超氧化物歧化酶在心肌再灌注性损伤中的作用

本实验在大鼠离体心脏灌注模型上,模拟心肌缺血再灌注过程(旷置30分钟,再灌注45分钟),结果发现:再灌注后,心脏频发室颤,组织水肿明显,脂质过氧化终产物丙二醛较正常时显著增加,冠脉流出液中乳酸脱氢酶呈一持续高水平释放,心肌组织ATP酶活性明显下降。再灌注开始后给予超氧化物歧化酶(SOD)。能够减轻以上心肌再灌注性损伤现象。这一结果提示:1)活性氧参与了心肌再灌注损伤现象的发生。2)再灌注开始后给予SOD仍有明显疗效,这为临床上在再灌注开始后应用SOD提供了一定的实验依据。     

Cardioprotective activity of endogenous and exogenous nitric oxide on ischaemia reperfusion injury in isolated guinea pig hearts

BACKGROUND: We evaluated the contribution of endogenous and exogenous nitric oxide (NO) in ischaemia reperfusion (IR) injury and histamine release in the isolated guinea pig heart. METHODS: Ischaemia reperfusion was performed in isolated Langendorff perfused guinea pig heart throughout the ligature of the left anterior descending coronary (LAD) artery for 20 min, and following the release of the ligature for a further 20 min. RESULTS: IR promoted a linear release of lactate dehydrogenase (LDH) and a preferential release of histamine in the reperfusion phase. The amount of nitrite (NO2-, one of the breakdown products of NO) released during IR was significantly lower than in the control hearts. These effects were accompanied by an increase in calcium levels and malonyl-dialdehyde (MDA) production in the left ventricle and by a decrease in cardiac mast cell metachromasia. Perfusion of the hearts with two inhibitors of the nitric oxide synthase pathway, namely N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M) or nitroarginine methylester (L-NAME, 10(-5) M) significantly enhanced histamine and LDH release; these effects were attenuated by co-infusion with L-arginine (10(-4) M) but not D-arginine (10(-4) M), while L-arginine (10(-4) M) alone had no effect. Perfusion of the heart with sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), glyceryl trinitrate (GTN), all at 10(-5) M, reduced histamine release, LDH release, calcium overload and MDA production induced by IR. These effects were amplified by concomitant perfusion with superoxide dismutase (SOD, 50 IU/ml). CONCLUSION: The endogenous production of NO provides significant myocardial protection from IR injury and histamine release. These effects were mimicked by various NO donors.     

Polymorphonuclear granulocytes induce myocardial dysfunction during ischemia and in later reperfusion of hearts exposed to low-flow ischemia

Polymorphonuclear granulocytes (PMNs) are known to contribute to reperfusion injury of the heart. However, whether PMNs compromise myocardial function of hearts exposed to a low-flow ischemia has not been determined. Moreover, not much is known about deleterious effects of PMNs at different times during ischemia and reperfusion. Isolated, working guinea pig hearts were subjected to 30 min of low-flow ischemia and reperfusion. Homologous PMNs were applied as 1-min boluses in the presence of thrombin during either ischemia or the first or fifth minute of reperfusion, and postischemic recovery of external heart work (REHW) and intracoronary PMN retention (PMNR) were quantified. In further experiments, the radical scavenger superoxide dismutase (SOD) was added. Compared with controls without PMNs (REHW, 92.4%), application of PMNs led to a significant loss of myocardial function, which was detected at all three examination times. Moreover, intracoronary PMNR increased significantly in comparison with that of controls with hearts not exposed to ischemia or reperfusion. On the other hand, addition of SOD significantly increased REHW. Intracoronary PMNR was not significantly changed by coapplication of SOD. We conclude that thrombin-stimulated PMNs applied at different times during ischemia and reperfusion significantly impaired cardiac function in hearts exposed to a low-flow ischemia.     

