Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice

Summary.  Background:  Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. Objectives:  To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. Methods:  We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. Results:  ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IκBα upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-κB (NF-κB) mediated cell signaling. Levels of nuclear activated NF-κB and proteins influenced by the NF-κB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). Conclusions:  We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-κB mechanism.     

Reduction of ischemia--reperfusion induced myocardial infarct size in rats by caffeic acid phenethyl ester (CAPE)

Myocardial ischemia--reperfusion (MI/R) represents a clinically relevant problem associated with thrombolysis, angioplasty, and coronary bypass surgery. MI/R injury is known to occur on restoration of coronary flow after a period of myocardial ischemia. Injury of myocardium caused by I/R includes cardiac contractile dysfunction, arrhythmias, as well as irreversible myocyte damage. Prevention of myocardial death in acute coronary syndromes is the immediate goal of therapy. The main factor concerned with the experimental generation of reperfusion damage is oxygen-derived free radicals. This MI/R injury has been shown to be salvaged by supplementing antioxidants to diseased hearts. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has antioxidant and anti-inflammatory properties, and may function in cardiac protection against I/R-induced damage. To test this hypothesis, we randomly assigned 14 male Wistar rats for necrosis experiments. To produce myocardial necrosis, the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion in anesthetized rats. CAPE (50 microM kg-1) was given intravenously 10 min before occlusion and continued during ischemia by infusion pump. The volume of infarct and the risk zone was determined by planimentry of each tracing and multiplying by the slice thickness. Infarct was normalized by expressing it as a percentage of the area at risk. Compared to control group, CAPE administration statistically reduced the myocardial infarct size/area of risk zone (50 +/- 4% and 32 +/- 6%, respectively) and the myocardial infarct size (23 +/- 3% and 9 +/- 4%, respectively) in rat model of ischemia-reperfusion. In conclusion, this result shows that CAPE is important in reducing I/R-induced myocardial damage.     

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.     

In vivo imaging of myocardial cell death using a peptide probe and assessment of long-term heart function

During acute myocardial infarction (AMI), both apoptosis and necrosis of myocardial cells could occur and lead to left ventricular (LV) functional decline. Here we determined whether in vivo imaging signals of myocardial cell death by ApoPep-1 (CQRPPR), a peptide probe that binds to apoptotic and necrotic cells through histone H1, at an early stage after AMI showed correlation with the long-term heart function. AMI was induced using a rat model of ischemia and reperfusion (I/R) injury. Fluorescence-labeled ApoPep-1 was administered by intravenous injection into rats 2 h after reperfusion. Ex vivo imaging of hearts isolated 2 h after peptide injection showed higher levels of near-infrared fluorescence (NIRF) signals at hearts of I/R rats than those of sham-operated rats. The fluorescent peptide was rapidly cleared from the blood and did not bind to red and white blood cells. Localization of fluorescent ApoPep-1 at the area of cell death was demonstrated by co-staining of myocardial tissue with TUNEL. The intensity of in vivo NIRF imaging signals by homing of ApoPep-1 to injured myocardium of I/R rats obtained 2 h after peptide injection (equivalent to 4 h after injury) showed strong and moderate correlation with the change in the LV ejection fractions (r² = 0.82) and the size of the fibrotic area (r² = 0.64), respectively, observed at four weeks after injury. These results suggest that ApoPep-1-mediated in vivo imaging signals of myocardial cell death, including both apoptosis and necrosis, at an early stage of AMI could be a potential biomarker for assessment of long-term outcome of heart function.     

Protective effect of caffeine administration on myocardial ischemia/reperfusion injury in rats

Many studies have examined the association between coffee consumption and risk of cardiovascular disease, but the results remain controversial. Caffeine is one of the main biologically active compounds of coffee. The aim of this study was to investigate the potential role of caffeine on myocardial ischemia/reperfusion (I/R) injury in the rats. We administered caffeine (25 mg/kg per day) or saline in rats for 4 weeks before myocardial ischemia/reperfusion operation. Compared with the sham group, caffeine treatment decreased ischemia-associated infarct size, serum creatine kinase, and lactate dehydrogenase 3-h reperfusion after 30-min ischemia. Myocardial neutrophil infiltration (assessed by myeloperoxidase activity) was significantly decreased compared with the control group. Meanwhile, caffeine reduced the myocardial apoptosis and suppressed the activation of caspase 3 during myocardial I/R. Importantly, we observed a strong poly(ADP-ribose) polymerase (PARP) activation during myocardial I/R, and caffeine administration inhibited PARP activation and attenuated the expression of PARP-related proinflammatory mediators such as inducible nitric oxide synthetase, IL-6, and TNF-α, all of which may be correlated with downregulated nuclear factor κB activity. We concluded that caffeine protected against myocardial I/R injury by inhibiting inflammation and apoptosis.     

