Reduction of oxidative stress by carvedilol: role in maintenance of ischaemic myocardium viability

OBJECTIVES: To differentiate the impact of the beta-blocking and the anti-oxidant activity of carvedilol in maintaining myocardium viability. METHODS: Isolated rabbit hearts, subjected to aerobic perfusion, or low-flow ischaemia followed by reperfusion, were treated with two doses of carvedilol, one dose (2.0 microM) with marked negative inotropic effect due to beta-blockage and the other (0.1 microM) with no beta-blockage nor negative inotropism. Carvedilol was compared with two doses of propranolol, 1.0 - without - and 5.0 microM - with negative inotropic effect. Anti-oxidant activity was measured as the capacity to counteract the occurrence of oxidative stress and myocardium viability as recovery of left ventricular function on reperfusion, membrane damage and energetic status. RESULTS: Carvedilol counteracted the ischemia and reperfusion induced oxidative stress: myocardial content of reduced glutathione, protein and non-protein sulfhydryl groups after ischaemia and particularly after reperfusion, was higher in hearts treated with carvedilol, while the myocardial content of oxidised glutathione was significantly reduced (0.30+/-0.03 and 0.21+/-0.02 vs. 0.39+/-0.03 nmol/mg prot, both P<0.01, in 0.1 and 2.0 microM). At the same time, carvedilol improved myocardium viability independently from its beta-blocking effect. On the contrary, propranolol maintained viability only at the higher dose, although to a lesser extent than carvedilol. This suggests that the effects of propranolol are dependent on energy saving due to negative inotropism. The extra-protection observed with carvedilol at both doses is likely due to its anti-oxidant effect. CONCLUSIONS: Our data show that the anti-oxidant activity of carvedilol is relevant for the maintenance of myocardium viability.     

New insights on myocardial pyridine nucleotides and thiol redox state in ischemia and reperfusion damage

OBJECTIVE: to investigate the changes of pyridine nucleotides and thiol redox state in cardiac tissue following ischemia and reperfusion. NADH/NAD and NADPH/NADP redox couples were specifically studied and the influence of NADPH availability on cellular thiol redox was also investigated. METHODS: isolated rabbit hearts were Langendorff perfused and subjected to a protocol of ischemia and reperfusion. An improved technique for extraction and selective quantitation of pyridine nucleotides was applied. RESULTS: ischemia and reperfusion induced an increase in diastolic pressure, limited recovery in developed pressure and loss of creatine phosphokinase. Creatine phosphate and ATP were decreased by ischemia and only partially recovered during reperfusion. NADH was increased (from 0. 36+/-0.04 to 1.96+/-0.15 micromol/g dry wt. in ischemia, P<0.001), whereas NADPH decreased during ischemia (from 0.78+/-0.04 to 0. 50+/-0.06 micromol/g dry wt., P<0.01) and reperfusion (0.45+/-0.03 micromol/g dry wt.). Furthermore, we observed: (a) release of reduced (GSH) and oxidised glutathione (GSSG) during reperfusion; (b) decreased content of reduced sulfhydryl groups during ischemia and reperfusion (GSH: from 10.02+/-0.76 to 7.11+/-0.81 nmol/mg protein, P<0.05, and to 5.48+/-0.57 nmol/mg protein; protein-SH: from 280.42+/-12.16 to 135.11+/-17.00 nmol/mg protein, P<0.001, and to 190.21+/-11.98 nmol/mg protein); (c) increased content in GSSG during reperfusion (from 0.17+/-0.02 to 0.36+/-0.02 nmol/mg protein, P<0.001); (d) increased content in mixed disulphides during ischemia (from 6.14+/-0.13 to 8.31+/-0.44 nmol/mg protein, P<0.01) and reperfusion (to 9.87+/-0.82 nmol/mg protein, P<0.01). CONCLUSIONS: under severe low-flow ischemia, myocardial NADPH levels can decrease despite the accumulation of NADH. The reduced myocardial capacity to maintain NADPH/NADP redox potential can result in thiol redox state changes. These abnormalities may have important consequences on cellular function and viability.     

The protective role of heat stress in the ischaemic and reperfused rabbit myocardium.

