不同浓度脂联素通过减轻氧化应激损伤保护缺血再灌注心肌

目的观察不同浓度脂联素对大鼠心肌缺血再灌注损伤及其所引起的氧化应激的影响,以探讨脂联素保护缺血再灌注心肌是否与减轻氧化应激有关。方法将80只健康SD大鼠随机分成假手术组、缺血再灌注组、低浓度脂联素组(60 ng/g)、中浓度脂联素组(120 ng/g)和高浓度脂联素组(180 ng/g),每组16只。假手术组只穿线,不结扎,旷置225 m in;缺血再灌注组冠状动脉前降支结扎45 m in后,再灌注180 m in;各脂联素组于缺血前30m in经股静脉给予不同浓度脂联素,再进行缺血再灌注。各组进一步随机分为两个亚组。亚组1(8只)在缺血45m in再灌注180 m in后,用Evans b lue-TTC双染法测定心肌梗死面积;亚组2(8只)在大鼠再灌注180 m in后对左心室内压及单位时间左心室内压变化值(±dp/dt)等血流动力学指标进行检测。实验结束后,在心尖处取血并取心肌组织,测定大鼠血清超氧化物歧化酶活性、心肌组织总一氧化氮合酶及一氧化氮含量。结果与假手术组比较,缺血再灌注组大鼠血清中超氧化物歧化酶活性及心肌组织中总一氧化氮合酶和一氧化氮含量明显下降,心肌梗死面积增大;与缺血再灌注组比较,各浓度脂联素组心肌梗死面积减小,心肌舒缩功能有所改善,大鼠血清超氧化物歧化酶活性及心肌组织总一氧化氮合酶和一氧化氮含量显著增加,并随脂联素浓度增加而增加。结论脂联素对缺血再灌注心肌细胞有保护作用,减少缺血再灌注心肌的梗死面积,改善心脏舒缩功能;其作用机制可能是通过增加缺血再灌注心肌组织总一氧化氮合酶和一氧化氮含量及血清超氧化物歧化酶活性,从而减轻氧化应激损伤。     

急性心肌梗死大鼠心脏一氧化氮合酶表达的改变

目的 通过建立大鼠心肌梗死模型，观察急性心肌梗死对大鼠心脏内皮型一氧化氮合酶mRNA和诱导型一氧化氮合酶蛋白表达的影响。方法48只健康成年SD大鼠（体重200～250g）随机分为假手术组和缺血组，取1、2、8和24h四个不同时间点观察。采用开胸结扎冠状动脉左前降支建立心肌缺血模型，逆转录聚合酶链反应检测大鼠心肌梗死后1、2及24h三个时段缺血心肌内皮型一氧化氮合酶mRNA的表达；免疫组织化学染色检测冠状动脉结扎后8h缺血心肌诱导型一氧化氮合酶蛋白的表达。结果冠状动脉结扎后2h，缺血组大鼠缺血心肌组织内皮型一氧化氮合酶mRNA表达下降（P〈0．05），并持续至结扎后24h；结扎后24h组内皮型一氧化氮mRNA的表达与结扎后2h组相比无显著性差异（P〉0．05）。冠状动脉结扎后8h，梗死区存活心肌组织细胞诱导型一氧化氮合酶蛋白大量表达，而假手术组未见诱导型一氧化氮合酶蛋白表达。结论正常大鼠心肌组织有内皮型一氧化氮合酶基因表达，无诱导型一氧化氮合酶蛋白表达。在心肌梗死早期缺血心肌内皮型一氧化氮合酶mRNA表达减少。心肌急性缺血刺激早期诱导大鼠缺血心肌组织诱导型一氧化氮合酶蛋白大量表达。     