链脲佐菌素诱导糖尿病大鼠NO变化对缺血再灌注损伤的影响

目的:探讨链脲佐菌素(STZ)诱导的不同周期糖尿病对心肌缺血/再灌注(I/R)损伤的影响及其与血浆一氧化氮(NO)变化的关系.方法:阻断和开放左冠状动脉前降支建立大鼠急性心肌I/R模型.分别用TTC染色测定大鼠心肌I/R后梗死面积;用硝酸还原酶法测定NO含量;用免疫印迹法定量分析代表细胞生存信号的磷酸化蛋白激酶B(P-Akt)的表达.结果:STZ处理后2周,糖尿病组(2WD+I/R)心肌梗死面积比相应周期对照组(2WC+I/R)明显缩小,STZ处理后16周(16WD+I/R),梗死面积比相应对照组(16WC+I/R)增加;血浆NO水平在2周糖尿病组中较对照组增高,但是在16周糖尿病组中较对照组显著减少;P-Akt在心肌的表达在2WD组比2WC组增加35%,在16WD组比16WD组明显减少.结论:STZ诱导的急、慢性期糖尿病对心肌I/R损伤呈现相反的作用.这可能是由于急、慢性期糖尿病相反的NO改变而引起的.     

Possible involvement of PKC-δ in the abrogated cardioprotective potential of ischemic preconditioning in hyperhomocysteinemic rat hearts

The present study has been designed to investigate the possible role of protein kinase C-delta (PKC-δ) in hyperhomocysteinemia-induced attenuation of cardioprotective potential of ischemic preconditioning (IPC). Rats were administered l-methionine (1.7 g/kg/day, p.o.) for 4 weeks to produce hyperhomocysteinemia. Isolated Langendorff perfused normal and hyperhomocysteinemic rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. Myocardial infarct size was assessed macroscopically using triphenyltetrazolium chloride (TTC) staining. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK) release to assess the degree of cardiac injury. Moreover, the oxidative stress in heart was assessed by measuring lipid peroxidation and superoxide anion generation. The ischemia-reperfusion (I/R) was noted to produce myocardial injury as assessed in terms of increase in myocardial infarct size, LDH and CK in coronary effluent and oxidative stress in normal and hyperhomocysteinemic rat hearts. In addition, the hyperhomocysteinemic rat hearts showed enhanced I/R-induced myocardial injury with high degree of oxidative stress as compared with normal rat hearts subjected to I/R. Four episodes of IPC (5 min each) afforded cardioprotection against I/R-induced myocardial injury in normal rat hearts as assessed in terms of reduction in myocardial infarct size, LDH, CK and oxidative stress. On the other hand, IPC mediated myocardial protection against I/R-injury was abolished in hyperhomocysteinemic rat hearts. Treatment with rottlerin (10 μM), a selective inhibitor of PKC-δ did not affect the cardioprotective effects of IPC in normal rat hearts; but its treatment significantly restored the cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts. The high degree of oxidative stress produced in hyperhomocysteinemic rat hearts during reperfusion may activate PKC-δ, which may be implicated in the observed paradoxically abrogated cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts.     

Aldehyde dehydrogenase 2 preserves mitochondrial morphology and attenuates hypoxia/reoxygenation-induced cardiomyocyte injury

BACKGROUND: Disturbance of mitochondrial fission and fusion (termed mitochondrial dynamics) is one of the leading causes of ischemia/reperfusion (I/R)-induced myocardial injury. Previous studies showed that mitochondrial aldehyde dehydrogenase 2 (ALDH2) conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes. However, whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.     

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.     