Mathematical and computational models of oxidative and nitrosative stress

The importance of nitric oxide (NO), superoxide (O2-), and peroxynitrite (ONOO-), interactions in physiologic functions and pathophysiological conditions such as cardiovascular disease, hypertension, and diabetes have been established extensively in in vivo and in vitro studies. Despite intense investigation of NO, O2-, and ONOO- biochemical interactions, fundamental questions regarding the role of these molecules remain unanswered. Mathematical models based on fundamental principles of mass balance and reaction kinetics have provided significant results in the case of NO. However, the models that include interaction of NO, O2-, and ONOO- have been few because of the complexity of these interactions. Not only do these mathematical and computational models provided quantitative knowledge of distributions and concentrations of NO, O2-, and ONOO- under normal physiologic and pathophysiologic conditions, they also can help to answer specific hypotheses. The focus of this review article is on the models that involve more than one of the 3 molecules (NO, O2-, and ONOO-). Specifically, kinetic models of O2- dismutase and tyrosine nitration and biotransport models in the microcirculation are reviewed. In addition, integrated experimental and computational models of dynamics of NO/O2-/ONOO- in diverse systems are reviewed.     

Involvement of superoxide and/or nitric oxide in renal tissue injury.

We examined the influence of superoxide (O2-) and/or nitric oxide (NO) on renal tissue injury estimated from the levels of lipid peroxidation and sulfhydryl (SH) oxidation. Pyrogallol, an O2- generator and precursor of hydrogen peroxide, produced marked tissue injury, but this was prevented by superoxide dismutase (SOD)/catalase (CAT). Hemoglobin (Hb), a NO scavenger, provided protection from tissue injury caused by sodium nitroprusside (SNP). The tissue injury produced by 3-morpholinosydnonimine (SIN-1), which is thought to form peroxynitrite (ONOO-) as a simultaneous O2- and NO generator, was blocked by SOD/CAT or Hb. On the other hand, protein-SH and nonprotein-SH were significantly increased by addition of SOD/CAT or Hb. These data suggest that the renal tissue injury induced by O2-, NO and ONOO- can be blocked by SH, suggesting an important protective role against these reactive oxygen species in the mechanism of tissue defense.     

Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO-) produced from the reaction of nitric oxide (NO.) with superoxide (O2(.-)). Since human spermatozoa are able to produce both NO. and O2(.-) during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105-14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO- (2.5-50 micromol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO- also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO- modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.     

大蒜素抑制脑缺血-再灌注诱导的海马神经元凋亡及其机制初探

目的：探讨大蒜素抗脑缺血-再灌注诱导的海马神经元凋亡作用及其机制。方法： 采用大鼠全脑缺血-再灌注模型，应用流式细胞仪检测海马神经元凋亡率；采用比色法测定海马组织NO和MDA含量及SOD活性。结果： I-R组大鼠海马神经元凋亡率、海马组织NO和MDA含量明显高于sham组，而SOD活性显著低于sham组；静脉给予大蒜素预处理可抑制上述变化，且呈剂量依赖性效应。结论： 大蒜素具有抑制全脑缺血-再灌注诱导的海马神经元凋亡作用及抗氧化作用；大蒜素的抗氧化作用可能是其抑制海马神经元凋亡的重要机制之一。     

Oxidative stress and adaptation of the infant heart to hypoxia and ischemia

The potential contribution of oxidative stress to cardioprotection in infants induced by adaptation to chronic hypoxia and by ischemic preconditioning is poorly understood. Under conditions of oxidative stress, reactive oxygen species and reactive nitrogen species may contribute to phenotypic changes in hearts adapted to chronic hypoxia and to the pathogenesis of myocardial injury during both ischemia/reperfusion and hypoxia/reoxygenation. Hearts from infant rabbits normoxic from birth can be preconditioned by brief periods of ischemia. In contrast, hearts from infant rabbits adapted to hypoxia from birth appear resistant to ischemic preconditioning. Chronically hypoxic infant rabbit hearts are already resistant to ischemia compared with age-matched normoxic controls, and thus additional cardioprotection by ischemic preconditioning may not be possible. Endothelial nitric oxide synthase (NOS3) protein and its product nitric oxide are increased, but not NOS3 message, in chronically hypoxic infant hearts to protect against ischemia. Chronic hypoxia from birth also increases cardioprotection of infant hearts by increasing association of heat shock protein 90 with NOS3. Normoxic infant hearts also generate more superoxide by an N(omega)-nitro-L-arginine methyl ester-inhibitable mechanism than chronically hypoxic hearts. Thus, NOS3 appears to be critically important in adaptation of infant hearts to chronic hypoxia and in resistance to subsequent ischemia by regulating the production of reactive oxygen and nitrogen species.     