Cells subjected to increases in temperature induce the expression of several proteins known as heat shock or stress proteins. This process enhances the cell's ability to overcome the effects of further stress. In this respect, the effects of heat stress have been reported to protect the hearts of rats following ischaemia and reperfusion. We have confirmed and extended this observation, not only using different indices of myocardial injury but also in another species, namely the rabbit. Animals were anaesthetized and the body temperature raised to 42 degrees C for a 15-min period. Controls were treated in the same way but without heating. Twenty-four hours later the rabbits were re-anaesthetized and the hearts removed for either heat stress protein analysis or perfusion with Krebs buffer using an isolated perfused heart apparatus. Hearts were subjected to 60 min of low flow (1 ml/min) ischaemia followed by 30 min of reperfusion. All hearts subjected to heat stress showed an enhanced recovery of function upon reperfusion as measured by improvements in developed pressure (27.3 +/- 3.6 vs 16.3 +/- 3.0 mmHg) and diastolic pressure (37.3 +/- 7.4 vs 54.7 +/- 3.1 mmHg). In addition, creatine kinase release, associated with reperfusion, was significantly reduced in the heat-stressed hearts (532 +/- 102 vs 1138 +/- 73 mU/min/g wet wt). Myocardial accumulation and release of oxidized glutathione, an index of oxidative stress, was significantly reduced in the heat-stressed group (0.003 +/- 0.003 vs 0.376 +/- 0.113 nmol/min/g wet wt). The improved metabolic status of the reperfused heat-stressed hearts was further demonstrated by a significant conservation in the levels of ATP (6.1 +/- 0.9 vs 2.8 +/- 0.8 mumol/g dry wt) and CP (36.9 +/- 6.4 vs 16.4 +/- 5.1 mumol/g dry wt). Finally, isolated mitochondrial function in terms of respiratory control index (RCI) was maintained in the heat-stressed hearts (9.2 +/- 0.9 vs 5.7 +/- 0.2) and overloading with calcium was reduced. These data extend the hypothesis that heat stress protects the heart following ischaemia and reperfusion in this in vitro model, in a way as yet undetermined.     

一氧化碳吸入对急性肺损伤大鼠肺组织细胞凋亡的影响及其机制

目的观察吸入低浓度一氧化碳(CO)对脂多糖(LPS)致急性肺损伤(ALI)大鼠肺组织细胞凋亡的影响,探讨可能机制。方法18只雄性SD大鼠随机均分为3组。ALI组静脉滴注LPS5mg/kg,正常对照组注入生理盐水,CO吸入组在LPS诱导肺损伤后持续吸入体积分数为2.5×10-4CO。观察3h后放血处死,取肺组织,半定量逆转录聚合酶链反应(RT PCR)测定血红素氧化酶1(HO1)表达,酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子α(TNFα)、白细胞介素6(IL6)和IL10的含量;化学比色法测定丙二醛(MDA)含量、髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)活性;光镜观察并盲法评分比较肺组织学;流式细胞仪检测细胞凋亡。结果ALI组HO1mRNA、TNFα、IL6、IL10、MDA、MPO、SOD、细胞凋亡与正常对照组[1.002±0.004、(0.47±0.06)pg/mg蛋白、(0.49±0.12)pg/mg蛋白、(0.42±0.08)pg/mg蛋白、(0.79±0.14)nmol/mg蛋白、(6.0±1.0)U/mg蛋白、(74±7)U/mg蛋白、(0.12±0.03)%]比较差异均有统计学意义(P<0.05或<0.01),肺损伤严重。CO吸入组TNFα、IL6、MDA、MPO和细胞凋亡[(0.91±0.25)pg/mg蛋白、(0.64±0.05)pg/mg蛋白、(1.02±0.23)nmol/mg蛋白、(7.2±1.6)U/mg蛋白、(1.60±0.34)%]显著低于ALI组[(1.48±0.23)pg/mg蛋白、(1.16±0.26)pg/mg蛋白、(1.27±0.33)nmol/mg蛋白、(8.2±1.5)U/mg蛋白、(3.18±0.51)%,P均<0.05];HO1mRNA、IL10和SOD表达[5.433±0.921、(0.26±0.07)pg/mg蛋白、(60±10)U/mg蛋白]显著高于ALI组[3.081±0.823、(0.15±0.03)pg/mg蛋白、(51±7)U/mg蛋白,P均<0.05],肺损伤减轻。结论吸入CO通过调控氧化/抗氧化、促炎/抗炎反应、抑制过度细胞凋亡和上调HO1表达,可能对LPS诱导ALI起保护作用。     

PEG-SOD and myocardial antioxidant status during ischaemia and reperfusion: dose-response studies in the isolated blood perfused rabbit heart.

