(interleukin-1β,IL-1β)和白介素-6(interleukin-6, IL-6)(二)将健康雄性SD大鼠随机分为溶剂对照组(control)、模型对照组(CCl4+N2)、富氢水处理组(CCl4+H2)。大鼠腹腔单次注射CCl4/橄榄油混合液(v：v=l：1)2 ml/kg bw制作急性肝损伤模型(生存分析实验大鼠腹腔注射CCl4/橄榄油混合液7.5 ml/kg bw)。在CCl4注射前10 min以及注射后每3小时腹腔分别给予5 ml/kg生理盐水、氮饱和生理盐水以及氢饱和生理盐水。CCl4注射12hr后,取血和肝组织检测如下指标(MAPK信号分子检测在CCl4注射3hr后取材)：1)HE染色和病理学检查；2)血清AST、ALT、LDH活性和DBIL、TBIL水平；3)TUNEL组织化学检查和组织Caspase-3活性；4)组织丙二醛(malondialdehyde, MDA)和谷胱甘肽(Glutathione, GSH);5)血清肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α);6)组织MAP激酶信号分子：JNK、ERK和p38 MAPK磷酸化水平。结果：(一)肠缺血再灌注后,组织学检查可见明显粘膜损伤,Chiu氏评分显著增加。血清DAO活性,小肠组织MDA、PC、MPO含量,血清TNF-α、IL-1β、IL-6水平显著增高。富氢水处理可以显著缓解小肠病理学损伤,并显著抑制以上指标的升高,而富氮水处理组与模型组未见明显差别。(二)富氢水处理可以显著延长大鼠CCl4中毒后的生存时间,减轻CCl4引起的大鼠肝脏病理学损伤,显著减少气球样变性细胞数量；显著降低CCl4引起的血清AST、ALT、LDH活性和DBIL、TBIL水平升高；显著减少CCl4引起的肝细胞凋亡,降低肝组织Caspase-3活性；显著抑制CCl4引起的肝组织MDA含量、血清TNF-a水平增高以及肝组织GSH含量的下降；显著降低肝组织JNK、ERK和p38MAPK磷酸化水平。结论：1)富氢水可以有效减轻肠缺血再灌注损伤,其保护作用可能与富氢水减轻再灌注损伤引起的氧化应激,缓解中性粒细胞浸润和降低炎性因子升高等机制有关。2)富氢水可以有效减轻CCl4诱导的肝损伤,其保护作用可能与富氢水抑制CCl4引起的肝细胞凋亡、减轻CCl4引起的氧化应激、降低TNF-α升高以及抑制MAP激酶信号通路激活等机制有关。3)本研究开发并应用了富氢盐水用于静脉注射和腹腔注射的新途径,为氢气的应用范围、作用机制、给药方式、临床应用前景提供了新的参考依据,并为烧伤常见临床危重病救治提供了新的可能策略和方向。更多还原

【Abstract】 Protective Effects and Mechanisms of Hydrogen-rich Saline on Intestinal Ischemia-reperfusion Injury and Carbon Tetrachloride Induced Hepatic Injury in RatsBackground:Intestinal ischemia/reperfusion (II/R) injury and drug/chemical induced liver injury are two common clinical problems faced in burn department and critical care medicine. II/R injury often occurs in severe burns, acute hemorrhagic, shock, sepsis, intestinal obstruction, mesenteric arterial occlusion by thrombi or embolisms, and so on. II/R is accompanied by decreased contractile activity, increased microvascular permeability and dysfunction of mucosal barrier, which could eventually cause systemic inflammatory response syndrome and remote organ damage, and even to multiple organ dysfunction syndrome (MODS). Drug or chemical induced liver injury is also a major health problem faced in clinical practice and there is still lack of effective therapeutic strategies or specific medicines for such liver diseases. The prevalence of drug or chemicals induced hepatotoxicity went up over the past decades following the drug development. In addition, liver is the main metabolic and detoxifying organ. It is prone to be injured as it is often exposed to environmental toxicants, drugs, and other xenobiotics or endogenous agents, so the protection of liver is the first task for us to conduct cyto-preservation measures. CCl4 is a potent toxicant for producing model of drug or chemical induced hepatic injury. CCl4 is also a common compound used in industry, and the treatment of CCl4 toxicity is an important subject in critical care and emergency medicine.Oxidative stress plays a very important role in the pathological process of intestinal ischemia/reperfusion injury and CCl4 induced hepatic injury. The excessive production and activation of radical oxygen species is the mechanism in common in these two models of injury. Reactive oxygen species (ROS) are reactive molecules that contain the oxygen atom. They are highly reactive due to the presence of unpaired valence shell electrons. Major ROS includes hydrogen peroxide, superoxide, and hydroxyl radical. Excessive ROS will damage DNA, lipids and proteins in cells. As we know, the hydroxyl radical is the most reactive product of ROS generated in cells. It is biologically important to eliminate hydroxyl radicals, because superoxide anion and hydrogen peroxide are detoxified by antioxidant defense enzymes, superoxide dismutase, and peroxidase or glutathione-peroxidase, respectively; however, no endogenous enzymatic pathway is known to neutralize hydroxyl radicals. Recently, Ohsawa et al. found that molecular hydrogen could selectively reduce·OH in vitro and inhalation of hydrogen could exert therapeutic antioxidant activity in a rat middle cerebral artery occlusion model. Therefore, in this study, we developed hydrogen saturated physiological saline (hydrogen-rich saline), which is easy and safe to apply. We investigated whether