缺血预处理前加用腺苷对未成熟兔心肌再灌注损伤的影响

目的和方法:建立3-4周龄幼兔离体心脏左心作功模型,心脏缺血期间均间断灌注心脏停搏液,研究缺血预处理前加用腺苷对未成熟兔再灌注心肌的保护作用并探讨其机制。结果:再灌注期,缺血预处理前加用腺苷的心脏的左室收缩压(LVSP)、左室舒张末压(LVEDP)、左室内压上升最大速率(+dp/dt_max)和左室内压下降最大速率(-dp/dt_max)恢复最佳,肌酸激酶(CK)漏出率低,心肌组织丙二醛(MDA)含量低、超氧化物歧化酶(SOD)活性高,心肌组织三磷酸腺苷和二磷酸腺苷(ATP+ADP)含量高;心肌含水量(MWC)低。结论:缺血预处理前加用腺苷能强化缺血预处理对未成熟兔再灌注心肌的保护作用。     

成纤维细胞生长因子和丹参对实验性心肌缺血再灌注损伤的预防作用

目的观察碱性成纤维细胞生长因子（bFGF）和丹参（SM）对缺血再灌注心肌组织脂质过氧化反应的预防作用。方法采用新西兰长耳大白兔,心脏左前支冠状动脉结扎,制作心肌缺血动物模型。观察经bFGF和SM预处理的缺血再灌注2 h的心肌组织中琥珀酸脱氢酶（SDH）、脂质过氧化代谢产物MDA含量;抗脂质过氧化酶性指标超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSHpx）活性和非酶性指标MDA、还原性谷胱甘肽（GSH）、一氧化氮（NO）含量。结果bFGF和SM联合预处理组心肌中SDH、SOD、GSHpx活性明显高于缺血再灌注组,抗氧化物GSH和NO的含量明显高于缺血再灌注组,心肌病理改变明显好于未用bFGF和SM组。而MDA的含量比未用SM和bFGF组低。尤其是bFGF和SM联合用药效果更为明显。结论bFGF和SM能有效地减轻心肌缺血再灌注所致的损伤,二者联合使用的抗氧化效果好于单独使用。     

小檗碱干预2型糖尿病模型大鼠脑缺血再灌注损伤

文题释义： 小檗碱(berberine,BBR)：是从黄连中提取的一种异喹啉类生物碱,具有多种生化和药理作用,在临床上得到广泛应用。已有文献证明,小檗碱具有降低高血糖、减轻胰岛素抵抗和抑制脂质合成的作用。缺血再灌注损伤：遭受一定时间缺血的组织细胞恢复血流(再灌注)后,组织损伤程度迅速增剧的情况。再灌注后有大量钙内流,并生成大量氧自由基,是广泛组织细胞损伤的主要发病机制。临床上多种疾病如迟发性神经元坏死、不可逆性休克、心肌梗死、脑梗死及器官移植排斥反应等的发生、发展都与缺血、再灌注有关。 背景：既往研究提示小檗碱可减轻脑缺血再灌注损伤。 目的：探讨小檗碱对2型糖尿病大鼠脑缺血再灌注损伤的影响及其分子机制。 方法：实验方案经海南省中医院伦理委员会批准。用低剂量链脲佐菌素30 mg/kg每隔1 d大鼠腹腔注射2次,处理8周建立2型糖尿病模型,选取平均血糖≥11.1 mmol/L造模成功的雄性SD大鼠90只,随机分为对照组、缺血/再灌注组和缺血/再灌注+小檗碱组,每组30只。对照组和缺血/再灌注组大鼠经生理盐水灌胃,缺血/再灌注+小檗碱组用小檗碱 200 mg/(kg·d)灌胃,处理7 d后,后2组采用大脑中动脉阻断2 h,再灌注12 h建立大脑中动脉缺血再灌注模型。苏木精-伊红染色和透射电镜观察脑梗死体积;采用ELISA检测梗死区超氧化物歧化酶、丙二醛和一氧化氮水平;采用TUNEL法检测脑细胞凋亡情况;Westernblot检测PI3K、Akt和磷酸化Akt(p-Akt)蛋白的表达。 结果与结论：①与缺血/再灌注组比较,缺血/再灌注+小檗碱组脑梗死体积明显减少(P < 0.05),超氧化物歧化酶水平明显升高,丙二醛和一氧化氮的表达明显降低;②与缺血/再灌注组比较,缺血/再灌注+小檗碱组脑梗死区细胞凋亡减少,Bcl-2表达增加,cleaved-Caspase3和Bax表达降低;③与缺血/再灌注组相比,小檗碱可使缺血/再灌注+小檗碱组PI3K和p-Akt的表达水平明显上调;④结果说明,小檗碱可通过激活PI3K-Akt信号通路,在2型糖尿病大鼠脑缺血模型中发挥抗凋亡作用,减轻脑缺血/再灌注损伤。ORCID: 0000-0003-3326-2624(符芸瑜) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