川芎嗪减轻大鼠心肌缺血再灌注损伤炎症反应及机制

目的观察川芎嗪预处理对大鼠心肌缺血再灌注损伤炎症反应的影响并探讨其可能的机制。方法 48只雄性SD大鼠随机分为假手术组、缺血再灌注组、川芎嗪组、左旋-硝基-精氨酸甲酯组以及川芎嗪+左旋-硝基-精氨酸甲酯组,假手术组左前降支近端穿线但不结扎,其余四组给予结扎前降支缺血35 min,再灌注120 min。光镜观察大鼠心肌组织结构变化,测定缺血心肌组织超氧化物歧化酶活性和丙二醛含量及髓过氧化物酶、白细胞介素1β、一氧化氮含量,逆转录聚合酶链反应和免疫印迹法测定心肌内皮型一氧化氮合酶mRNA和蛋白的表达水平。结果与缺血再灌注组相比,川芎嗪预处理能减少心肌白细胞浸润,增加心肌组织超氧化物歧化酶活性,降低丙二醛含量及髓过氧化物酶活性,降低白细胞介素1β水平,增加一氧化氮含量及心肌内皮型一氧化氮合酶mRNA和蛋白的表达水平,左旋-硝基-精氨酸甲酯显著抑制上述指标的变化并取消了川芎嗪所致的内皮型一氧化氮合酶mRNA和蛋白表达水平的增加。结论川芎嗪预处理能减少大鼠心肌缺血再灌注损伤的炎症反应,其机制可能与上调内皮型一氧化氮合酶表达,增加内源性一氧化氮水平有关。     

一氧化氮在心肌缺血再灌注时对心肌功能的影响

动物实验发现心肌缺血／再灌注损伤能降低心肌细胞Ｃａ＾２＋依赖的一氧化氮合酶的活性,导致内皮细胞和心肌细胞功能障碍,使细胞合成一氧化氮的能力受损。心肌缺血所导致的心功能障碍与心肌组织内一氧化氮含量减少有关,缺血后再灌注一氧化氮合成前体左旋精氨酸,可改善因缺血引起的左室舒缩功能障碍,而一氧化氮合酶抑制剂可以对心功能产生负性肌力效应,影响缺血后心功能的恢复。     

一氧化氮在心肌缺血再灌注时对心肌功能的影响

动物实验发现心肌缺血／再灌注损伤能降低心肌细胞 Ｃａ２ ＋ 依赖的一氧化氮合酶的活性,导致内皮细胞和心肌细胞功能障碍,使细胞合成一氧化氮的能力受损。心肌缺血所导致的心功能障碍与心肌组织内一氧化氮含量减少有关,缺血后再灌注一氧化氮合成前体左旋精氨酸,可改善因缺血引起的左室舒缩功能障碍,而一氧化氮合酶抑制剂可对心功能产生负性肌力效应,影响缺血后心功能的恢复     

氟伐他汀对正常血脂兔心肌缺血再灌注损伤的保护作用

目的　观察氟伐他汀对正常血脂兔的心肌缺血再灌注损伤有无保护作用及其可能原因。方法　将2 4只标准饲养的日本大耳白兔随机分为假手术组、缺血再灌注组和氟伐他汀组,氟伐他汀组在行缺血再灌注术前给予氟伐他汀10mg/ (kg·d)干预一周。建立心肌缺血再灌注模型,监测血流动力学变化;检测乳酸脱氢酶和肌酸激酶活性;以Evans蓝和TTC双重染色方法测量心肌梗死面积。取局部梗死区及对应部位心肌检测诱导型一氧化氮合酶活性的表达。结果　与缺血再灌注组比较,氟伐他汀组心肌梗死面积、乳酸脱氢酶1及肌酸激酶活性均显著减小(P <0 .0 5及P <0 .0 1) ;缺血即刻始氟伐他汀组各时间点较缺血再灌注组左心室舒张末压减小(P <0 .0 5 ) ,左心室内压变化最大速率(±dp/dtmax)增大(P <0 .0 5 ) ;缺血再灌注组及氟伐他汀组总一氧化氮合酶活性均显著高于假手术组(P <0 .0 5 ) ,缺血再灌注组诱导型一氧化氮合酶活性与总一氧化氮合酶活性比值显著大于假手术组和氟伐他汀组(P <0 .0 1)。结论　氟伐他汀可以保护正常血脂兔的心肌缺血再灌注损伤,其机制可能部分与调节一氧化氮合酶活性的表达有关。     

没食子酸对缺血再灌注损伤大鼠的保护作用

目的探讨没食子酸(GA)对大鼠心肌缺血/再灌注(I/R)损伤的保护作用及其机制。方法 32只雄性Wistar大鼠随机分为假手术组、缺血/再灌注组(模型组)、阳性药丹参组(DS)(100 mg/kg)组、GA(20 mg/kg)组,每组8只,结扎左冠状动脉前降支40 min,松扎再灌注120 min制备大鼠心肌I/R损伤模型,检测GA对血清中MDA和SOD以及NO的影响,同时以光镜观察心肌细胞病理组织形态学变化,免疫组化法检测心肌细胞诱导型一氧化氮合酶(iNOS)的蛋白表达。结果 GA组与模型组比较,MDA含量明显降低(P0.01),SOD活力增强(P0.01),NO含量降低(P0.01)。HE染色组织形态学观察显示,与模型组比较,GA组心肌细胞形态明显改善,炎细胞浸润减轻。免疫组化法检测结果显示,GA组心肌细胞iNOS表达量低于模型组(P0.05)。结论 GA可能通过抑制iNOS,减少氧自由基生成,对大鼠心肌I/R损伤产生保护作用。     