We have previously shown that the polyethylene glycol conjugated superoxide dismutase (SOD), which has a plasma half-life of more than 24 h, protects the blood perfused rabbit heart against injury during ischaemia and reperfusion. However, the profile for the dose-dependency of protection was bell-shaped with loss of efficacy below 6000 and above 30,000 U/kg. In the present study, isolated rabbit hearts, perfused with blood from support rabbits, were subjected to a 2 min infusion with St Thomas' Hospital cardioplegic solution followed by 60 min of global ischaemia (37 degrees C) and 60 min of reperfusion. PEG-SOD was administered 1 h or 12-24 h before ischaemia. We assessed the effect of PEG-SOD on ischaemia- and reperfusion-induced changes in: (i) the tissue content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) and (ii) the activity of CuZn-SOD, Mn-SOD and glutathione peroxidase and reductase (GPD and GRD). Ischaemia and reperfusion reduced tissue GSH content by 70% and increased GSSG content by 400% (from their fresh aerobic values of 13.1.9 and 0.09 +/- 0.01 nmol/mg protein, respectively). PEG-SOD, given intravenously at various doses to donor and support rabbits 1 h or 12-24 h before ischaemia, protected against these changes with a bell-shaped dose-response relationship. Thus, with 0, 3000, 6000, 12,000, 30,000 and 60,000 U/kg, GSH content was 4.1 +/- 0.4, 4.8 +/- 0.4, 8.5 +/- 0.5, 12.3 +/- 1.6, 12.3 +/- 1.6 and 5.0 +/- 0.5 nmol/mg protein in the 1 h pretreatment group and 4.1 +/- 0.4, 4.2 +/- 0.5, 10.4 +/- 1.5, 11.2 +/- 1.1, 11.4 +/- 0.7 and 4.7 +/- 0.6 nmol/mg protein in the 12-24 h pretreatment group (means +/- S.E.M.). For GSSG the corresponding values were 0.36 +/- 0.04, 0.34 +/- 0.03, 0.12 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.41 +/- 0.03 nmol/mg protein for the 1 h group and 0.36 +/- 0.04, 0.35 +/- 0.02, 0.15 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.34 +/- 0.02 nmol/mg protein for the 12-24 h group. Ischaemia and reperfusion had no effect on tissue MDA content or CuZn-SOD, GDP and GRD activity, and in general, PEG-SOD also lacked significant effect on any of these variables at any dose studied. However, Mn-SOD activity was severely reduced by ischaemia and reperfusion (from 42 +/- 7 U/mg protein in fresh aerobic controls to 6 +/- 1 U/mg protein at the end of reperfusion).(ABSTRACT TRUNCATED AT 400 WORDS)     

Arrhythmias and alpha 1-adrenoceptor binding characteristics of the guinea-pig perfused heart during ischaemia and reperfusion

Guinea-pig isolated hearts were perfused by the Langendorff method. Low flow (10%) global ischaemia for 30 min induced ventricular tachycardia (VT) and fibrillation (VF) in 87.5 and 37.5% respectively of the hearts. The onset times for VT and VF were 15.7 +/- 1.0 and 23.5 +/- 1.6 min respectively. On reperfusion the incidences of VT and VF were 81.3 and 75.0% and occurred after 16.0 +/- 1.5 and 35.0 +/- 4.9 s of reperfusion. In those hearts exhibiting arrhythmias, [3H]-prazosin binding to alpha 1-adrenoceptors of ventricular membrane fractions was measured and compared with normally perfused time-matched controls. There was no significant change in dissociation constant (KD) or density Bmax, of binding throughout the control perfusion period. Similarly, the KD (0.38 +/- 0.06 nM) and Bmax (15.5 +/- 1.4 fmol/mg protein) values obtained during ischaemia did not differ significantly from the corresponding control values (0.48 +/- 0.05 nM and 15.8 +/- 1.5 fmol/mg protein). Also, at 1 min of reperfusion the KD (0.42 +/- 0.04 nM) and Bmax (19.3 +/- 2.0 fmol/mg protein) values were not significantly different from the time-matched controls (0.38 +/- 0.09 nM and 20.1 +/- 2.6 fmol/mg protein). The same result was obtained if the crude membrane fraction pelleted by the initial slow spin was used. Thus, although cardiac arrhythmias are induced by ischaemia and reperfusion of the guinea-pig isolated perfused heart and previous studies have shown these to be susceptible to alpha-adrenoceptor blockade, they are not accompanied by an increase in alpha 1-adrenoceptor affinity or density.     