缺血预处理对大鼠压疮缺血再灌注损伤保护作用的实验研究

目的比较不同缺血处理方法对大鼠皮肤及皮下组织的保护作用,为临床判断压疮损伤的程度及压疮干预效果提供理论依据。方法将60只SD大鼠随机分为对照组（S组）、缺血再灌注组（RI组）和缺血预处理组（IPC组）,每组20只．建立大鼠压疮缺血再灌注损伤处理模型,通过肉眼观察受压程度,检测血清氧自由基的水平,以判断压疮缺血再灌注损伤程度。结果RI组和IPC组大鼠出现I期压疮的发生率为100％。IPC组血清一氧化氮、丙二醛含量高于S组,明显低于RI组．超氧化物歧化酶活性低于S组,明显高于RI组。结论缺血预处理对压疮缺血再灌注损伤具有保护作用。     

Advanced glycation end products accelerate ischemia/reperfusion injury through receptor of advanced end product/nitrative thioredoxin inactivation in cardiac microvascular endothelial cells

The advanced glycation end products (AGEs) are associated with increased cardiac endothelial injury. However, no causative link has been established between increased AGEs and enhanced endothelial injury after ischemia/reperfusion. More importantly, the molecular mechanisms by which AGEs may increase endothelial injury remain unknown. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and incubated with AGE-modified bovine serum albumin (BSA) or BSA. After AGE-BSA or BSA preculture, CMECs were subjected to simulated ischemia (SI)/reperfusion (R). AGE-BSA increased SI/R injury as evidenced by enhanced lactate dehydrogenase release and caspase-3 activity. Moreover, AGE-BSA significantly increased SI/R-induced oxidative/nitrative stress in CMECs (as measured by increased inducible nitric oxide synthase expression, total nitric oxide production, superoxide generation, and peroxynitrite formation) and increased SI/R-induced nitrative inactivation of thioredoxin-1 (Trx-1), an essential cytoprotective molecule. Supplementation of EUK134 (peroxynitrite decomposition catalyst), human Trx-1, or soluble receptor of advanced end product (sRAGE) (a RAGE decoy) in AGE-BSA precultured cells attenuated SI/R-induced oxidative/nitrative stress, reduced SI/R-induced Trx-1 nitration, preserved Trx-1 activity, and reduced SI/R injury. Our results demonstrated that AGEs may increase SI/R-induced endothelial injury by increasing oxidative/nitrative injury and subsequent nitrative inactivation of Trx-1. Interventions blocking RAGE signaling or restoring Trx activity may be novel therapies to mitigate endothelial ischemia/reperfusion injury in the diabetic population.     

Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia. A histochemical study.

