【作者】 陈涵；

【导师】 王强； 孙学军； 孙延平；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 研究背景肠缺血再灌注损伤常见于腹主动脉瘤手术、小肠移植、心肺分流术、肠绞窄、新生儿坏死性小肠结肠炎等情况,亦发生于感染性休克和低血容量性休克。肠缺血再灌注损伤不仅会影响肠道的吸收功能,更会引起肠粘膜屏障功能损伤,导致肠内细菌和毒素移位到体循环,引起网状内皮系统发生系列反应,进而导致大量相关介质及细胞因子的释放,甚至诱发全身炎症反应综合征(Systemic inflammatory response syndrome,SIRS)和多器官功能衰竭(Multiple organ failure,MOF),因此小肠被称为“创伤后多脏器功能衰竭的起源”或“二次打击的起源”。活性氧自由基的产生是小肠缺血再灌注损伤的一个重要机制。再灌注时产生过量的氧自由基,氧自由基的作用靶点主要是脂质、蛋白质、核酸等生物大分子。细胞膜、线粒体膜等膜性结构富含不饱和脂肪酸,特别易于遭受氧自由基攻击,导致脂质过氧化,损伤生物膜,引起细胞坏死或凋亡。氢被认为是一种新型的抗氧化剂,具有选择性抗氧化作用。氢在疾病防治领域的应用基础研究近几年方兴未艾,在脑、心肌、肝脏缺血、糖尿病、肠移植损伤、慢性肝炎、动脉粥样硬化等许多疾病和损伤中都发挥重要作用。近期有研究报道,动物呼吸2%的氢就可以有效清除自由基,显著改善脑缺血再灌注损伤,并采用化学反应、细胞学手段证明,氢溶解在液体中可以选择性中和羟自由基和亚硝酸阴离子,而后两者是氧化损伤的重要介质,体内缺乏其代谢途径。高压氧被广泛用于治疗一氧化碳中毒、减压病、动脉气体栓塞,并作为辅助治疗手段治疗各种氧供障碍的疾病。高压氧预处理较多用于对脑、脊髓、肝脏和心肌的缺血耐受研究,结果表明可明显减轻脑、脊髓、肝脏和心肌的缺血再灌注损伤。高压氧预处理能激发机体内源性保护机制,从而减轻其后所受到的组织缺血、创伤等因素引起的组织损伤,起到较好的预防作用,在临床应用中比较有价值。目前,高压氧预处理对肠缺血再灌注损伤的效应尚未明确。目标1.通过构建肠缺血再灌注模型,观察氢水治疗肠缺血再灌注损伤的有效性,并进行初步的机制探讨。2.研究高压氧预处理对小肠缺血再灌注损伤的影响,并探讨介导其效应的机制。方法1.采用肠系膜上动脉无创性暂时阻断45min,之后恢复肠系膜血流再灌注60min的方法构建大鼠肠缺血再灌注损伤模型。2.再灌注60min后,快速剪取0.5cm长空肠环,洗去内容物,置入离体组织灌流浴槽用于肠张力测定。3.取大鼠远端空肠制备肠组织匀浆样本,测定样本丙二醛含量和髓过氧化物酶活性。4.血清细胞因子水平检测采用双抗体夹心ELISA法。5.TUNEL法测定肠上皮细胞的凋亡情况。6.免疫组化检测肠上皮细胞PCNA的表达和分布。结果1.第一部分：与缺血再灌注组和缺血再灌注+生理盐水组对比,氢水组大鼠肠管对KCl的反应性显著升高(P<0.01),提示再灌注前30min经尾静脉注射氢水(6 ml／kg)能显著改善缺血再灌注诱导的肠收缩功能下降。与假手术组对比,缺血再灌注组和缺血再灌注+生理盐水组大鼠肠组织MDA含量、MPO活性、血清TNF-α和IL-6水平显著增加(P<0.05),而氢水治疗能明显降低大鼠肠组织MDA含量、MPO活性、血清TNF-α和IL-6水平(P<0.05)。氢水组大鼠肠隐窝PCNA阳性表达细胞数显著高于缺血再灌注组和缺血再灌注+生理盐水组水平(P<0.01),显示再灌注前30min经尾静脉注射氢水(6 ml/kg)能显著促进肠隐窝上皮细胞的增殖。假手术组大鼠肠上皮凋亡细胞很少。与假手术组对比,缺血再灌注组和缺血再灌注+生理盐水组大鼠肠上皮TUNEL阳性细胞数显著增多(P<0.01)。而氢水组大鼠肠上皮TUNEL阳性细胞数则明显低于缺血再灌注组和缺血再灌注+生理盐水组水平(P<0.01),显示再灌注前30min经尾静脉注射氢水(6 ml／kg)能显著抑制缺血再灌注损伤诱导的肠上皮细胞凋亡。2.第二部分：与缺血再灌注组对比,高压氧预处理组大鼠肠管对KCl的反应性显著升高(P<0.01),提示高压氧预处理能显著改善缺血再灌注诱导的肠收缩功能下降。与假手术组对比,缺血再灌注组大鼠肠组织MDA含量和MPO活性显著增高(P<0.05),而高压氧预处理能明显降低大鼠肠组织MDA含量和MPO活性(P<0.05)。假手术组大鼠肠上皮凋亡细胞很少。与假手术组对比,缺血再灌注组大鼠肠上皮TUNEL阳性细胞数显著增多(P<0.01)。而高压氧预处理组大鼠肠上皮TUNEL阳性细胞数则明显低于缺血再灌注组水平(P<0.01),显示高压氧预处理能显著抑制缺血再灌注损伤诱导的肠上皮细胞凋亡。结论1.氢水对肠缺血再灌注损伤有明显的保护作用,其机制可能是抑制肠缺血再灌注诱导的氧化应激损伤、炎症反应和肠上皮细胞凋亡,促进肠上皮细胞增殖。2.高压氧预处理可能通过其抗氧化、抗炎、抗凋亡效应从而减轻大鼠肠缺血再灌注损伤。更多还原

【Abstract】 BackgroundThe intestinal ischemia-reperfusion (I/R) injury is a devastating syndrome which occurs in a variety of clinical settings including abdominal aortic aneurysm, small intestinal transplantation, cardiopulmonary bypass, strangulated hernia, and neonatal necrotizing enterocolitis. I/R injury of the intestine also occurs in septic and hypovolemic shock. The consequence of intestinal I/R injury not only alternates absorptive function of intestine, but also may cause intestinal bacteria colony shifting, and even lead to the multiple organ failure (MOF). Overproduction of toxic hydroxyl radicals has been shown to play an important role in the pathogenesis of intestinal I/R injury, which thereby promoting the lipid peroxidation, DNA oxidation, thiyl radical formation and mitochondrial depolarization and eventually leading to cellular apoptosis and necrosis. Toxic hydroxyl radicals can damage cellular membrane and subcellular structures, which contain large amounts of phospholipids and protein, resulting in lipid peroxidation and sequentially structural and metabolic alterations, and leading to cell death and necrosis.Hydrogen (H2), which could react with hydroxyl radical to produce water, has been considered as a novel antioxidant and has been demonstrated recently to have high protective properties in human, animal, and in vivo and in vitro studies, including the protective effect on lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance, transplantation induced intestinal graft injury, chronic liver inflammation, arteriosclerosis, myocardial ischemia-reperfusion (I/R) injury, acute oxidative stress and focal brain I/R injury, and chronic allograft nephropathy. A recent study provided evidence that inhaled H2 gas markedly protect the brain against I/R injury and stroke in cell-free systems by selectively reducing the hydroxyl radical, the most cytotoxic one of reactive oxygen species (ROS), which is commonly produced under various pathological conditions including in intestinal