Ischemia and Reperfusion: Effect of Fructose-1,6-Bisphosphate

Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the “oxygen paradox” in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.     

氧化苦参碱对大鼠缺血再灌注心肌损伤的保护机制研究

目的:探讨氧化苦参碱(OMT)对大鼠缺血再灌注心肌损伤(MIRI)的保护机制。方法:随机将60只成年Wistar大鼠分成对照组、MIRI组和OMT组,每组20只,除对照组外,其他两组结扎30 min后松解结扎线灌注60 min。结扎前10 min,OMT组股静脉输入苦参注射液120 mg/kg,对照组、MIRI组则输入等容量生理盐水。造模后,记录两组心率(HR)、左心室收缩压(LVSP)、左室内压最大上升或下降速率(+dp/dt_(max)或-dp/dt_(min))及血清乳酸脱氢酶(LDH),检测两组心肌组织中一氧化氮(NO)、丙二醛(MDA)、一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)水平。结果:与MIRI组相比,OMT组HR、LVSP和+dp/dt_(max)、-dp/dt_(min)均显著升高(P0.05),且OMT组上述指标与对照组比较,差异均无统计学意义(均P0.05)。与MIRI组相比,OMT组的NO、NOS、SOD、GSH-PX水平均显著升高,而MDA、血清LDH水平显著降低,比较差异均有统计学意义(均P0.05),且OMT组上述指标与对照组比较,差异均无统计学意义(均P0.05)。结论:OMT对MIRI大鼠具有心肌保护作用,其机制可能与提高抗氧自由基活性、改善微循环及舒张冠脉血管有关。     

Lipid Peroxidation, Tissue Necrosis, and Metabolic and Mechanical Recovery of Isolated Reperfused Rat Heart as a Function of Increasing Ischemia

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coen-zymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intra-ventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 μmollg dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 μmol/g dry weight and 0.069 μmol/ 30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.     

Detection of Hydroxyl Radical in Intact Cells of Chlorella Vulgaris

Using ESR with 5,5-dimethyl-l-pyrroline N-oxide (DMPO) as a spin-trapping reagent, we measured the levels of free radical species generated from living cells of Chlorella vulgaris var. vulgails (IAM C-534). To investigate the production of free radicals in the living Chlorella vulgaris cells, the influence of DMPO toward the intact cells of the Chlorella vulgaris using the O₂ evolution rate was first studied as a guide. Since the 0₂ evolution rate was not changed by DMPO, it was judged that DMPO has no toxicity toward the intact cells of Chlorella vulgaris.

Only hydroxyl radicals (-OH) were detected as the DMPO-OH adduct in the suspension of intact cells of Chlorella vulgaris irradiated with visible light. Moreover, since production of -OH was inhibited by some hydroxyl radical scavengers such as KI and ethanol, production of -OH was proved to be due to hydroxyl radicals. It was also clear that the intensity of OH increased with increasing irradiation intensity of visible light. Therefore, it was suggested that -OH might be one of the photoinhibition factors of the intact Chlorella vulgaris cells in severe light conditions.     

Glutathione and Ascorbate During Ischemia and Postischemic Reperfusion in Rat Brain

Thirty minutes of total cerebral ischemia (decapitation) decreased total glutathione (GSH + GSSG) by 7% but had no detectable effect on the concentration of oxidized glutathione (GSSG), reduced ascorbate, or total ascorbate. In a model of reversible, bilateral hemispheric ischemia (four-vessel occlusion) no changes in glutathione or ascorbate were detected after 30 min of ischemia. During 24 h of reperfusion following such an insult no detectable change in total ascorbate, reduced ascorbate, or oxidized glutathione was noted; however, total brain glutathione declined by 25%. The findings are discussed in relation to the hypothesis that the deleterious effects of ischemia are due to an increase in free radical production which in turn leads to increased lipid peroxidation.     

Spin Trapping Evidence for Radical Generation by Isolated Hearts and Cultured Heart Cells

The spin trap 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) has been applied to monitor the generation of free oxy-radicals in samples derived from isolated hearts and heart cells. · OH was trapped in the effluent of isolated hearts in the early phase of reperfusion following an ischemia time of only 10min. Radical detection was possible even when the cardioactive DMPO was added to the effluent after draining off the heart, demonstrating that the short-lived · OH was generated by components released from the affected heart. These results support the hypothesis that radicals are of relevance for reperfusion injury.