This paper describes a histochemical study of superoxide generation in buffer-perfused, isolated rat hearts during the first 2 minutes of reperfusion after 60 minutes of warm ischemia. Superoxide radical production was demonstrated by a modification of Karnovsky's manganese/diaminobenzidine technique, in which superoxide oxidizes Mn++ to Mn ions, which in turn oxidize diaminobenzidine to form amber, osmiophilic polymers, observable by light or electron microscopy. Isolated hearts were rendered ischemic, reperfused with oxygen equilibrated buffer containing Mn++ and diaminobenzidine, fixed by perfusion with Trump's solution, and processed for light and electron microscopy. The method consistently demonstrated evidence of superoxide generation near the luminal surfaces of arterial, capillary, and venular endothelial cells during the first 2 minutes of reoxygenation after ischemia. The histochemical reaction was absent or markedly reduced in non-manganese-treated or nonischemic hearts, as well as in hearts perfused with calcium-free or oxygen-free buffers. The histochemical differences were statistically significant on quantitative morphometric analysis. These results provide direct, visual evidence of the existence and endothelial localization of a burst of superoxide radicals in intact, postischemic myocardium and suggest the pathophysiologic importance of calcium-dependent endothelial cell activation in the initiation of reperfusion injury.     

Characterization of in vivo tissue redox status, oxygenation, and formation of reactive oxygen species in postischemic myocardium

The current study aims to characterize the alterations of in vivo tissue redox status, oxygenation, formation of reactive oxygen species (ROS), and their effects on the postischemic heart. Mouse heart was subjected to 30 min LAD occlusion, followed by 60 min reperfusion. In vivo myocardial redox status and oxygenation were measured with electron paramagnetic resonance (EPR). In vivo tissue NAD(P)H and formation of ROS were monitored with fluorometry. Tissue glutathione/glutathione disulfide (GSH/GSSG) levels were detected with high-performance liquid chromatography (HPLC). These experiments demonstrated that tissue reduction rate of nitroxide was increased 100% during ischemia and decreased 33% after reperfusion compared to the nonischemic tissue. There was an overshoot of tissue oxygenation after reperfusion. Tissue NAD(P)H levels were increased during and after ischemia. There was a burst formation of ROS at the beginning of reperfusion. Tissue GSH/GSSG level showed a 48% increase during ischemia and 29% decrease after reperfusion. In conclusion, the hypoxia during ischemia limited mitochondrial respiration and caused a shift of tissue redox status to a more reduced state. ROS generated at the beginning of reperfusion caused a shift of redox status to a more oxidized state, which may contribute to the postischemic myocardial injury.     

Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis

A subset of familial cases of amyotrophic lateral sclerosis are linked to missense mutations in copper/zinc superoxide dismutase type 1. Patients with missense mutations in copper/zinc superoxide dismutase type 1 develop a paralytic disease indistinguishable from sporadic amyotrophic lateral sclerosis through an unknown toxic gain of function. Nitric oxide reacts with the superoxide anion to form the strong oxidant, peroxynitrite, which participates in neuronal injury in a variety of model systems. Peroxynitrite is an alternate substrate for copper/zinc superoxide dismutase type 1, causing catalytic nitration of tyrosine residues in other proteins. Mutations in copper/zinc superoxide dismutase type 1 may disrupt the active site of the enzyme and permit greater access of peroxynitrite to copper, leading to increased nitration by peroxynitrite of critical cellular targets. To investigate whether neuronal-derived nitric oxide plays a role in the pathogenesis of familial amyotrophic lateral sclerosis, we examined the effects of three different nitric oxide synthase inhibitors: a non-selective nitric oxide synthase inhibitor, nitro-L-arginine methyl ester; a relatively selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole; and a novel highly selective neuronal nitric oxide synthase inhibitor, AR-R 17,477, in transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 (Gly-->Ala at position 93; G93A) containing a high transgene copy number and a low transgene copy number. AR-R 17,477, but not nitro-L-arginine methyl ester or 7-nitroindazole, significantly prolonged survival in both the high and low transgene transgenic mice. To determine whether neuronal nitric oxide synthase is involved in the pathogenesis resulting from the familial amyotrophic lateral sclerosis copper/zinc superoxide dismutase type 1 mutation, we produced mice with the copper/zinc superoxide dismutase type 1 mutation which lack the neuronal nitric oxide synthase gene. The transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background do not live significantly longer than transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1. Western blot analysis indicates the presence of two neuronal nitric oxide synthase-like immunoreactive bands in spinal cord homogenates of the neuronal nitric oxide synthase null mice, and residual neuronal nitric oxide synthase catalytic activity ( > 7%) is detected in the spinal cord of the transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background. This amount of residual activity probably does not account for lack of protection afforded by the disrupted neuronal nitric oxide synthase gene in the familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 mice. Immunological nitric oxide synthase is not detected in the copper/zinc superoxide dismutase type 1 mutant mice at several different ages, thus excluding immunological nitric oxide synthase as a contributor to the pathogenesis of familial amyotrophic lateral sclerosis. Levels of neuronal nitric oxide synthase as well as Ca2+-dependent nitric oxide synthase catalytic activity in the copper/zinc superoxide dismutase type 1 mutant mice do not differ from wild type mice. Endothelial nitric oxide synthase levels may be decreased in the copper/zinc superoxide dismutase type 1 mutant mice. Together, these results do not support a significant role for neuronal-derived nitric oxide in the pathogenesis of familial amyotrophic lateral sclerosis transgenic mice.     

Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus.

Macro and microvascular diseases are the principal causes of morbidity and mortality in patients with type I and II diabetes mellitus. Growing evidence implicates reactive nitrogen species (RNS), such as peroxynitrite (ONOO-), derived from nitric oxide (NO) and superoxide anion (O2*-), are important in diabetes. The mechanisms by which diabetes increases RNS, and those by which RNS modifies vascular function, are poorly understood. The authors recently discovered that physiologically relevant concentrations of ONOO- oxidize the zinc thiolate center in endothelial nitric oxide synthase (eNOS). In active eNOS dimers, a tetracoordinated zinc ion is held by four thiols, two from each 135-kDa monomer. Because it remains partially positively charged, the zinc thiolate center is subject to attack by the ONOO-. This oxidant disrupts the zinc thiolate center, releasing zinc, and oxidizing the thiols. Upon thiol reduction, eNOS dimers dissociate into monomers. This modification of eNOS results in reduced NO bioactivity and enhanced endothelial O2*- production, which reacts with NO, further generating ONOO- (eNOS uncoupling). In addition, the authors' studies also demonstrate that low concentrations of ONOO- selectively nitrate and inactivate prostacyclin synthase (PGIS), which not only eliminates the vasodilatory, growth-inhibiting, antithrombotic, and antiadhesive effects of prostacyclin (PGI2), but also increases release of the potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents, prostaglandin H2 (PGH2) and thromboxane A2 (TxA2). In diabetic mice and rats, eNOS is uncoupled resulting in an increased tyrosine nitration of PGIS. The authors' studies indicate that in diabetes the synthetic enzymes of the two major endogenous vasodilators undergo oxidative inactivation by different mechanisms, which are, however, tightly interdependent.     

