环孢菌素A拮抗心肌缺血/再灌注损伤的作用

目的:研究环孢菌素A(CsA)拮抗小型猪心肌缺血/再灌注损伤(MI/RI)的作用及可能的机制。方法:经皮球囊封堵冠状动脉左前降支制备小型猪MI/RI模型。将存活的动物随机分为3组:即对照组(n=4)、CsA组(n=6)及他可英司(FK-506)组(n=6),分别静滴生理盐水100 ml、25 mg/kg CsA及1 mg/kg FK-506。所有动物均经90 min缺血和3 h再灌注。通过病理检查评估心肌梗死(MI)面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果:CsA组MI的面积比对照组[(7.5±0.6)cm2vs.(10.5±2.6)cm2]和FK-506组[(7.5±0.6)cm2vs.(9.6±2.7)cm2]明显减少(P0.01);CsA组心肌细胞的凋亡率(%)比对照组[(11.9±1.88)%vs.(22.3±1.66)%]和FK-506组[(11.9±1.88)%vs.(19.2±1.82)%]明显下降(P0.01)。透射电子显微镜检查显示,CsA组能维持线粒体的形态,线粒体坍塌的百分率为(20%±7%),比对照组(53%±12%)和FK-506组(47%±9%)明显减少(P0.01)。结论:CsA可能对MI/RI具有拮抗作用,其机制可能是通过抑制线粒体膜通透性转换孔(mPTP),保持线粒体形态完整而实现,此种效应不依赖于钙调磷酸酶抑制途径。     

环孢菌素A拮抗心肌缺血／再灌注损伤的作用

目的：研究环孢菌素A（CsA）拮抗小型猪心肌缺血／再灌注损伤（MI／RI）的作用及可能的机制。方法：经皮球囊封堵冠状动脉左前降支制备小型猪MI／RI模型。将存活的动物随机分为3组：即对照组（n=4）、CsA组（n=6）及他可英司（FK-506）组（n=6）,分别静滴生理盐水100ml、25mg／kgCsA及1mg／kgFK-506。所有动物均经90rain缺血和3h再灌注。通过病理检查评估心肌梗死（MI）面积。用免疫组化染色法检测心肌细胞凋亡。用透射电子显微镜观察各组心肌细胞线粒体的形态。结果：CsA组MI的面积比对照组[（7．5±0．6）cm。粥．（10．5±2．6）cm。]和FK-506组[（7．5±0．6）cm。掷．（9．6±2．7）cm。]明显减少（P〈0．01）;CsA组心肌细胞的凋亡率（％）比对照组[（11．9±1．88）％郴．（22．3±1．66）％]和FK-506组[（11．9±1．88）％郴．（19．2±1．82）％]明显下降（JP〈0．01）。透射电子显微镜检查显示,CsA组能维持线粒体的形态,线粒体坍塌的百分率为（20％±7％）,比对照组（53％±12％）和FK-506组（47％±9％）明显减少（P〈0．01）。结论：CsA可能对MI／RI具有拮抗作用,其机制可能是通过抑制线粒体膜通透性转换孔（mPTP）,保持线粒体形态完整而实现,此种效应不依赖于钙调磷酸酶抑制途径。     

Myocardial reperfusion injury in neuronal nitric oxide synthase deficient mice

BACKGROUND: A substantial amount of data suggesting that endothelial cell nitric oxide synthase (eNOS) plays a cardioprotective role in animal models of ischemia-reperfusion injury has amassed. We have previously demonstrated that eNOS-deficient (-/-) mice exhibit significantly larger myocardial infarcts than do wild-type mice. Few investigations have examined the neuronal form of nitric oxide synthase in the heart. The two constitutive isoforms have been demonstrated to play differing roles in studies of cerebral ischemia-reperfusion. OBJECTIVE: To characterize the role of neuronal nitric oxide synthase (nNOS) in myocardial ischemia-reperfusion injury. METHODS: Wild-type and nNOS -/- mice were subjected to 20 min of coronary artery occlusion and 120 min of reflow. RESULTS: We found no significant difference between the two groups in terms of infarct size. Microscopic cross-sections from both groups were examined for infiltration of polymorphonuclear leukocyte. Hearts of nNOS -/- mice exhibited significantly (P < 0.05) more polymorphonuclear leukocytes than did hearts of wild-type mice. CONCLUSION: Despite the fact that eNOS plays a cardioprotective role in the ischemic-reperfused myocardium, we observed no change in size of myocardial infarcts when nNOS was genetically disrupted.     

