Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Peroxidative injury of the mitochondrial respiratory chain during reperfusion of hypothermic rat liver

Mitochondrial dysfunction in ischemic liver has been demonstrated to be due to decrease in the intramitochondrial level of ATP and the subsequent disruption of the proton barrier of the inner membrane (Watanabe, F., Hashimoto, T. and Tagawa, K. (1985) J. Biochem. 97, 1229-1234). In this study, another injury process, impairment of the electron-transfer system, which occurred during reoxygenation of ischemic liver, was studied during reperfusion of cold preserved liver and during cold incubation of isolated rat-liver mitochondria. The sites of the respiratory chain that were sensitive to peroxidative damage were ubiquinone-cytochrome c oxidoreductase and NADH-ubiquinone oxidoreductase. These enzymic activities decreased with increase in lipid peroxidation. Incubation of submitochondrial particles with t-butyl hydroperoxide or with an NADPH-dependent peroxidation system decreased the enzymic activities of the electron-transport system. These data strongly suggested that lipid peroxidation during reoxygenation of ischemic liver impaired the electron-transfer system. Thus, mitochondria of ischemic liver suffer from two different types of injury: increase in proton permeability during anoxia, and decrease in enzymic activities of the electron-transport system during reoxygenation.     

Mitochondrial uncoupling protein-2 mediates steatotic liver injury following ischemia/reperfusion

Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemi-a/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.     

Ghrelin reduces injury of hippocampal neurons in a rat model of cerebral ischemia/reperfusion

Ghrelin, an acyl-peptide gastric hormone and an endogenous ligand for growth hormone secretagogue (GHS) receptor 1a (GHS-R 1a) exerts multiple functions. It has been reported that synthetic GHS-hexarelin reduces injury of cerebral cortex and hippocampus after brain hypoxia-ischemia in neonatal rats. However, the effect of ghrelin in tolerance of the brain tissues to cerebral ischemia/reperfusion (I/R) injury has not been studied. The aim of the present study was to examine whether ghrelin have potential protective effect on hippocampal neurons of rats against I/R injury. I/R injury was induced by a modified four-vessel occlusion model. Ghrelin was administered intraperitoneally after the insult. Histological damage of the neurons was determined with hematoxylin-eosin (H&E) staining and assay of the neuronal apoptosis was performed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). The results showed that I/R decreased the number of surviving neurons and induced apoptosis of the neurons in CA1 area of the hippocampus in rats. In contrast, administration of ghrelin significantly increased the number of surviving neurons and reduced the number of TUNEL-positive apoptotic neurons in the equivalent areas after I/R. In conclusion, the present data provide evidence for the first time that ghrelin can exert a neuroprotective role in vivo in the tolerance of hippocampal neurons to I/R injury, and that the mechanism underlying this effect involves an anti-apoptotic property of ghrelin.     

Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria

Free fatty acids (FFA) are known to uncouple oxidative phosphorylation in mitochondria. However, their mechanism of action has not been elucidated as yet. In this study we have investigated in detail the patterns of uncoupling by the FFA oleate and palmitate in rat liver mitochondria and submitochondrial particles. The patterns of uncoupling by FFA were compared to uncoupling induced by the ionophores valinomycin (in the presence of K+) and gramicidin (in the presence of Na+) and the proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP). The most striking difference in the pattern of uncoupling relates to the effect on the proton electrochemical potential gradient, delta mu H. Uncoupling by ionophores, particularly valinomycin, is associated with and most likely caused by a major reduction of delta mu H. In contrast, uncoupling by FFA is not associated with a significant reduction of delta mu H, indicating another mechanism of uncoupling. We suggest the use of the term decouplers for uncoupling agents such as FFA and general anesthetics that do not collapse the delta mu H [Rottenberg, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3313-3317]. The protonophore CCCP and to some extent the ionophore gramicidin indicate a mixed mode of uncoupling since their effect on delta mu H is moderate when compared to that of valinomycin. Another distinguishing feature of uncouplers that collapse the delta mu H is their ability to stimulate ADP-stimulated respiration (state 3) further. Decouplers such as FFA and general anesthetics do not stimulate state 3 respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Daidzein administration in vivo reduces myocardial injury in a rat ischemia/reperfusion model by inhibiting NF-kB activation