Kallikrein-modified mesenchymal stem cell implantation provides enhanced protection against acute ischemic kidney injury by inhibiting apoptosis and inflammation

Mesenchymal stem cells (MSCs) migrate to sites of tissue injury and serve as an ideal vehicle for cellular gene transfer. As tissue kallikrein has pleiotropic effects in protection against oxidative organ damage, we investigated the potential of kallikrein-modified MSCs (TK-MSCs) in healing injured kidney after acute ischemia/reperfusion (I/R). TK-MSCs secreted recombinant human kallikrein with elevated vascular endothelial growth factor levels in culture medium, and were more resistant to oxidative stress-induced apoptosis than control MSCs. Expression of human kallikrein was identified in rat glomeruli after I/R injury and systemic TK-MSC injection. Engrafted TK-MSCs exhibited advanced protection against renal injury by reducing blood urea nitrogen, serum creatinine levels, and tubular injury. Six hours after I/R, TK-MSC implantation significantly reduced renal cell apoptosis in association with decreased inducible nitric oxide synthase expression and nitric oxide levels. Forty-eight hours after I/R, TK-MSCs inhibited interstitial neutrophil and monocyte/macrophage infiltration and decreased myeloperoxidase activity, superoxide formation, p38 mitogen-activated protein kinase phosphorylation, and expression of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1. In addition, tissue kallikrein and kinin significantly inhibited H2O2-induced apoptosis and increased Akt phosphorylation and cell viability in cultured proximal tubular cells. These results indicate that implantation of kallikrein-modified MSCs in the kidney provides advanced benefits in protection against ischemia-induced kidney injury by suppression of apoptosis and inflammation.     

Eupatilin inhibits the apoptosis in H9c2 cardiomyocytes via the Akt/GSK-3β pathway following hypoxia/reoxygenation injury

Eupatilin, a pharmacologically active flavone derived from the Artemisia plant species, has been reported to have anti-oxidant, anti-inflammatory, anti-allergic, and neuroprotective activities against cerebral ischemia/reperfusion (I/R). However, the role of eupatilin in myocardial I/R injury remains unclear. In the present study, we aimed to investigate the potential molecular mechanisms against hypoxia/reoxygenation (H/R) induced cardiomyocytes apoptosis in vitro. Our results showed that eupatilin markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. Eupatilin also suppressed oxidative stress and apoptosis in H9c2 cells after myocardial I/R injury. Furthermore, eupatilin obviously increased the phosphorylation of Akt and GSK-3β in H9c2 cells. Our results suggested that eupatilin could provide significant cardioprotection against myocardial I/R injury, and the potential mechanisms might involve inhibition of cardiomyocyte apoptosis through activating the Akt/GSK-3β signaling pathway.     

Cardioprotective effects of ozone oxidative preconditioning in an in vivo model of ischemia/reperfusion injury in rats

Abstract Background. Several studies have demonstrated the beneficial effects of ozone oxidative preconditioning in several pathologies characterized by cellular oxidative and inflammatory burden. The present study was designed to investigate the cardioprotective effects of oxidative preconditioning in ischemia/reperfusion (I/R) injury. Methods. Rats were randomly assigned into five groups. Groups 1 and 2 were normal and I/R groups, respectively. Two of the other groups received two different doses of ozone therapies by rectal insufflations. The last group received vehicle (oxygen). Rats were subjected to myocardial I/R (40min/10min). Heart rate and ventricular arrhythmias were recorded during I/R progress. At the end of reperfusion, plasma creatine kinase-MB (CK-MB) activity and total nitrate/nitrite (NO(x)) were determined. In addition, lactate, adenine nucleotides, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and myeloperoxidase (MPO) activity were estimated in the heart left ventricle. Histological examination was also performed to visualize the protective cellular effects. Results. Both doses of ozone therapy were equally protective in reducing CK-MB release. However, the higher dose was more effective in reducing oxidative stress, lactate accumulation, elevated MPO activity and plasma NO(x) as well as preserving myocardial adenine nucleotides. Histological examination also revealed better improvement with a higher dose of ozone therapy compared to the I/R group. Conclusion. Ozone therapy can afford significant cardioprotection against biochemical and histological changes associated with I/R injury.     

Adipocytokines, cardiovascular pathophysiology and myocardial protection

Reducing myocardial damage resulting from ischaemia-reperfusion (I/R) is vital in ensuring patient recovery and survival. It relies upon the activation of the so-called Reperfusion Injury Salvage Kinase (RISK) pathway. Experimentally various treatments, both mechanical and chemical, have been shown to protect the myocardium against I/R injury. Chemical facilitators of myocardial preservation include endogenous factors such as insulin, erythropoietin and glucagon-like peptide 1. The adipocytokines, products of white adipose tissue, are important peptide hormones with respect to metabolic control and satiety, and were formerly considered in the context of obesity and metabolic disease. More recently, however, evidence has been presented indicating that the adipocytokines play significant roles in cardiac function and, as we have suggested, in myocardial protection. To date leptin, adiponectin, apelin and visfatin have all been shown to protect against I/R injury. Significantly, the protection afforded by these peptides involves the activation of kinases which are key elements of the mechanisms underlying tissue preservation, including the RISK pathway components PI3K-Akt and p44/42, and inhibition of the mitochondrial permeability transition pore (MPTP). In this article we examine the roles played by the adipocytokines in cardiovascular function and disease. In particular, we focus on the evidence that these peptides promote myocardial survival, much of it having been obtained in this laboratory. To conclude, we discuss some future directions in the field, including the prospects for some of the adipocytokines finding application as therapeutic agents in myocardial infarction.     