S-nitroso human serum albumin reduces ischaemia/reperfusion injury in the pig heart after unprotected warm ischaemia

AIMS: Uncoupled endothelial nitric oxide synthase (eNOS) is a major contributor to vascular reactive oxygen species generation in ischaemia/reperfusion (I/R) injury. Supplementation of NO by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) may inhibit uncoupling of eNOS (feedback inhibition). METHODS AND RESULTS: Pigs (n = 14; 33.1 +/- 1.7 kg) were continuously monitored for heart rate (HR), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and coronary flow (CF). Infusion of either human serum albumin (n = 8; controls) or S-NO-HSA (n = 6) lasted 60 min (0.1 micromol/kg/h) starting 15 min prior to ischaemia. After clamping the aorta under cardiopulmonary bypass (CPB), the hearts underwent 15 min of warm, unprotected ischaemia (37 degrees C). Reperfusion lasted 150 min (30 min under CPB; 15 min weaning; additional 105 min reperfusion). In biopsies from non-ischaemic hearts and myocardial biopsies taken after 150 min of reperfusion, high-energy phosphates were measured and the calcium ionophore-stimulated release of NO, superoxide, and peroxynitrite (ONOO(-)) were monitored with nanosensors. Compared with non-ischaemic hearts, the NO level decreased from 930 +/- 25 to 600 +/- 15 nmol/L (P < 0.001) while the superoxide level increased from 45 +/- 5 to 110 +/- 10 nmol/L (P < 0.001) after ischaemia. S-NO-HSA restored the NO level to 825 +/- 20 nmol/L, shifted favourably the [NO]/[ONOO(-)] balance (a marker of eNOS uncoupling) from 1.36 +/- 0.06 (ischaemia) to 3.59 +/- 0.18, significantly improved CF (65 +/- 10 vs. control, 43 +/- 5 mL/min, P < 0.05), MAP (57 +/- 5 vs. 39 +/- 3 mm Hg, P < 0.01), LVSP (106 +/- 5 vs. 81 +/- 4 mm Hg, P < 0.01) and phosphocreatine (PCr) content (41.5 +/- 7.3 vs. 18.0 +/- 5.6 micromol/g protein; P < 0.01) at 150 min of reperfusion. CONCLUSION: Long-lasting release of NO by S-NO-HSA prevented uncoupling of eNOS and thereby improved systolic and diastolic function, myocardial perfusion, and the energetic reserve of the heart after I/R injury.     

Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition

OBJECTIVE: Diminishing oxidative stress may protect the heart against ischaemia-reperfusion injury by preventing opening of the mitochondrial permeability transition (MPT) pore. The general anaesthetic agent propofol, a free radical scavenger, has been investigated for its effect on the MPT and its cardioprotective action following global and cardioplegic ischaemic arrest. METHOD: Isolated perfused Wistar rat hearts were subjected to either warm global ischaemia (Langendorff) or cold St. Thomas' cardioplegia (working heart mode) in the presence or absence of propofol. MPT pore opening was determined using [3H]-2-deoxyglucose-6-phosphate ([3H]-DOG-6P) entrapment. The respiratory function of isolated mitochondria was also determined for evidence of oxidative stress. RESULTS: Propofol (2 micrograms/ml) significantly improved the functional recovery of Langendorff hearts on reperfusion (left ventricular developed pressure from 28.4 +/- 6.2 to 53.3 +/- 7.3 mmHg and left ventricular end diastolic pressure from 52.9 +/- 4.3 to 37.5 +/- 3.9 mmHg). Recovery was also improved in propofol (4 micrograms/ml) treated working hearts following cold cardioplegic arrest. External cardiac work on reperfusion improved from 0.42 +/- 0.05 to 0.60 +/- 0.03 J/s, representing 45-64% of baseline values, when compared to controls (P < 0.05). Propofol inhibited MPT pore opening during reperfusion, [3H]-DOG-6P entrapment being 16.7 vs. 22.5 ratio units in controls (P < 0.05). Mitochondria isolated from non-ischaemic, propofol-treated hearts exhibited increased respiratory chain activity and were less sensitive to calcium-induced MPT pore opening. CONCLUSION: Propofol confers significant protection against global normothermic ischaemia and during cold cardioplegic arrest. This effect is associated with less opening of mitochondrial MPT pores, probably as a result of diminished oxidative stress. Propofol may be a useful adjunct to cardioplegic solutions in heart surgery.     