诱导型一氧化氮合酶在衰老大鼠心肌对缺血再灌注损伤敏感性增加中的作用

目的探讨衰老大鼠心肌组织过氧亚硝基阴离子(ONOO-)的来源及其在衰老大鼠心肌对缺血再灌注损伤敏感性增加中的作用。方法选取雄性成年SD大鼠和衰老SD大鼠,随机分为3组,成年缺血再灌注组(缺血30 min,再灌注24 h)、衰老缺血再灌注组(缺血30 min,再灌注24 h),衰老缺血再灌注+1 400W组,缺血30 min,再灌注24 h,缺血前24 h及再灌注前25 h分别腹腔注射诱导型一氧化氮合酶(iNOS)的特异性阻断剂1 400W。采用Evans blue和TTC双染法检测心梗面积;用ELISA法检测心肌组织硝基酪氨酸(NT)含量;用Western-blot蛋白印迹法检测大鼠心肌组织中iNOS的蛋白表达水平。结果与成年缺血再灌注组相比,衰老缺血再灌注组心梗面积增大(P0.05);心肌组织NT含量较成年缺血再灌注组明显增高(7.29±0.1 vs 4.61±0.1,P0.05);iNOS表达增高(P0.05);与衰老缺血再灌注组比较,衰老缺血再灌注+1 400W组NT含量减少(3.2±0.1 vs 7.29±0.1,P0.05);心肌梗死面积减小。结论衰老大鼠对心肌缺血再灌注损伤的敏感性增加可能与衰老大鼠心脏中iNOS表达升高有关,催化生成大量NO进而生成毒性的ONOO-,从而损伤心肌。     

诱导型一氧化氮合酶和解偶联蛋白-2对心肌缺血预适应的作用

目的 探讨诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）和解偶联蛋白-2（uncoupling protein-2,UCP2）对大鼠心肌缺血预适应的保护机制。方法 结扎左冠状动脉复制大鼠心肌缺血再灌注模型。预适应组行3次缺血5min,再灌注10min的预处理,分别于预处理0,6,12,24和48h（分别为0,6,12,24和48h亚组）后行30min缺血及120min再灌注：对照组开胸后不结扎左冠状动脉,电镜观察心肌超微结构,据Rainio评分标准进行心肌超微结构损伤程度的半定量分析。采用Western Blot及比色法检测心肌UCP2活性及iNOS活性。结果 预适应各亚组UCP2活性均增高（P〈0．05）,0h亚组UCP2表达水平最高（P〈0．01）,24小时亚组和48小时亚组心肌iNOS活性显著升高（P〈0．05）。结论 UCP2和iNOS共同参与了大鼠心肌缺血预适应心肌保护作用。     

替罗非班对大鼠心肌缺血再灌注后无复流及一氧化氮合酶活性和一氧化氮含量的影响

目的研究替罗非班对大鼠心肌缺血再灌注后无复流及一氧化氮合酶(NOS)活性、一氧化氮(NO)含量的影响,探讨替罗非班改善心肌缺血再灌注后无复流的作用机制。方法雄性Wistar大鼠,随机分为假手术组、对照组和替罗非班组。建立急性心肌缺血再灌注无复流模型,用硫黄素S活体染色,观察大鼠心肌无复流范围;伊文斯蓝、氯化三苯基四氮唑(TTC)染色评估大鼠心肌缺血及梗死范围;紫外分光光度计测定缺血再灌注120min时缺血区心肌内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)、总一氧化氮合酶(tNOS)活性及NO含量。结果缺血再灌注后120min,替罗非班组大鼠与对照组大鼠心肌缺血范围相似[(43.13±5.69)%比(39.98±3.75)%,P>0.05],但无复流范围及梗死范围明显小于对照组[(34.36±6.04)%比(52.09±6.89)%,P<0.01;(80.41±8.48)%比(90.13±5.72)%,P<0.05);对照组大鼠心肌的eNOS活性低于假手术组,iNOS、tNOS活性及NO含量高于假手术组(P<0.01);替罗非班组大鼠心肌的iNOS活性及NO含量高于假手术组(P<0.05,P<0.01),eNOS、tNOS活性与假手术组差异无统计学意义。与对照组比较,替罗非班组大鼠心肌的eNOS活性高于对照组(P<0.05),iNOS活性及NO含量低于对照组(P<0.05,P<0.01),tNOS活性较对照组有降低的趋势,但差异无统计学意义。结论大鼠心肌缺血90min再灌注120min可发生无复流现象;替罗非班可缩小无复流及梗死范围,其机制可能与保护内皮功能有关。     