AimsWe tested the hypothesis that daidzein may reduce myocardial damage by both inhibiting the release of cytokines and limiting the nuclear translocation of NF-kB.Main methodsMale Sprague–Dawley rats were anesthetized, and the left anterior descending coronary artery (LAD) was ligated for 25 min. Twenty-four hours after reperfusion was established, the hemodynamics and infarct size were examined.Key findingsTreatment with daidzein (10 mg/kg, i.p.) 1 h prior to the ischemia/reperfusion procedure (I/R) reduced the infarct size by 52.8% (P < 0.05). Daidzein also significantly improved I/R-induced myocardial contractile dysfunction by improving the left ventricular diastolic pressure and the positive and negative maximal values of the first derivative of the left ventricular pressure. In addition, daidzein reduced the plasma levels of TNF-α and IL-6 in I/R rats and decreased malondialdehyde levels, myeloperoxidase activity, catalase activity and neutrophil infiltration in I/R rat myocardium. Interestingly, daidzein inhibited I/R-induced myocardial apoptosis by decreasing DNA strand breaks and cleaved caspase-3 activity. Furthermore, daidzein inhibited both the nuclear translocation of NF-kB in I/R rat hearts and the H₂O₂-induced activation of NF-kB-luciferase activity in human umbilical vein endothelial cells.SignificanceThis study reveals that the administration of daidzein in vivo attenuates I/R-induced myocardial damage via inhibition of NF-kB activation, which in turn may suppress inflammatory cytokine expression.     

Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo

To determine the role of neutrophils in the pathogenesis of hepatic ischemia/reperfusion injury, livers from male Fischer rats were subjected to 45 min of no-flow ischemia followed by reperfusion for up to 24 h. Two phases of liver injury were identified, an initial phase during the first hour of reperfusion and a later progression phase with 80 +/- 3% hepatocyte necrosis and an 80-fold increase of neutrophil infiltration in the liver after 24 h. Pretreatment with a monoclonal antibody against neutrophils, which caused consistent neutropenia, protected the liver from reperfusion injury as indicated by 28 +/- 10% necrosis, and 84% reduction of hepatic neutrophil accumulation and a complete recovery of the hepatic ATP content. Our data suggest that the later progression phase of reperfusion injury after hepatic no-flow ischemia is mediated mainly by neutrophils.     

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart

Gypenosides (GP) are the predominant components of Gynostemma pentaphyllum, a Chinese herb medicine that has been widely used for the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated to exert protective effects on the liver and brain against ischemia-reperfusion (I/R) injury, yet whether it is beneficial to the heart during myocardial I/R is unclear. In this study, we demonstrate that pre-treatment with GP dose-dependently limits infarct size, alleviates I/R-induced pathological changes in the myocardium, and preserves left ventricular function in a rat model of cardiac I/R injury. In addition, GP pre-treatment reduces oxidative stress and protects the intracellular antioxidant machinery in the myocardium. Further, we show that the cardioprotective effect of GP is associated with the preservation of mitochondrial function in the cardiomyocytes, as indicated by ATP level, enzymatic activities of complex I, II, and IV on the mitochondrial respiration chain, and the activity of citrate synthase in the citric acid cycle for energy generation. Moreover, GP maintains mitochondrial membrane integrity and inhibits the release of cytochrome c from the mitochondria to the cytosol. The cytoprotective effect of GP is further confirmed in vitro in H9c2 cardiomyoblast cell line with oxygen-glucose deprivation and reperfusion (OGD/R), and the results indicate that GP protects cell viability, reduces oxidative stress, and preserves mitochondrial function. In conclusion, our study suggests that GP may be of clinical value in cytoprotection during acute myocardial infarction and reperfusion.     

Energy charge restoration, mitochondrial protection and reversal of preservation induced liver injury by hypothermic oxygenation prior to reperfusion

Background

We investigated the benefit of two different techniques for resuscitating marginally preserved liver grafts, unexpectedly subjected to long storage times.

Methods

Rat livers were cold-stored for 22 h (CS22). Some grafts were subsequently subjected to 90 min of hypothermic reconditioning by venous systemic oxygen persufflation (VSOP) or oxygenated hypothermic machine perfusion (HMP). Livers stored for only 6 h (CS6) served as reference. Viability of the livers was assessed thereafter by warm reperfusion in vitro.

Results

VSOP and HMP significantly increased endischemic tissue energy charge, and abrogated cellular enzyme loss upon reperfusion even significantly below control values. Ammonia clearance and bile production were more than 3-fold improved to similar values as CS6. Hypothermic reconditioning by both techniques induced mitochondrial chaperone expression (HSP70 family) and significantly improved early resumption of oxygen utilisation upon reperfusion.

Conclusion

Viability of long preserved liver grafts can be augmented by transient hypothermic reconditioning using either machine perfusion or gaseous oxygen persufflation, both preventing initial mitochondrial dysfunction and subsequent tissue injury.     

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain.     