By omitting antioxidants commonly used for incubation media of cultured cells, it was possible for the first time to demonstrate the formation of · OH in the incubation solution of cardiac cells.     

The Effect of Vitamin E-Deficiency in Isolated Rat Heart on the Cellular Defence System Against Free Radicals During Normal Reperfusion After Hypoxic, Ischemic and Ca2+-Free Perfusion

We investigated whether vitamin E plays a role in the protection against potential free radical formation and related biochemical changes in hypoxic, ischemic and Ca²⁺-depleted rat heart upon normal reperfusion.

In the heart of normally fed rats a decrease in the activity of superoxide dismutase and the capacity of the glutathione system, factors of the cellular protective mechanisms against free radicals, occurred upon exposure to the above mentioned treatments. This decrease was not further enhanced if vitamin E-deficient rat hearts were treated. Vitamin E-deficiency, however, led to detectable peroxidation of lipids if Ca²⁺-depleted or hypoxic hearts were reperfused. Lipid peroxidation was measured as the formation of thiobarbituric acid reactive material, which is readily formed during this process. Reflow after ischemia did not induce lipid peroxidation either in normal or in vitamin E-deficient rat heart.

Since changes in Ca²⁺ -homeostasis are thought to be primarily responsible for the Ca²⁺-reperfusion injury, a role for Ca²⁺-ions in lipid peroxidative processes, either directly or indirectly, seems indicated. Furthermore the results imply that even a sharp and extensive decrease of reduced glutathione, as seen upon Ca²⁺ -repletion after a period of Ca²⁺ -depletion, does not necessarily induce peroxidation of lipids in heart tissue. Obviously, vitamin E is very important in the protection of cardiac membranes. Replenishment of the water-soluble protective factors in the heart seems, however, more important during above mentioned treatments, especially since repair of the vitamin E-free radical is dependent on water-soluble factors.     

Spin Trapping Evidence for Radical Generation by Isolated Hearts and Cultured Heart Cells

The spin trap 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) has been applied to monitor the generation of free oxy-radicals in samples derived from isolated hearts and heart cells. · OH was trapped in the effluent of isolated hearts in the early phase of reperfusion following an ischemia time of only 10min. Radical detection was possible even when the cardioactive DMPO was added to the effluent after draining off the heart, demonstrating that the short-lived · OH was generated by components released from the affected heart. These results support the hypothesis that radicals are of relevance for reperfusion injury.

By omitting antioxidants commonly used for incubation media of cultured cells, it was possible for the first time to demonstrate the formation of · OH in the incubation solution of cardiac cells.     

Effect of MCI-186 on Postischemic Reperfusion Injury in Isolated Rat Heart

MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a newly developed antioxidant which has been shown to reduce brain edema in cerebral ischemia through inhibition of the lipoxygenase pathway of arachidonic acid. However, its effect on myocardial reperfusion injury after prolonged ischemia has not yet been demonstrated. We compared the mode of the effect of MCI-186 and recombinant human CuZn superoxide dismutase (rh-SOD) in isolated perfused rat hearts subjected to 60-min ischemia followed by 60-min reperfusion. Left ventricular developed pressure (LVDP), necrotic area and the release of creatine phosphokinase (CPK) and endogenous CuZn superoxide dismutase (endoge-SOD) were measured to evaluate myocardial damage. The decrease in left coronary flow (CBF) was measured as an index of the damage of left coronary circulation. MCI-186 (17.5 mg/L) was perfused for 10 min in the MCI group and rh-SOD (70 mg/L) was perfused during the reperfusion period in the SOD group starting 5 min prior to reperfusion. The release patterns of CPK and endoge-SOD were analyzed to elucidate the difference in the mode of protection of MCI-186 and rh-SOD. The LVDP remained higher in both MCI and SOD groups than that of control (76 ± 1, 77 ± 2 and 69 ± 1% of preischemic value, respectively). The necrotic area was significantly attenuated in both MCI and SOD groups compared with that in the control group (16 ± 1,14 ± 1 and 32 ± 170, respectively, p<0.05). Total CPK release was lower in both MCI and SOD groups thfn in the control (78 ± 7, 100 ± 2 and 116 ± 4 × 10³ units/g myocardium respectively). The decrease in CPK release was more marked in the MCI group than that in the SOD group (p<0.05). The reduction in CBF was significantly attenuated by the treatment with rh-SOD or MCI-186, but the effect was much higher in the SOD group than in the MCI group (69 ± 5, 58 ± 2, and 48 ± 2% in SOD, MCI and control groups, respectively). The release pattern of endoge-SOD was identical to that of CPK and thus this did not distinguish the mode of effect of MCI-186 from that of rh-SOD. These results indicate that MCI-186 reduces reperfusion injury in isolated perfused hearts with prolonged ischemia and the effect is more closely related to the reduction of myocyte damage than the preservation of the coronary circulation.     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.     