Intracellular glutathione and bronchoalveolar cells in fibrosing alveolitis: effects of N-acetylcysteine.

Extracellular glutathione deficiency and exaggerated oxidative stress may contribute to the pathogenesis of fibrosing alveolitis (FA). High-dose N-acetylcysteine (NAC) supplementation partially reverses extracellular glutathione depletion and oxidative damage, but effects on intracellular glutathione are unknown. Intracellular total glutathione (GSHt) and activation of bronchoalveolar lavage cells (BAC) obtained from 18 FA patients (9 males, aged 52+/-2 yrs), before and after 12 weeks of oral NAC (600 mg t.i.d.), were assessed. Eight healthy nonsmokers (2 males, aged 36+/-6 yrs) served as a control group. Intracellular GSHt was decreased in FA (1.57+/-0.20 nmol 1x10(6) BAC(-1) versus 2.78+/-0.43 nmol x 10(6) BAC(-1)). After NAC treatment, the intracellular GSHt content increased (1.57+/-0.20 versus 1.87+/-0.19 nmol x 1 x 10(6) BAC(-1)). The spontaneous oxidative activity of BAC decreased after NAC treatment (2.7+/-0.8 versus 1.0+/-0.2 nmol x 1 x 10(6) BAC(-1) x h(-1)). Interleukin-8 concentration (82.1+/-31.5 versus 80.0+/-22.6 pg x mL bronchoalveolar fluid (BALF), nonsignificant (NS)) and myeloperoxidase activity (1.93+/-0.64 versus 1.55+/-0.47 mU x mL(-1) BALF, NS) did not change significantly, but were found to be inversely correlated to intracellular GSHt. In conclusion, high-dose N-acetylcysteine supplementation increases intracellular glutathione levels slightly. This increase is associated with a mild reduction of oxidative activity but not with a reduction of bronchoalveolar cell activation in these patients.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

细胞穿透肽PEP-1介导人铜,锌-超氧化物歧化酶转导入大鼠离体心脏及其对缺血再灌注损伤的保护作用

目的 采用离体心脏灌注模型,研究细胞穿透肽PEP-1介导入铜,锌-超氧化物歧化酶(Cu,Zn-SOD,SOD1)穿透心肌组织能力及其对心脏缺血再灌注损伤的保护作用.方法 构建的原核表达载体pET15b-SOD1和pET15b-PEP-1-SOD1,分别转化大肠杆菌,表达和纯化SOD1和PEP-1-SOD1融合蛋白.采用Langendorff灌流系统,大鼠离体心脏停灌30 min后复灌60 min建立缺血再灌注损伤模型.大鼠随机分为对照组,100μmol/L SOD1蛋白预处理组,25、50、100μmol/L PEP-1-SOD1蛋白预处理组.免疫荧光及Western blot检测PEP-1-SOD1的转导效果,超氧化物歧化酶(SOD)试剂盒检测转导的目的 蛋白的SOD活性.测定心肌组织丙二醛(MDA)含量和复灌后15 min内冠状动脉流出液肌酸激酶(CK)活性.脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测心肌细胞凋亡,复灌60 min时红四氮唑(TTC)染色测定心肌梗死面积.结果 免疫荧光及Western blot检测均显示PEP-1-SOD1以剂量依赖性方式转导入离体灌注的心肌组织内,SOD1蛋白预处理组心肌组织内未见到目的 蛋白.对照组,SOD1组和25、50、100 μmol/L PEP-1-SOD1处理组的SOD活性分别为(10.06±0.77),(10.59±0.71)和(32.29±1.42)、(43.16±1.16)、(55.14±1.59)U/mg蛋白,MDA含量分别为(1.48±0.19),(1.39±0.11)和(1.01±0.14)、(0.73±0.13)、(0.50±0.06)nmol/mg蛋白,CK活性分别为(1.73±0.58),(1.68±0.14)和(1.40±0.28)、(0.97±0.39)、(0.61±0.56)U/mg蛋白,心肌细胞凋亡率(AI)分别为(17.25±0.75)%,(16.63±1.07)%和(11.50±0.57)%、(6.50±0.63)%、(4.13±0.52)%,心肌梗死面积百分比分别为(55.13±2.18)%,(52.13±2.59)%和(33.88±2.06)%、(25.50±2.16)%、(15.38±1.14)%.与对照组和SOD1组比较,PEP-1-SOD13种剂量组的SOD活性、MDA含量、CK活性、心肌细胞凋亡率及心肌梗死面积百分比指标差异均有统计学意义(均P<0.01).说明PEP-1-SOD1蛋白预处理组SOD活性以剂量依赖性方式显著高于SOD1组,MDA含量、CK活性及心肌细胞凋亡率均明显低于SOD1组,心肌梗死面积小于SOD1组.结论 PEP-1肽介导SOD1蛋白能直接以天然活性的形式高效穿透进入离体心肌组织,转导的PEP-1-SOD1融合蛋白对心肌缺血再灌注损伤具有明显的保护作用.     