大鼠肢体缺血预处理对肝缺血/再灌注损伤的保护作用

目的：研究肢体缺血预处理对大鼠肝缺血/再灌注损伤是否具有保护作用。方法：雄性SD大鼠32只,随机分为对照组（S组）;缺血/再灌注组（I/R组）;经典缺血预处理组（IPC组）;肢体缺血预处理组（远端缺血预处理组,RPC组）。S组仅行开腹,不作其他处理;IPC组以肝缺血5min作预处理;RPC组以双后肢缺血5min,反复3次作预处理,2个预处理组及I/R组均行肝缺血1h再灌注3h。取血用于血清谷丙转氨酶（ALT）与血清谷草转氨酶（AST）检测。切取肝组织用于测定湿干比（W/D）、中性粒细胞（PMN）计数及观察显微、超微结构的变化。结果：与I/R组比较,IPC组,RPC组ALT,AST,W/D值,及PMN计数均明显降低（P〈0.01）,肝脏的显微及超微结构损伤减轻。结论：肢体缺血预处理对大鼠肝脏I/R损伤有明显的保护作用,强度与经典缺血预处理相当,其机制可能与抑制肝脏炎症反应、减轻肝脏水肿、改善肝组织微循环有关。     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22°C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22°C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22°C and from 56 to 67 kJ/mol in the range from 22 to 37°C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.     

Distinct regulation of brain mitochondrial carrier protein-1 and uncoupling protein-2 genes in the rat brain during cold exposure and aging

Mitochondrial uncoupling proteins (UCPs) have been postulated to be regulators of thermogenesis, energy balance, and oxidative stress. Brain mitochondrial carrier protein-1 (BMCP1) is a new member of the UCP family, but little is known about the gene regulation and the role of BMCP1 in the central nervous system. In the present study, we first cloned BMCP1 cDNA encoding 325 amino acids from rat brain. The BMCP1 mRNA showed a distinct distribution pattern compared with that of UCP2 gene in human brain. Cold exposure did not affect the mRNA levels of BMCP1 and UCP2 in rat whole brain, but did increase the expression of UCP2 in the spinal cord. The mRNA level of BMCP1 in the brain of 26-month-old rats was decreased by 30% and that of UCP2 increased by 60% compared with the levels in 6-month-old rats. These results suggest differential roles of BMCP1 and UCP2 in thermoregulation and aging.     

Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation

Previous studies have indicated that pyruvate is able to reduce ischaemia/reperfusion (I/R) injury in a variety of tissues, but a full understanding of the effects is lacking. In this current preliminary study, magnetic resonance spectroscopy (MRS) was used to investigate the biochemical effects of differing concentrations of pyruvate (3 and 15mM) on liver metabolism during the cold hypoxic preservation period itself, in order to gain insight into possible mechanisms. Hepatic lactate, alanine, and succinate levels were increased in livers preserved with 15mM pyruvate added to the University of Wisconsin (UW) solution and were generally elevated (but to a lesser degree) in livers flushed with 3mM pyruvate, compared to those cold stored in UW alone. Further, from enzymatic assays of adenine nucleotides, 15mM levels of pyruvate were found to maintain higher ATP levels during short periods (up to 4h) of cold hypoxic storage than in UW stored livers, whilst energy charge ratios (after 4 and 24h) were also higher (P<0.01 in each case). This may arise from enhanced glycolysis secondary to an improved redox status in the pyruvate-treated livers, as evident by the increase in the levels of lactate.     

Melatonin reduces oxidative damage to skin and normalizes blood coagulation in a rat model of thermal injury

This study was designed to determine the effect of melatonin treatment on the glutathione (GSH) and lipid peroxidation (LPO) levels in the skin as well as prothrombin time (PT) and fibrin degradation products (FDPs) in the blood of rats with thermal injury. Under ether anaesthesia, the shaved dorsum of the rats was exposed to 90 degrees C bath for 10 s to induce burn injury. Rats were decapitated either 3 or 24 hours after burn injury. Melatonin (10 mg/kg) was administered i.p. immediately after burn injury to same animals. In the 24 hour burn group, melatonin injections were repeated for two more occasions 8 and 16 h after burn injury. In the control group the same protocol was applied except that the dorsum was exposed to a 25 degrees C water bath for 10 s. Severe skin scald injury (30% of total body surface area) caused a significant decrease in PT at post burn 3 and 24 hours. FDPs was not increased at post burn 3 hour but was significantly increased at post burn 24 hour. GSH levels were significantly depressed at post burn 3 hour but were not changed at post burn 24 hour. LPO levels were significantly increased both at post burn 3 and 24 hours. Skin protein levels were significantly reduced at post burn 24 hour as evidenced by electrophoresis. Treatment of rats with melatonin normalized PT levels both at post burn 3 and 24 hours. FDP decreased at post burn 24 hour due to melatonin treatment. GSH levels significantly increased as a result of melatonin treatment both at post burn 3 and 24 hours melatonin treatment. LPO levels were not changed by melatonin at post burn 3 hour; however, the melatonin significantly decreased LPO values at post burn 24 hours. In conclusion, exogenously administered melatonin reduced skin oxidant damage and normalized the activated blood coagulation induced by thermal trauma.