Inhibiting protease-activated receptor 4 limits myocardial ischemia/reperfusion injury in rat hearts by unmasking adenosine signaling

Harnessing endogenous cardioprotectants is a novel therapeutic strategy to combat ischemia/reperfusion (I/R) injury. Thrombin causes I/R injury, whereas exogenous adenosine prevents I/R injury. We hypothesized that blocking thrombin receptor activation with a protease-activated receptor (PAR) 4 antagonist would unmask the cardioprotective effects of endogenous adenosine. The protective role of two structurally unrelated PAR4 antagonists, trans-cinnamoyl-YPGKF-amide (tc-Y-NH(2)) and palmitoyl-SGRRYGHALR-amide (P4pal10), were evaluated in two rat models of myocardial I/R injury. P4pal10 (10 microg/kg) treatment before ischemia significantly decreased infarct size (IS) by 31, 21, and 19% when given before, during, and after ischemia in the in vivo model. tc-Y-NH(2) (5 microM) treatment before ischemia decreased IS by 51% in the in vitro model and increased recovery of ventricular function by 26%. To assess whether the cardioprotective effects of PAR4 blockade were due to endogenous adenosine, isolated hearts were treated with a nonselective adenosine receptor blocker, 8-sulfaphenyltheophylline (8-SPT), and tc-Y-NH(2) before ischemia. 8-SPT abolished the protective effects of tc-Y-NH(2) but did not affect IS when given alone. Adenosine-mediated survival pathways were then explored. The cardioprotective effects of tc-Y-NH(2) were abolished by inhibition of Akt (wortmannin), extracellular signal-regulated kinase 1/2 [PD98059 (2'-amino-3'-methoxyflavone)], nitric-oxide synthase [N(G)-monomethyl-l-arginine (l-NMA)], and K(ATP) channels (glibenclamide). PD98059, l-NMA, and glibenclamide alone had no effect on cardioprotection in vitro. Furthermore, inhibition of mitochondrial K(ATP) channels [5-hydroxydecanoic acid (5-HD)] and sarcolemmal K(ATP) channels (sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl)(methylcarbamothioyl)amide; HMR 1098) abolished P4pal10-induced cardioprotection in vivo. Thrombin receptor blockade by PAR4 inhibition provides protection against injury from myocardial I/R by unmasking adenosine receptor signaling and supports the hypothesis of a coupling between thrombin receptors and adenosine receptors.     

Targeting myocardial reperfusion injuries with cyclosporine in the CIRCUS Trial – pharmacological reasons for failure

The mitochondrial permeability transition (mPTP) is a key feature of cardiac cell death in ischaemia‐reperfusion injury (I/R). The mPTP blocker, cyclosporine A (CsA), has been shown to give protection against reperfusion‐induced myocardial necrosis and troubles generated by acute coronary artery repermeabilization. Nevertheless, the results of the CIRCUS trial (Does Cyclosporine Improve Clinical Outcome in ST‐Elevation Myocardial Infarction Patients) seem to go against this hypothesis. Pharmacological reasons linked to CsA pharmacokinetics and pharmacodynamics could be suggested. First, it could be explained by a limited diffusion of the drug in the area at risk, due to the only inclusion of patients with a TIMI 0 or 1 coronary blood flow in the anterior territory and the absence of collateral perfusion. Second, to explain a low tissue diffusion of the compound, blood cell capture and high metabolism could be suggested. Moreover, CsA is highly metabolized by cytochrome P450 3A4 (CYP3A4), a polymorphic enzyme leading to variations of C_max and AUC between 10–20% in patients using CsA. Finally, CsA blocks calcineurin, a protein implied in I/R damage but calcineurin inhibition could contribute to protection towards I/R damage only when Rcan1, a calcineurin natural inhibitor, expression is low. The results of the CIRCUS trial are disappointing and could contribute to the withdrawal of the mPTP blockade pharmacological strategy as a way to protect the myocardium from I/R lesions. Nevertheless, many pharmacological insights could have contributed to an increased variability and, as a consequence, an important reduction of the pharmacological power of the study.     