七氟醚预处理对缺血再灌注大鼠心肌活性氧产生的影响及其发挥心肌保护作用的机制

目的: 观察七氟醚预处理对缺血再灌注大鼠心肌活性氧以及一氧化氮(NO)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、过氧化氢酶(CAT)活性的影响,进一步探讨活性氧在七氟醚预处理减轻心肌缺血再灌注损伤中的作用。方法: 60只SD大鼠随机分为8组。在体用2%七氟醚预处理30 min 后结扎冠状动脉前降支30 min,然后再灌注120 min。以心肌梗死面积和凋亡指数反映心肌损伤情况,心肌梗死面积用氯化三苯基四氮唑染色显示,细胞凋亡用TUNEL染色显示。活性氧用活性氧荧光探针二氢乙啶测量。使用活性氧清除剂N-(2-巯基丙酰基)甘氨酸(2-MPG)以及一氧化氮合酶抑制剂N^ω-硝基-L-精氨酸甲酯(L-NAME)作为阻滞剂,用酶标仪测定心肌匀浆的NO、SOD、GPx和CAT活性,并进一步分析阻断活性氧及NO产生对七氟醚减轻心肌缺血再灌注损伤的影响。结果: 与对照组比较,七氟醚预处理在缺血再灌注前诱导活性氧产生(12.0±0.8 vs 2.6±0.5, P<0.05),在缺血再灌注后减少活性氧产生(16.2 ±0.9 vs 24.9±1.3, P<0.05);在缺血再灌注前,2-MPG减少七氟醚预处理心肌活性氧产生(5.1±0.7 vs 12.0±0.8, P<0.05),在缺血再灌注后2-MPG+七氟醚预处理组与缺血再灌注组比较无显著差别(24.9±1.4 vs 24.9±1.3, P>0.05);与对照组比较,七氟醚预处理同样诱导NO产生(34.5±3.2 vs 15.9±1.4, P<0.05),增加SOD(1.5±0.5 vs 0.6±0.2, P<0.05)、GPx(22.8±2.5 vs 12.7±2.2, P<0.05)和CAT(15.5±1.8 vs 11.2±1.4, P<0.05)的活性;2-MPG消除了七氟醚对NO、SOD、CPx和CAT的诱导作用和对缺血再灌注心肌的保护作用; L-NAME同样消除七氟醚预处理对SOD、GPx和CAT的诱导作用和心肌保护作用。结论: 七氟醚预处理减少心肌梗死面积和凋亡指数;七氟醚预处理产生的亚损伤量的活性氧和NO诱导缺血再灌注心肌SOD、GPx和CAT的产生,进而抑制缺血再灌注活性氧的产生和心肌损伤。     

Functional significance of inducible nitric oxide synthase induction and protein nitration in the thermally injured cutaneous microvasculature

Increased nitric oxide (NO) production after burn injury is well established. However, there is little information relating to the reactions that occur as a consequence of NO generation under such circumstances. We have investigated the synthesis and function of NO in a rat model of local cutaneous thermal injury. We show that NO levels are elevated from 3 hours after injury with a concomitant increase in protein nitration. A selective inducible nitric oxide synthase (iNOS) inhibitor (1400W) significantly attenuated NO synthesis, protein nitration, and neutrophil accumulation in this model, but had no effect on edema formation. The results also indicate that NO synthesis and protein nitration occurred independently of neutrophil accumulation because these parameters were unaffected by depletion of circulating neutrophils. 3-Chlorotyrosine, a marker of neutrophil/myeloperoxidase-mediated protein damage was significantly increased from 1 hour after burn. Our observations provide evidence for the involvement of reactive species in the inflammatory response after burn. The use of selective iNOS inhibitors may represent a novel approach for the management of human burn injuries.     

Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease

Nitric oxide (NO) and reactive oxygen species (ROS) are important mediators in the pathogenesis of inflammatory bowel disease (IBD). NO in IBD can be either harmful or protective. NO can react with superoxide anions (O2.-), yielding the toxic oxidizing agent peroxynitrite (ONOO-). Peroxynitrite induces nitration of tyrosine residues (nitrotyrosine), leading to changes of protein structure and function. The aim of this study was to identify the cellular source of inducible nitric oxide synthase (iNOS) and to localize superoxide anion-producing cells in mucosal biopsies from patients with active IBD. Additional studies were performed to look at nitrotyrosine formation as a measure of peroxynitrite-mediated tissue damage. For this, antibodies against iNOS, endothelial NOS (eNOS), and nitrotyrosine were used. ROS-producing cells were detected cytochemically. Inflamed mucosa of patients with active IBD showed intense iNOS staining in the epithelial cells. iNOS could not be detected in non-inflamed mucosa of IBD patients and control subjects. eNOS was present in blood vessels, without any difference in the staining intensity between IBD patients and control subjects. ROS-producing cells were increased in the lamina propria of IBD patients; a fraction of these cells were CD15-positive. Nitrotyrosine formation was found on ROS-positive cells. These results show that iNOS is induced in epithelial cells from patients with active ulcerative colitis or Crohn's disease. Nitration of proteins was detected only on the ROS-producing cells at some distance from the iNOS-producing epithelial cells. These findings indicate that tissue damage during active inflammation in IBD patients is probably more related to ROS-producing cells than to NO. One may speculate that NO has a protective role when during active inflammation other mucosal defence systems are impaired.