administration of hydrogen-rich saline exerted protective effect in the models of intestinal ischemia/reperfusion injury and CCl4 induced hepatic injury.Methods:1. The SD rats were randomly divided into four groups:(1) sham-operated plus vehicle physiological saline treatment; (2) intestinal ischemia/reperfusion plus vehicle physiological saline treatment; (3) intestinal ischemia/reperfusion plus hydrogen-rich saline treatment; (4) intestinal ischemia/reperfusion plus nitrogen-rich saline treatment. A rat small intestinal I/R model was adapted by clamping the superior mesenteric artery maintaining for 45 minutes and removing the clamp for 120 minutes reperfusion. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was given via jugular venous cannula infusion at 10 minutes before reperfusion initiation, respectively. 120 minutes after reperfusion initiation, blood was drawn and intestinal tissue samples were collected. The following markers are measured:1) HE staining and histopathological observation;2)Serum diamine oxidase (DAO), tissue malondialdehyde (MDA) and myeloperoxidase (MPO)level;3) Serum tumor necrosis factor-a (TNF-a), interleukin-1β(IL-1β), and interleukin-6 (IL-6).2. The SD rats were randomly divided into three groups:Control, CCl4+ nitrogen-rich saline and CC14+ hydrogen-rich saline. CC14 mixed with olive oil (1:1 v/v, 2 ml/kg) was injected intraperitoneally for hepatic injury model. (For survival analysis experiment, Rats were injected with a lethal dose of CCl4 mixed with olive oil (1:1 v/v,7.5 ml/kg) intraperitoneally.) Hydrogen-rich saline (5 ml/kg) or nitrogen-rich saline of equivalent volume was given intraperitoneally 10 minutes prior to the injection of CCl4 and every 3 hours after the administration of CCl4. The blood and liver tissue samples were collected 12 hours after CCl4 administration (For MAP kinase tested liver tissues were collected 3 hours after CCl4 administration). The following markers are measured:1) HE staining and histopathological observation;2) Serum AST, ALT, LDH activities and DBIL, TBIL levels;3) TUNEL histochemical assay and tissue caspase-3 activity;4) Tissue malondialdehyde (MDA) and glutathione (GSH) level;5) Serum tumor necrosis factor-α(TNF-α);6) Tissue MAP kinase signal molecules:JNK, ERK and p38 MAPK phosphorylation.Result:1. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-α, IL-1βand IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury while no significant reduction was observed in the nitrogen-rich saline treated animals.2. Hydrogen-rich saline administration significantly attenuated the CCl4-induced hepatic injury, with the alleviation of pathological hepatic lesions and the reduction of elevated serum hepatic enzyme activities and bilirubin levels, and prolonged the survival time of CCl4-intoxicated rats. In addition, hydrogen-rich saline ameliorated the hepatocytes apoptosis, as well as lowered the increase of hepatic caspase-3 activity in CCl4-treated rats. Furthermore, the increases of malondialdehyde concentrations as well as the reduction of glutathione levels in liver induced by CCl4 were diminished by hydrogen-rich saline treatment. Moreover, the elevated serum TNF-a levels and the activation of JNK, ERK and p38 MAPK induced by CCl4 was downregulated by hydrogen-rich saline treatment.Conclusion:1) Hydrogen-rich saline protected the small intestine against I/R injury possibly by relief of oxidative stress, attenuating PMN infiltration and reduction of inflammatory mediators elevation.2) Hydrogen-rich saline has a protective effect on CCl4 induced hepatotoxicity in rats and the hepatoprotective effects of hydrogen-rich saline may be mediated through anti-oxidative, anti-inflammatory and anti-apoptotic pathways, as well as the inhibition of MAP kinase activation.3) This study developed and applied hydrogen-rich saline for intravenous and intraperitoneal delivery. It supplies evidence for effects, mechanisms, delivery approach, and potential for clinical application of hydrogen. And it supplies potential measurement for treating critical illness patients in burn department.更多还原