外源性热休克蛋白70基因对心肌细胞损伤的保护作用

目的：探讨热休克蛋白70（HSP70）基因转染对心肌细胞急性实验性缺氧／复氧损伤的保护作用及其机制。方法：进行大鼠乳鼠原代心肌细胞培养,随机分为4组,对照组、缺氧／复氧（A／R）组、热休克处理后缺氧／复氧（A／R＋HS）组和pCDNAHSP70质粒转导后缺氧／复氧（A／R＋pCDNA HSP70）组。用脂质体包裹pCDNAHSP70质粒,并转导入心肌细胞内。反转录-聚合酶链反应（RT-PCR）检测HSP70mRNA表达,Westernblot检测HSP70蛋白表达。以心肌细胞存活率（MTT法）、培养液中乳酸脱氢酶（I．DH）活性、心肌细胞超微结构改变（透射电镜）及Ca^2＋负荷来检测心肌细胞损伤程度。结果：与A／R组相比,A／R＋HS组和A／R’pCDNAHSP7。组细胞存活率显著提高;LDH活性明显降低;细胞超微结构明显改善;心肌细胞Ca^2＋负荷明显减轻。A／R组HSP70mRNA和蛋白含量均较A／R＋HS组和A／R＋pCDNAHSP70组显著减低（P〈0．01）,而A／R＋pCDNA HSP70组的含量与A／R＋HS组相比显著增多（P〈0．01）。结论：通过基因转染使HSP70基因高表达能对抗缺氧／复氧对心肌细胞的损伤,这一作用与其能抗细胞Ca^2＋负荷有关。     

心肌缺血/再灌注损伤后瘦素的改变及机制初探

目的 观察大鼠心肌缺血/再灌注(I/R)损伤对瘦素(Leptin)、内皮素(ET)、C-反应蛋白(CRP)表达的影响,探讨Leptin在心肌I/R损伤中的作用.方法 将50只SD大鼠按随机数字表法分为假手术组、缺血组及I/R 1、2、3 h组,每组10只.以结扎左冠状动脉(冠脉)前降支45 min、再通1、2、3 h建立大鼠心肌I/R损伤模型;假手术组仅穿线、不结扎冠脉.各组于相应时间点取左股动脉血,检测血清Leptin、ET、CRP的浓度;取心肌组织行苏木素-伊红(HE)染色和免疫组化,观察心肌组织病理改变及Leptin蛋白表达水平.结果 缺血组血清Leptin含量(μg/L)显著低于假手术组(4.69±1.67比6.48±2.02,P＜0.05),随再灌注时间的延长,Leptin水平逐渐升高,至I/R 3 h时已恢复到损伤前水平[(6.59±2.58)μg/L].缺血组血清ET水平(ng/L)显著高于假手术组(110.58±37.86比80.74±34.43,P＜0.05),I/R 1、2、3 h组血清ET水平均显著低于缺血组(35.87±13.56、31.98±10.88、34.56±14.37比110.58±37.86,均P＜0.05).缺血组血清CRP水平(mg/L)显著高于假手术组(13.12±4.82比3.24±1.72,P＜0.01),随再灌注时间延长,CRP水平逐渐升高,I/R 1、2、3 h组均显著高于缺血组(18.37±6.48、24.30± 9.51、27.08±8.32比13.12±4.82,均P＜0.05).HE染色显示,缺血心肌细胞发生坏死、脱落,肌间质轻度充血、水肿;I/R损伤后心肌细胞呈灶性凝固性坏死,肌间质重度充血.免疫组化显示,心肌Leptin蛋白表达呈损伤后早期降低、后期升高的整体趋势.结论 在心肌I/R损伤时血清及心肌组织中Leptin水平早期明显减少,恢复期缓慢升高,提示其可能作为机体的一种应激保护因子,对抗I/R引起的心肌损伤,并可能与ET的先升后降、CRP的升高有一定的关系.     