Effects of diltiazem on the energy metabolism of the isolated rat heart submitted to ischaemia: a 31P NMR study

The possible direct attenuating modification by diltiazem (DZ) 10(-6) M of ischaemia-induced metabolic damage was studied by 31P NMR spectroscopy (at 101.3 MHz) on retrogradely perfused rat hearts submitted to a 24 min, normothermic (37 degrees C), global low-flow ischaemia (1% of the pre-ischaemic spontaneous coronary flow), followed by a 30 min reperfusion. The presence of DZ 10(-6) M altered neither the heart rate and the left intraventricular pressure under normoxic conditions, nor the extent of ATP and CP depletion during ischaemia, whilst the intramyocardial Pi accumulation during ischaemia was significantly reduced (by about 30%). The intracellular acidification induced by ischaemia was initially less in the presence of DZ, but the pH values reached by the end of ischaemia were somewhat lower than in control (albeit not significantly so): 5.85 +/- 0.07 v. 6.00 +/- 0.07 (Means +/- S.E.M.). On reperfusion, DZ-treated hearts exhibited a greater oxidative phosphorylation capacity than did control hearts. Indeed, NMR spectroscopy revealed a prompter, greater and durable rephosphorylation of creatine together with a simultaneous more rapid and furthermore sharp drop in Pi content in DZ-treated hearts. Moreover, although NMR spectroscopy did not reveal any significant difference in ATP alteration on reperfusion in DZ-treated hearts as compared with controls, biochemical measurements indicated slightly higher ATP content at the end of reperfusion and, more particularly, a better recovery of the adenylate charge: 0.81 +/- 0.03 v. 0.72 +/- 0.03, means +/- S.E.M. (Pre-ischaemic value 0.90-0.91). The intracellular pH differed insignificantly from its pre-ischaemic value at the end of reperfusion in DZ-treated hearts (7.08), while remaining below initial values in controls (7.00). From these results, it is inferred that, at relatively low concentration (10(-6) M), DZ exerts a direct beneficial effect on the energy metabolism of the ischaemic heart without preserving high-energy phosphate compounds during ischaemia and, most importantly, without reducing the extent of the concomitant intracellular acidification.     

N-acetylcysteine attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis

AIM To evaluate attenuating properties of N-acetylcysteine (NAC) on oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis (NASH).METHODS Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 8) was free accessed to regular dry rat chow (RC) for 6 wk.Group 2 (NASH, n = 8) was fed with 100% fat diet for 6 wk. Group 3 (NASH NAC20, n = 9) was fed with 100% fat diet plus 20 mg/kg per day of NAC orally for 6 wk. All rats were sacrificed to collect blood and liver samples at the end of the study.RESULTS The levels of total glutathione (GSH)and hepatic malondialdehyde (MDA) were increased significantly in the NASH group as compared with the control group (GSH; 2066.7 ± 93.2 vs 1337.5 ± 31.5 μmol/L and MDA; 209.9± 43.9 vs 3.8 ±1.7 μmol/g protein, respectively, P ＜ 0.05). Liver histopathology from group 2 showed moderate to severe macrovesicular steatosis, hepatocyte ballooning, and necroinflammation.NAC treatment improved the level of GSH (1394.8 ± 81.2 μmol/L, P ＜ 0.05), it did not affect MDA (150.1 ± 27.0 μmol/g protein), but led to a decrease in fat deposition and necroinflammation.CONCLUSION NAC treatment could attenuate oxidative stress and improve liver histology in rats with NASH.     

Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia

Alterations in energy substrate utilization during reperfusion of ischemic hearts can influence the functional recovery of the myocardium. Energy substrate preference by the reperfused myocardium, however, has received limited attention. Therefore, we measured oxidation rates of glucose and palmitate during reperfusion of ischemic hearts. Isolated working rat hearts were perfused with 1.2 mM palmitate and 11 mM [14C]glucose, 1.2 mM [14C]palmitate and 11 mM glucose, or 11 mM [14C]glucose alone, at an 11.5 mm Hg preload and 80 mm Hg afterload. Hearts were subjected to 60-minute aerobic perfusion or 25-minute global ischemia followed by 60-minute aerobic reperfusion. Steady-state oxidative rates of glucose or palmitate were determined by measuring 14CO2 production. In hearts perfused with glucose alone, oxidative rates during reperfusion were not significantly different than nonischemic hearts (1,008 +/- 335 vs. 1,372 +/- 117 nmol [14C]glucose oxidized/min/g dry wt, respectively). In the presence of palmitate, glucose oxidation was markedly reduced in reperfused and nonischemic hearts (81 +/- 11 and 101 +/- 15 nmol [14C]glucose oxidized/min/g dry wt, respectively). Palmitate oxidation rates were not significantly different in reperfused compared with nonischemic hearts (369 +/- 55 and 455 +/- 50 nmol [14C]palmitate oxidized/min/g dry wt, respectively). [14C]Palmitate was incorporated into myocardial triglycerides to a greater extent in reperfused ischemic hearts than in nonischemic hearts (26.0 and 13.8 mumol/g dry wt, respectively). Under the perfusion conditions used, palmitate provided over 90% of the ATP produced from exogenous substrates. Addition of the carnitine palmitoyltransferase I inhibitor, ethyl 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir, 10(-6) M), during reperfusion stimulated glucose oxidation and improved mechanical recovery of ischemic hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessing experimental models in myocardial injury: lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion

BACKGROUND: Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-alpha-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases mu- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-alpha. METHODS: Isolated hearts (guinea pig) were subjected to global ischaemia (15 min) and reperfused. Calpain activity was determined by zymography. Calpastatin (inhibitor) and troponin I (substrate) were quantified by western blotting. Immunohistology of hearts and a human mast cell line (HMC-1) was used to localise expression of TNF-alpha and TACE. Shedding of TNF-alpha was assessed in Mono Mach, Jurkat-T, HMC-1 and peripheral blood leucocytes with and without oxidative stress. RESULTS: Neither of the ubiquitous calpains (mu- and m-calpain) was significantly activated by brief ischaemia/reperfusion, nor were calpastatin and troponin degraded more than in extracts of control hearts. Cardiac TNF-alpha immunoreactivity was localised to mast cells. None of the tested cell lines shed TNF-alpha in response to non-toxic amounts of oxidants. However, HMC-1 cells showed poor expression of proTNF-alpha, while TACE was abundant. CONCLUSIONS: Although the severity of ischaemia in the current model may have been insufficient, activation of calpain by ischaemia/reperfusion cannot be demonstrated simply in the Langendorff-mode perfused isolated heart. Mast cells are the prime source of myocardial TNF-alpha. A suitable whole-cell model remains to be found to demonstrate acute oxidative activation of TACE.     