肝炎患者血清一氧化氮和一氧化氮合酶水平变化及其临床意义

目的 探讨血清一氧化氮水平、总一氧化氮合酶和诱导型一氧化氮合酶活力与病毒性肝炎的关系.方法 用生化方法 检测对照组、中度慢性乙型肝炎、肝硬化、慢性重症肝炎及乙戊肝重叠感染患者血清一氧化氮、总一氧化氮合酶和诱导型一氧化氮合酶水平.结果 中度慢性乙型肝炎、肝硬化及乙戊肝重叠感染患者血清一氧化氮水平、总一氧化氮合酶和诱导型一氧化氮合酶活力与对照组比较明显升高,慢性重症肝炎患者血清一氧化氮水平、总一氧化氮合酶和诱导型一氧化氮合酶活力与对照组比较明显降低(P＜0.05).结论 一氧化氮、总一氧化氮合酶和诱导型一氧化氮合酶在病毒性肝炎发病中具有重要作用,并在一定程度上与肝损害程度相关.     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice

Mitochondrial nitric oxide synthase (mtNOS) produces nitric oxide (NO) to modulate mitochondrial respiration. Besides a constitutive mtNOS isoform it was recently suggested that mitochondria express an inducible isoform of the enzyme during sepsis. Thus, the mitochondrial respiratory inhibition and energy failure underlying skeletal muscle contractility failure observed in sepsis may reflect the high levels of NO produced by inducible mtNOS. The fact that mtNOS is induced during sepsis suggests its relation to inducible nitric oxide synthase (iNOS). Thus, we examined the changes in mtNOS activity and mitochondrial function in skeletal muscle of wild-type (iNOS(+/+)) and iNOS knockout (iNOS(-/-)) mice after sepsis. We also studied the effects of melatonin administration on mitochondrial damage in this experimental paradigm. After sepsis, iNOS(+/+) but no iNOS(-/-) mice showed an increase in mtNOS activity and NO production and a reduction in electron transport chain activity. These changes were accompanied by a pronounced oxidative stress reflected in changes in lipid peroxidation levels, oxidized glutathione/reduced glutathione ratio, and glutathione peroxidase and reductase activities. Melatonin treatment counteracted both the changes in mtNOS activity and rises in oxidative stress; the indole also restored mitochondrial respiratory chain in septic iNOS(+/+) mice. Mitochondria from iNOS(-/-) mice were unaffected by either sepsis or melatonin treatment. The data suggest that inducible mtNOS, which is coded by the same gene as that for iNOS, is responsible for mitochondrial dysfunction during sepsis. The results also suggest the use of melatonin for the protection against mtNOS-mediated mitochondrial failure.     

Nitric oxide and cardioprotection during ischemia-reperfusion

Coronary artery reperfusion is widely used to restore blood flow in acute myocardial infarction and limit its progression. However, reperfusion of ischemic myocardium results in reperfusion injury and persistent ventricular dysfunction even when achieved after brief periods of ischemia. Normally, small amounts of nitric oxide (NO) generated by endothelial NO synthase (eNOS) regulates vascular tone. Ischemia-reperfusion triggers the release of oxygen free radicals (OFRs) and a cascade involving endothelial dysfunction, decreased eNOS and NO, neutrophil activation, increased cytokines and more OFRs, increased inducible NO synthase (iNOS) and marked increase in NO, excess peroxynitrite formation, and myocardial injury. Low doses of NO appear to be beneficial and high doses harmful in ischemia-reperfusion. eNOS knock-out mice confirm that eNOS-derived NO is cardioprotective in ischemia-reperfusion. iNOS overexpression increases peroxynitrite but did not cause severe dysfunction. Increased angiotensin II (AngII) after ischemia-reperfusion inactivates NO, forms peroxynitrite and produces cardiotoxic effects. Beneficial effects of angiotensin-converting-enzyme inhibition and AngII type 1 (AT₁) receptor blockade after ischemia-reperfusion are partly mediated through AngII type 2 (AT₂) receptor stimulation, increased bradykinin and NO. Interventions that enhance NO availability by increasing eNOS might be beneficial after ischemia-reperfusion.     