一氧化氮合酶在N-乙酰半胱氨酸调控大鼠心肌冷缺血-再灌注损伤中的变化

目的:观察一氧化氮合酶(NOS)在N-乙酰半胱氨酸(NAC)调控大鼠心肌冷缺血-再灌注损伤中的变化。方法:将健康雄性Lewis大鼠60只随机分为3组。(1)对照组:摘取供心前30 min,经供体大鼠下腔静脉注射生理盐水0.5 mL;(2)供体预处理组:摘取供心前30 min,经供体大鼠下腔静脉注射NAC300 mg/kg,受体大鼠不作预处理;(3)受体预处理组:移植前30 min,经受体大鼠下腔静脉注射NAC300 mg/kg,供体大鼠不作预处理。将冷藏于4℃HTK液18 h的供心移植至受体大鼠腹腔,建立同种异体心脏移植模型。于再灌注24 h后取供心采用免疫组化方法检测诱导型一氧化氮合酶/内皮型一氧化氮合酶(iNOS/eNOS)蛋白表达水平,以免疫组化评分(IHS)表示。采用Real time-PCR法检测iNOS/eNOS mRNA表达。结果:与对照组相比,供、受体预处理组的iNOS蛋白表达降低,IHS评分分别为3.00±0.15、1.50±0.22、1.63±0.26,P<0.05;而eNOS蛋白表达升高,IHS评分分别为2.00±0.21,3.60±0.16,3.40±0.26,P<0.05。与对照组相比,受体预处理组iNOS mRNA表达降低(0.43±0.17对1.00±0.41,P<0.05),供体预处理组eNOS mRNA表达升高(3.06±1.47对1.00±0.65,P<0.05)。结论:NOS参与了NAC预处理减轻移植大鼠心肌缺血-再灌注损伤的过程。     

Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins

BACKGROUND/AIMS: Bacillus Calmette Guerin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice. METHODS: Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice. RESULTS: iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-gamma serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately. CONCLUSIONS: These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.     

Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism

Although the inducible isoform of NO synthase (iNOS) mediates late preconditioning (PC), it is unknown whether iNOS gene transfer can replicate the cardioprotective effects of late PC, and the role of this protein in myocardial ischemia is controversial. Thus, the cDNA for human iNOS was cloned behind the Rous sarcoma virus (RSV) promoter to create adenovirus (Ad) 5/iNOS lacking E1, E2a, and E3 regions. Intramyocardial injection of Ad5/iNOS in mice increased local iNOS protein expression and activity and markedly reduced infarct size. The infarct-sparing effects of Ad5/iNOS were at least as powerful as those of ischemic PC. The increased iNOS expression was associated with increased cyclooxygenase-2 (COX-2) protein expression and prostanoid levels. Pretreatment with the COX-2-selective inhibitor NS-398 completely abrogated the infarct-sparing actions of Ad5/iNOS, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection. We conclude that gene transfer of iNOS (an enzyme commonly thought to be detrimental) affords powerful cardioprotection the magnitude of which is equivalent to that of late PC. This is the first report that upregulation of iNOS, in itself, is sufficient to reduce infarct size. The results provide proof-of-principle for gene therapy against ischemia/reperfusion injury, which increases local myocardial NO synthase levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. The data also reveal the existence of a close coupling between iNOS and COX-2, whereby induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS. We propose that iNOS and COX-2 form a stress-responsive functional module that mitigates ischemia/reperfusion injury.     