The cardioprotective effects of preconditioning with endotoxin, but not ischemia, are abolished by a peroxisome proliferator-activated receptor-gamma antagonist

We investigated whether endogenous ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) protect the heart against ischemia-reperfusion (I/R) injury. The selective PPAR-gamma antagonist GW9662 (2-chloro-5-nitrobenzanilide) was used in rat models of 1) regional myocardial I/R, 2) ischemic preconditioning, and 3) delayed cardioprotection by endotoxin. We also investigated the effects of the selective cyclooxygenase-2 inhibitor, parecoxib, on ischemic preconditioning and delayed cardioprotective effects of endotoxin. Male Wistar rats were anesthetized with sodium thiopentone. Animals were subjected to either 15 or 25 min of regional myocardial I/R and pretreated with the PPAR-gamma agonist ciglitazone (0.3 mg/kg), the PPAR-gamma antagonist GW9662 (1 mg/kg), or GW9662 and ciglitazone. Animals were also subjected to either 1) ischemic preconditioning alone, ischemic preconditioning, and pretreated with either GW9662 or parecoxib (20 mg/kg) or 2) lipopolysaccharide (LPS) (1 mg/kg) alone, LPS, and pretreated with ciglitazone, GW9662, or parecoxib (20 mg/kg). Myocardial infarct size was determined by p-nitroblue tetrazolium staining. The PPAR-gamma antagonist GW9662 (1 mg/kg) abolished the cardioprotection afforded by the potent PPAR-gamma agonist ciglitazone (0.3 mg/kg). Neither GW9662 nor parecoxib affected the cardioprotective effects of ischemic preconditioning. Pretreatment with ciglitazone did not provide additional cardioprotection to LPS-treated animals. Both GW9662 and parecoxib abolished the delayed cardioprotective effects of endotoxin. Thus, we propose that 1) endogenous ligands of PPAR-gamma are being generated by myocardial ischemia in sufficient amounts to attenuate myocardial I/R injury, and 2) that cyclooxygenase-2 metabolites contribute to (or even account for) the cardioprotective effects of endotoxin (second window of protection) by acting as endogenous PPAR-gamma ligands.     

Antiarrhythmic effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia/reperfusion injury in rats

OBJECTIVES: The present study was designed to determine the antiarrhythmic effect of caffeic acid phenethyl ester (CAPE), an active component of propolis, which exhibits antioxidant properties, in rats subjected to myocardial ischemia and ischemia-reperfusion (I/R) injury. DESIGN AND METHODS: Rats were subjected to 30 min coronary artery occlusion for evaluating the effect of CAPE on the myocardial ischemia injury. While in the myocardial I/R injury study, the coronary artery was ligated for a 5-min period of ischemia followed by a 30-min period of reperfusion. Animals were pretreated with or without CAPE before coronary artery ligation and the severity of myocardial ischemia- and I/R-induced arrhythmias and mortality were compared. RESULTS: Pretreatment of CAPE (0.1 and 1 microg/kg) not only reduced both the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) but also decreased the mortality during the myocardial ischemia and I/R injury period. CONCLUSIONS: Our results suggest that CAPE is a potent antiarrhythmic agent with cardioprotective effects in myocardial ischemia and I/R injury rats.     

Ischemic postconditioning protects myocardium from ischemia/reperfusion injury through attenuating endoplasmic reticulum stress

Ischemic postconditioning (I-postC) is a newly discovered endogenous protective phenomenon capable of protecting the myocardium from I/R injury. The cardioprotective mechanisms of I-postC involve protein synthesis and preventing an increase in cytosolic calcium. Endoplasmic reticulum (ER) is a principal site for secretory protein synthesis and calcium storage. Myocardial I/R causes ER stress and perturbations of ER function. The purpose of the present study was to determine whether I-postC's attenuation of I/R injury involves reductions in ER stress through mitogen-activated protein kinase (MAPK) pathway. In the present study, models of rat myocardial I/R and hypoxia/reoxygenation (H/R) of neonatal rat cardiomyocytes were used. Myocardial infarct size was measured by triphenyltetrazolium chloride staining, and flow cytometry was used to quantitate cardiomyocyte apoptosis. Calreticulin expression and activation of caspase 12, p38 MAPK, and c-Jun NH2-terminal kinase (JNK) in myocardium or cardiomyocytes were detected by Western blots. It is found that I-postC protects the I/R heart against myocardial infarction, and hypoxic postconditioning protects neonatal cardiomyocytes from H/R-induced apoptosis. Ischemic postconditioning suppressed I/R-induced ER stress, as shown by a decrease in calreticulin expression and caspase 12 activation. Hypoxic postconditioning up-regulates p38 MAPK phosphorylation and down-regulates JNK phosphorylation in cardiomyocytes subjected to H/R. These results indicate that I-postC protects myocardium from I/R injury by suppressing ER stress, and that p38 MAPK and JNK pathways are associated with the I-postC-induced suppression of ER stress.