SIRT6在缺血再灌注损伤中的作用机制及研究进展

缺血性损伤后恢复血液供应会导致缺血再灌注（ischemia reperfusion, IR）损伤,这会导致组织损伤进一步加剧。IR损伤伴随着一系列机制,包括谷氨酸兴奋性毒性、钙超载、氧化应激、炎症和细胞凋亡,最终导致细胞死亡。IR损伤过程均由Sirtuins家族调控,在Sirtuins家族中,特异性定位于细胞核中的SIRT6可以促进对DNA损伤和氧化应激的抵抗,抑制基因组的不稳定性,在代谢稳态中发挥作用,同时SIRT6在人重要脏器中处于高度表达状态。但SIRT6在IR损伤中研究较少,结合国内外最新的研究进展,对SIRT6在IR损伤中的作用进行了回顾性的总结和分析,希望对国内外学者对于SIRT6在IR损伤中的研究提供一些参考依据。     

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron – providing protection to the ischemic heart via preconditioning (PC).PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper.During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4–8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 g/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 g/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin – the major iron-storage protein.It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.     

针刺联合依达拉奉对缺血再灌注大鼠视网膜自由基代谢的影响

目的:研究针刺联合依达拉奉对大鼠视网膜缺血再灌注时自由基代谢的影响.方法:大鼠随机分为五组:假手术组、模型组、针刺组、依达拉奉组和联合(针刺+依达拉奉)组.建立缺血再灌注模型,于造模后1d,行视网膜电流图(ERG)检查,取视网膜组织,测定各组MDA含量及T-SOD、GSH-Px、CAT活性的变化.结果:①ERG a、b波振幅相对恢复率:联合组、针刺组及依达拉奉组与模型组相比,a、b波振幅相对恢复率升高,差异有显著性(P＜0.05),与针刺组和依达拉奉组相比,联合组作用更为显著(P＜0.05).②MDA含量及T-SOD、GSH-Px、CAT活性:联合组、针刺组及依达拉奉组与模型组相比,MDA显著降低,SOD、GSH-Px、CAT显著升高,差异有显著性(P＜0.05),联合组作用显著优于针剌组或依达拉奉组(P＜0.05).结论:针刺联合依达拉奉具有干预自由基代谢作用,明显减轻缺血再灌注引起的视网膜氧化应激损伤,对视网膜功能起保护作用.     

Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two-vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3- and 2,5-DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3- and 2,5-DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3- and 2,5-DHBA levels increased by 2.5- and 2.8-fold, respectively. Levels of 2,3-DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5-DHBA levels persisted for 2 h. The increases in 2,3- and 2,5-DHBA levels observed following hyperthermic ischemia were significantly higher (3.8- and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical-induced injury in the development of ischemic damage.     