神经调节蛋白-1对大鼠心肌缺血/再灌注损伤保护作用的研究

目的:探讨神经调节蛋白-1(NRG-1)对大鼠心肌缺血/再灌注(I/R)损伤的保护作用及其潜在机制。方法:雄性,SD大鼠,分三组:假手术组(n=8)、心肌(I/R)组(n=8)和NRG-1+I/R组(n=9)。通过结扎冠状动脉左前降支45 min,再灌注3 h建立在体大鼠心肌I/R模型。用伊文氏蓝/2,3,5-三苯基氯化四氮唑(TTC)染色法测定心肌梗死范围。用脱氧核糖核苷酸末端转移酶介导的d UTP缺口末端标记法(TUNEL)染色法检测心肌细胞凋亡指数;免疫荧光法测定线粒体膜电位水平;用Western blot方法检测线粒体细胞色素c的转位、凋亡相关因子Bcl-2和Bax的表达;caspase 3试剂盒检测caspase 3活性;用透射电镜观察心肌组织线粒体超微结构。结果:与假手术组相比,I/R组心肌细胞凋亡显著增加[(23.2±3.8)vs.(3.0±1.3)%,P0.01],心肌线粒体膜电位降低[(209.1±13.6)vs.(336.8±10.3)m V,P0.05],细胞色素c向胞浆发生转位,caspase 3活性显著升高[(20.1±3.6)vs.(8,3±1.5),P0.01]。与单纯I/R组比较,NRG-1给药显著降低I/R大鼠心肌的梗死面积/危险区面积[(28.6±9.2)vs.(51.7±7.8)%,P0.01],减少心肌细胞凋亡指数[(11.9±3.5)vs.(23.2±3.8)%,P0.01],升高Bcl-2/Bax蛋白表达比值[(1.647±0.172)vs.(0.490±0.080),P0.01],升高线粒体膜电位[(327.2±15.4)vs.(209.1±13.6)m V,P0.05],抑制细胞色素c向胞浆的转位[(0.207±0.055)vs.(0.483±0.075),P0.01],降低心肌caspase 3活性[(9.3±2.6)vs.(20.1±3.6),P0.01],改善线粒体超微结构。结论:NRG-1具有抗心肌I/R损伤的保护作用,其机制部分通过抑制线粒体介导的心肌细胞凋亡途径实现。     

Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase

OBJECTIVE: The role of nitric oxide (NO) in myocardial ischemia/reperfusion injury remains controversial as both NO donors and NO synthase (NOS) inhibitors have shown to be protective. We generated transgenic (TG) mice that overexpress endothelial NOS (eNOS) exclusively in cardiac myocytes to determine the effects of high cardiac NO levels on ischemia/reperfusion injury and cellular Ca(2+) homeostasis. Wild-type (WT) mice served as controls. METHODS: Hearts were perfused in vitro and subjected to 20 min of total no-flow ischemia and 30 min of reperfusion (n=5 per group). Left ventricular function, cGMP levels and intracellular Ca(2+) transients (Ca(2+)(i)) were determined. RESULTS: Left ventricular pressure was reduced (maximum, -33%) and basal cardiac cGMP was increased (twofold) in TG hearts, and the changes were reversed by NOS blockade with N(G)-nitro-L-arginine methyl ester (L-NAME). Relative to baseline, recovery of reperfusion contractile function was significantly better in hearts from TG (98%) than WT (51%) mice, and L-NAME abolished this effect. Heart rate and coronary perfusion pressure were not different between groups. Systolic and diastolic Ca(2+)(i) concentrations were similar in WT and TG hearts, but Ca(2+)(i) overload during early reperfusion tended to be less in TG hearts. Kinetic analysis of pressure curves and Ca(2+)(i) transients revealed a faster left ventricular diastolic relaxation and abbreviated aequorin light signals in TG hearts at baseline and during reperfusion. CONCLUSIONS: High levels of NO/cGMP strongly protect against ischemia/reperfusion injury, the protection is largely independent of changes in Ca(2+)(i) modulation, but relates to reduced preischemic performance. Myocyte-specific NO augmentation may aid in studies of the (patho)physiological roles of cardiac-derived NO.     