The protective effect of desferal on rat myocardial mitochondria is not prolonged after withdrawal of desferal

Summary Reperfusion of ischaemic myocardium is necessary to sustain tissue viability (without it the tissue becomes necrotic), but reperfusion, on the other hand, can damage cells which have survived ischaemia. There is now considerable evidence that oxygen radicals, especially hydroxyl radicals produced via the Haber-Weiss and Fenton reactions, are responsible for reperfusion damage. Various investigators have reported that desferal, an iron chelator, has a beneficial effect on the myocardium during ischaemia and reperfusion. The aim of this study was two-fold: i) whether superoxide anions in the absence of LMWI can impair mitochondrial function, and ii) whether the protective effect of desferal on the mitochondrial function persists after withdrawal of desferal. Experiments were done on isolated rat hearts subjected to normothermic ischaemic cardiac arrest (NICA), with or without desferal, followed by 15-min reperfusion with desferal, followed by 15-min perfusion without desferal, or a hypoxanthine/xanthine oxidase medium that generates superoxide anions (with or without desferrioxamine (desferal) in the perfusate). Mitochondrial function (QO₂ (state 3), ADP/O and OPR) as well as LMWI were measured. Our results indicated that i) superoxide anions and/or hydrogen peroxide can, independently of LMWI, damage the mitochondria, and ii) withdrawal of desferal after the respiratory burst resulted in the same or more severe mitochondrial damage than without any desferal.Abbreviation list LMWI Low molecular weight iron - NICA normothermic ischaemic cardiac arrest - QO₂ (state 3) nmol oxygen consumed in the presence of ADP/mg mitochondrial protein/min - ADP/O nmol ADP consumed/nmol oxygen consumed - OPR oxidative phosphorylation rate, nmol ADP consumed/mg mitochondrial protein/min     

Effects of myocardial catecholamine depletion on cellular electrophysiology and arrhythmias during ischaemia and reperfusion

The effect of myocardial catecholamine depletion on cellular electrophysiology and arrhythmias was assessed in Langendorff perfused guinea pig hearts during ischaemia and reperfusion. Myocardial noradrenaline was reduced to 0.17 +/- 0.04 microgram X g-1 by intracardiac injection of 6-hydroxydopamine (450 mg X kg-1 in six doses over 20 days) compared with 1.5 +/- 0.2 microgram X g-1 in vehicle injected controls. Myocardial catecholamine depletion significantly reduced the incidence of ventricular tachycardia and fibrillation during 30 min of global ischaemia and subsequent reperfusion. Myocardial catecholamine depletion prolonged action potential duration and refractory period during control perfusion and blunted ischaemia induced reduction in action potential amplitude, Vmax, and duration, but accentuated the prolongation in conduction time and QRS width. Catecholamine depletion abolished or attenuated reperfusion induced shortening of action potential duration and refractory period. Catecholamine depletion increased myocardial glycogen levels from 2.47 +/- 0.3 mg X g-1 wet weight to 4.39 +/- 0.3 mg X g-1; fasting animals for 48 h prior to study reversed this with no attenuation of the electrophysiological or antiarrhythmic action. These results provide further evidence that release of endogenous myocardial catecholamines contributes to the electrophysiological changes and arrhythmias associated with myocardial ischaemia and reperfusion.     

Alpha 1 adrenoceptors in the ischaemic and reperfused myocardium

The [3H]-prazosin binding activity of membranes isolated from aerobically perfused, ischaemic and reperfused cat and rat hearts was investigated. Alpha 1 adrenoceptors in membranes from aerobically perfused cat hearts had a KD of 0.363 +/- 0.067 nM and a Bmax of 68.8 +/- 7.0 fmol/mg protein; 30 min normothermic global ischaemia caused an increase (P less than 0.01) in density (Bmax, 111.4 +/- 9.3 fmol/mg protein), without any change in affinity (KD 0.430 +/- 0.069 nM). Post-ischaemic reperfusion (15 min) caused the Bmax to return to control values, with no change in KD. Membranes isolated from aerobically perfused rat hearts contained a single population of high affinity alpha 1 adrenoceptor binding sites, with a KD of 0.092 +/- 0.02 nM and a Bmax of 43.02 +/- 2.49 fmol/mg protein. Neither global nor low-flow (0.1 ml/min) ischaemia for either 30 or 60 min, nor post-ischaemic reperfusion, caused any change in either the affinity or density of these binding sites. In both species, and under all the above experimental conditions, the selectivity of the alpha 1 adrenoceptor binding sites was maintained, with phentolamine much greater than rauwolscine in displacing bound [3H]-prazosin. These results show that the ability of ischaemia to increase alpha 1 adrenoceptor density in cardiac membranes is species specific, and that it can occur as a direct (as opposed to a reflex) response to ischaemia.