PBN spin trapping of free radicals in the reperfusion-injured heart. Limitations for pharmacological investigations

Post-ischemic reperfusion causes cardiac dysfunction and radical-induced lipid peroxidation (LPO) detectable by ESR spin trapping. This study deals with the applicability of the spin trap technique to pharmacological investigations during myocardial reperfusion injury. The use of the spin trap phenylbutylnitrone (PBN, 3 mM) in isolated rat hearts demonstrated the release of alkoxyl radicals (a_N = 1.39 mT, a_H = 0.19 mT) formed particularly within the first 15 min of reperfusion following 30 min of ischemia. The decline of radicals, after 10 min of reperfusion, was accompanied by recovery of function in 80% of the hearts. The radical concentration in the coronary effluent (maximum after 7.5 min) was reduced by the infusion of 1 mM mercaptopropionylglycine (MPG, 2.7 ± 0.5 U/ml, p < 0.001) or 5 M vitamin E (11.7 ± 0.8 U/ml, p < 0.001), compared to the (PBN-containing) control (29.7 ± 4.3 U/ml). Moreover, functional recovery (left ventricular developed pressure, LVDP 91.6 ± 20% of pre-ischemic level, p < 0.05) was improved by the hydrophilic radical scavenger MPG, compared to the (PBN-containing) control (LVDP 50.5 ± 15.7% of baseline). PBN alone led to higher functional recovery (p < 0.05) and reduced VF (duration of ventricular fibrillation; 7.10 ± 0.36 min/30 min, p < 0.05), compared to the untreated (PBN-free) control (LVDP 26.6 ± 11.8%; VF 19.42 ± 3.64 min/30 min). The Ca antagonist verapamil (0.1 M), MPG, and the lipophilic vitamin E showed cardioprotection in the absence of PBN: post-ischemic recovery of LVDP was 25.4 ± 6.8% (p < 0.05), 39.6 ± 12.7% (p < 0.05) and 52.4 ± 2.6% (p < 0.01), respectively, compared to the corresponding untreated control (13.3 ± 6.6%). Whereas verapamil and vitamin E were able to protect the heart when present alone, they offered no additive effect in the presence of PBN. Therefore, PBN can be used to estimate the radical scavenger properties of an agent in the heart. However, because of the protective properties of PBN itself, the results of simultaneous investigations of the effects of other compounds, such as Ca antagonists or lipophilic radical scavengers, on heart function may be limited.     

Studies on the Effects of Antioxidants and Inhibitors of Radical Generation on Free Radical Production in the Reperfused Rat Heart Using Electron Spin Resonance Spectroscopy

Reperfusion of the heart after a period of ischaemia can precipitate ventricular arrhythmias and lead to an exacerbation of tissue injury. Direct evidence to suggest the involvement of free radicals has been obtained using electron spin resonance (esr) spectroscopy and the spin trap N-tert. butyl-α-phenyl nitrone (PBN). In the present study, we have used esr spectroscopy and PBN to examine the individual effects of superoxide dismutase (SOD), catalase. allopurinol or desferal on radical production in the isolated. reperfused rat heart. A burst of radical production was observed in the control group during the first 5 minutes of reperfusion; the peak occurred during the first minute, when signal intensity had increased by almost 300%. but returned to the baseline by 15 minutes of reperfusion. The esr signals were consistent with the trapping of either alkoxyl or carbon-centered radicals (a_N = 13.6 and a_H = 1.56G). In the desferal-treated group, a burst of radical production was observed during the first five minutes of reperfusion; this was maximal during the second minute, when signal intensity had increased by almost 200%, but had returned to the baseline value by 30 minutes of reperfusion. In the SOD-treated group, a burst of radical production was observed during the first 10 minutes of reperfusion; signal intensity was maximal during the tenth minute of reperfusion, when signal intensity had increased by almost 200%. but had returned to the baseline value by 30 minutes of reperfusion. In the allopurinol- and catalase-treated groups, no significant burst of radical production could be detected. These data further support the concept that cytotoxic, oxygen-derived species are formed upon reperfusion and that hydrogen peroxide and/or hy-droxyl radicals, are likely to be involved.     

Augmentation of Nitric Oxide,Superoxide, and Peroxynitrite Production During Cerebral Ischemia and Reperfusion in the Rat

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, N-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.     