Inhibition of N-ethylmaleimide-sensitive factor protects against myocardial ischemia/reperfusion injury

Exocytosis of endothelial granules promotes thrombosis and inflammation and may contribute to the pathophysiology of early reperfusion injury following myocardial ischemia. TAT-NSF700 is a novel peptide that reduces endothelial exocytosis by inhibiting the ATPase activity and disassembly activity of N-ethylmaleimide-sensitive factor (NSF), a critical component of the exocytic machinery. We hypothesized that TAT-NSF700 would limit myocardial injury in an in vivo murine model of myocardial ischemia/reperfusion injury. Mice were subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. TAT-NSF700 or the scrambled control peptide TAT-NSF700scr was administered intravenously 20 minutes before the onset of ischemia. Myocardial ischemia/reperfusion caused endothelial exocytosis, myocardial infarction, and left ventricular dysfunction. However, TAT-NSF700 decreased von Willebrand factor levels after myocardial ischemia/reperfusion, attenuated myocardial infarct size by 47%, and preserved left ventricular structure and function. These data suggest that drugs targeting endothelial exocytosis may be useful in the treatment of myocardial injury following ischemia/reperfusion.     

Adiponectin protects against myocardial ischaemia-reperfusion injury via AMP-activated protein kinase, Akt, and nitric oxide

AIMS: Cardiovascular disease and type 2 diabetes mellitus are associated with low plasma concentration of adiponectin. The aim of this study was to investigate whether adiponectin exerts cardioprotective effects during myocardial ischaemia-reperfusion and whether this effect is related to the production of nitric oxide (NO). METHODS AND RESULTS: Isolated rat hearts were subjected to 30 min of either global or local ischaemia followed by 60 min of reperfusion. The hearts received vehicle, adiponectin (3 microg/mL), the NO-synthase inhibitor nitro-l-arginine (L-NNA) (0.1 mM), or a combination of adiponectin and L-NNA at the onset of ischaemia. Haemodynamics, infarct size, and expression of endothelial NO-synthase (eNOS), AMP-activated protein kinase (AMPK), and Akt were determined. Adiponectin significantly increased left ventricular function and coronary flow during reperfusion in comparison with the vehicle group. Co-administration of L-NNA abrogated the improvement in myocardial function induced by adiponectin. Infarct size following local ischaemia-reperfusion was 40 +/- 6% of the area at risk in the vehicle group. Adiponectin reduced infarct size to 19 +/- 2% (P < 0.01). L-NNA did not affect infarct size per se but abolished the protective effect of adiponectin (infarct size 40 +/- 5%). Phosphorylation of eNOS Ser1177, AMPK Thr172, and Akt Ser 473 was increased in the adiponectin group (P < 0.05). CONCLUSION: Adiponectin protects from myocardial contractile dysfunction and limits infarct size following ischaemia and reperfusion by a mechanism involving activation of AMPK and production of NO.     

内皮型一氧化氮合酶基因多态性与急性心肌梗死的相关性探讨

目的　探讨内皮型一氧化氮合酶 (eNOS)基因多态性与急性心肌梗死 (AMI)的相关性。方法　依据eNOS基因外显子 7G894T位点设计引物 ,通过巢式聚合酶链反应 (PCR)扩增目的片段 ,限制性内切酶消化目的片段 ,琼脂糖凝胶电泳 ,紫外透射分析仪检测 ,计数 10 7例AMI病人及 81例健康者基因型及突变基因频率 ,通过χ2 检验有无统计学意义。结果　eNOS基因外显子 7的 894位点有 3种基因型 :GG、GT、TT。AMI组 10 7例中2 5例发生G894T突变 ,纯合子TT 9例 ,杂合子GT 16例。对照组 81例中 13例发生G894T突变 ,均为杂合子。两组等位基因纯合子突变具有非常显著统计学意义 ,x2 =5 4 2 9,P <0 0 5 ,两组等位基因总突变率 (纯合子 +杂合子 )无明显统计学意义 ,x2 =1 5 2 9,P >0 0 5。结论　eNOS基因 894位点TT型突变与AMI发病密切相关 ,是AMI发病的危险因子