I/R injury.Hyperbaric oxygenation (HBO) has been widely used as a primary therapy in patients with carbon monoxide poisoning, decompression sickness, and arterial gas embolism, and it has been used as an adjunctive therapy for the treatment of various diseases accompanied by impaired oxygen delivery. Interestingly, HBO preconditioning has also shown promising results in some models of ischemia, including I/R injury of brain, spinal cord, liver, and myocardium. However, protective effects of HBO preconditioning has not been verified concerning intestinal ischemia-reperfusion injury.Objective1. To determine the protective effect of hydrogen-rich saline on intestinal I/R injury and the underlying related mechanisms using a rat mesenteric ischemia-reperfusion injury model.2. To investigate whether HBO preconditioning plays a role in intestinal I/R injury and its possible mechanisms.Methods1. Intestinal I/R injury was induced in Sprague-Dawley rats using bulldog clamps in superior mesenteric artery by 45 min ischemia followed by 1 h reperfusion.2. Following reperfusion, segments of terminal jejunum were rapidly taken and transferred into isolated organ bath and responses to KCl were recorded.3. Samples of terminal jejunum were taken for measuring malondialdehyde(MDA) and myeloperoxidase(MPO).4. Cytokine levels in serum were determined by highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.5. Apoptosis in intestinal epithelium was determined with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL).6. Expression and distribution of proliferating cell nuclear antigen (PCNA) were detected with immunohistochemistry.Results1. In part one, compared with I/R or I/R + saline group, the intestinal smooth muscle contractility in the hydrogen-rich saline treatment animals recovered more significantly (P < 0.01). The intestinal MDA content and MPO activity increased significantly in intestinal I/R and I/R + saline groups when compared with sham-operated group (P< 0.05). More importantly, treatment of rats with hydrogen-rich saline at a dose of 6 ml/kg significantly attenuated the increasing in intestinal MDA level and MPO activity (P< 0.05). Serum TNF-αand IL-6 concentrations were significantly elevated in intestinal I/R and I/R + saline animals when compared with those in sham-operated animals. Hydrogen-rich saline administration lowered the I/R-induced elevation of serum TNF-a and IL-6 concentrations. In intestinal crypts, hydrogen-rich saline administration significantly elevated the positive rate of PCNA, compared with I/R or I/R + saline group (P< 0.01). There were few apoptotic cells in sham-operated group. The apoptotic index in I/R and saline-treated groups markedly increased compared with sham-operated group (P< 0.01). When rats treated with hydrogen-rich saline, fewer TUNEL-positive cells were seen and the apoptotic index significantly decreased as compared to the I/R or I/R + saline-treated group (P< 0.01).2. In part two, compared with I/R group, the intestinal smooth muscle contractility in the HBO preconditioning animals recovered more significantly (P< 0.01). The intestinal MDA content and MPO activity increased significantly in intestinal I/R group when compared with sham-operated group (P< 0.05). More importantly, treatment of rats with HBO preconditioning significantly attenuated the increasing in intestinal MDA level and MPO activity (P< 0.05). There were few apoptotic cells in sham-operated group. The apoptotic index in I/R group markedly increased compared with sham-operated group (P< 0.01). When rats treated with HBO preconditioning, fewer TUNEL-positive cells were seen and the apoptotic index significantly decreased as compared to the I/R group(P< 0.01).Conclusions1. Hydrogen treatment has a protective effect against intestinal I/R injury. This protective effect is possibly due to its ability to inhibit I/R-induced oxidative stress, inflammation, apoptosis and to promote epithelial cell proliferation.2. HBO preconditioning protects the small intestine against I/R injury mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic activities.更多还原