刺槐素对大鼠心肌缺血再灌注损伤的保护作用与机制研究

摘要 目的：探讨刺槐素对大鼠心肌缺血再灌注损伤(MIRI)的作用以及可能的作用机制。方法：对24只Sprague-Dawley (SD)大鼠进行随机分组,分为：假手术组、模型组、刺槐素给药组、刺槐素+AG490给药组,每组6只,通过结扎冠状动脉左前降支,缺血30 min,再灌注120 min复制心肌缺血再灌注损伤模型。利用氯化三苯基四氮唑测定心肌梗死面积,紫外分光光度计和酶联免疫法检测血清中肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）的活性,蛋白印迹法分别检测心肌组织中Bcl-2、Bax、Stat3和p-Stat3蛋白相对表达水平。结果：与假手术组比较,模型组大鼠血清中CK-MB、LDH活性明显升高（P＜0.01）,心肌梗死面积百分比显著增加（P＜0.01）,p-Stat3/Stat3比率、Bcl-2/Bax比率显著下降（P＜0.01）;与模型组相比,刺槐素给药组中CK-MB、LDH的活性,以及心肌梗死面积百分比显著降低（P＜0.01）,Bcl-2/Bax比率和p-Stat3/Stat3比率显著提高（P＜0.05）。然而在刺槐素+AG490药物组中刺槐素对于受损心肌的保护作用被AG490消除。结论：刺槐素可减轻MIRI大鼠心肌损伤,发挥心肌保护作用,其机制可能与活化Jak2/Stat3信号通路进而抑制心肌细胞凋亡有关。     

心宁片对糖尿病合并心肌缺血再灌注损伤的保护作用和机制研究

目的:研究心宁片对糖尿病合并心肌缺血再灌注损伤的保护作用及其作用机制。方法:腹腔注射STZ加高脂高糖饲料喂养诱导二型糖尿病小鼠模型,随机分为假手术组、模型组及心宁片高、中、低剂量组(心宁片10、20和30 mg·kg~(-1)),每组10只。在此基础上,制作心肌缺血再灌注模型。测定小鼠体内血糖和血脂水平,测定缺血再灌注后心肌酶(LDH和CK-MB)、心梗面积以及AMPK磷酸化水平。结果:心宁片能够有效的控制糖尿病小鼠体内血糖和血脂水平,减轻胰岛素抵抗情况。心宁片能够减轻缺血再灌注引起的心肌梗死,降低LDH和CK-MB水平,减少MDA水平。同时,还发现心宁片能够促进AMPK蛋白磷酸化。采用AMPK特异性抑制剂Compound C抑制AMPK后,LDH水平显著升高,心宁片的心肌保护作用减弱。结论:心宁片能够保护糖尿病合并缺血再灌注损伤,其机制可能是通过AMPK信号通路。     

Myocardial protection by Protykin,a novel extract of trans-resveratrol and emodin

Protykin is an all-natural, high potency standardized extract of trans-resveratrol (20%) and emodin (10%) derived from the dried rhizome of Polygonum cuspidatum. Previous studies have demonstrated free radical scavenging and anti-inflammatory activities of resveratrol. Since free radicals play a crucial role in the pathogenesis of myocardial ischemia/reperfusion injury, we examined whether Protykin could preserve the heart during ischemic arrest. Sprague—Dawley rats were divided into two groups: experimental group was gavaged Protykin (100 mg/kg body wt) dissolved in corn oil for three weeks, while the control group was gavaged corn oil alone. After three weeks, rats were sacrificed, isolated hearts perfused via working mode, were made globally ischemic for 30 min followed by 2 h of reperfusion. Left ventricular functions were continuously monitored and malonaldehyde (MDA) (presumptive marker for oxidative stress) formation were estimated. At the end of each experiment, myocardial infarct size was measured by TTC staining method. Peroxyl radical scavenging activity of Protykin was determined by examining its ability to remove peroxyl radical generated by 2,2′-azobis (2-amidinopropane) dihydrochloride, while hydroxy radical scavenging activity was tested with its ability to reduce 7-OH^·-coumarin-3-carboxylic acid. The results of our study demonstrated that the Protykin group provided cardioprotection as evidenced by improved post-ischemic left ventricular functions (dp, dp/dt_max) and aortic flow as compared to control group. This was further supported by the reduced infarct size in the Protykin group. Formation of MDA was also reduced by Protykin treatment. In vitro studies demonstrated that Protykin possessed potent peroxyl and hydroxyl radical scavenging activities. The results of this study indicate that Protykin can provide cardioprotection, presumably by virtue of its potent free radical scavenging activity.