Inhibition of Inducible Nitric Oxide Synthase Prevents Hepatic, but Not Pulmonary, Injury Following Ischemia-Reperfusion of Rat Liver

The aim of this study was to investigate the contribution of inducible nitric oxide synthase (iNOS)-derived nitric oxide on the liver and lung injury following hepatic ischemia-reperfusion (I/R) using a novel and potent iNOS inhibitor, ONO-1714. Rats were subjected to 90 min of partial hepatic ischemia followed by 3, 6, 12, and 24 hr of reperfusion. Expression of iNOS mRNA peaked at 3 hr of reperfusion in the liver and lung. Plasma nitric oxide levels were increased fourfold at 24 hr of reperfusion and plasma ALT was increased, reaching a peak at 12 hr of reperfusion; both were significantly inhibited by ONO-1714. Histological examination revealed extensive liver damage, whereas this was not seen in the ONO-1714 group. Lung injury was not significantly changed in groups with versus without ONO-1714. Nitrotyrosine expression was seen in regions similar to those of the histological injuries of the liver, while this staining was absent in the ONO-1714 group. These data show that generation of peroxynitrite could be involved in the pathogenesis of liver injury but not lung injury after hepatic I/R. Inhibition of iNOS could be applied for attenuation of liver injury following hepatic I/R.     

iNOS Expression in Oral and Gastrointestinal Tract Mucosa

It is known that the overproduction of nitric oxide (NO) by nitric oxide synthase (NOS) occurs during the progression of various inflammatory diseases in intestinal tract. NOS inhibitors or inducible nitric oxide synthase (iNOS) gene expression inhibitors should be considered as potential anti-inflammatory agents, as NO synthesized by iNOS is related to various pathophysiological processes including inflammation. In order to understand the relationship between iNOS and pathological reactions such as the inflammatory process and malign transformation clearly, the existence and amount of constitutive expression should be determined. It is crucial to comprehend the harmful and protective amounts of iNOS expressions in order to clarify the relationship between iNOS and pathological processes. Evidently, only after this inspection is it possible to utilize iNOS as a marker and treatment instrument during the diagnosis and treatment of malign transformation and the inflammatory process.     

肝癌组织中一氧化氮合酶及其基因表达的研究

目的研究肝癌组织中一氧化氮合酶(iNOS)及其基因表达与肝癌发生发展的关系。方法用免疫组化和原位杂交的方法对21例肝癌及癌旁组织中的诱导型一氯化氮合酶(iNOS)及其基因表达进行原位检测和观察。结果:NOS 阳性反应物质呈黄色或棕黄色,位于细胞浆中。非癌殖织(肉眼观距癌组织边缘>1.5)多呈阴性或弥漫弱阳性,但部分非癌组织中可见 iNOS 呈阳性的细胞呈点状分布;癌旁组织多呈阳性,提示 iNOS 表达与肝组织癌变有关。癌组织核心多呈阴性或弥漫弱阳性,但分化中和差的癌组织核心也分别有一例 NOS 呈强阳性;周边癌组织呈局灶阳性,侵入纤维组织中的弥敢癌细胞星强阳性,提示 NOS 的表达与肝癌组织的侵润能力有关。肝癌组织 iNOSmRNA 阳性细胞的分布与 iN-OS 蛋白的表达基本相似。结论 iNOS 蛋白及其基因表达与肝组织癌变及肝癌侵润能力有关。     

iNOS/NO体系在慢性肝炎及肝硬化患者中的表达

目的:探讨内源性NO在慢性病毒性肝炎-肝硬化发展进程中的作用.方法:采用硝酸还原酶法比色测定外周静脉及门静脉血浆中iNOS活性,并用免疫组织化学和RT-PCR方法观察肝组织iNOS蛋白及RNA的表达.结果:在慢性肝炎患者及肝硬化患者门静脉血与外周血中iNOS活性与对照组相比均明显升高(F=102.793,25.052,P<0.01),且门静脉血iNOS活性增高更为明显.慢性肝炎组及肝硬化组中iNOS蛋白的灰阶值均低于正常对照组(F=46.796,P<0.05),表明iNOS表达增强.慢性肝炎组、肝硬化组iNOS mRNA的表达均分别显著高于正常对照组(F=26.832,P<0.01),且随着肝脏病变的加重表达逐渐增加.结论:iNOS/NO体系在慢性肝炎-肝硬化发生发展中起着保持血管舒张状态的重要作用.