4-hydroxy-2-nonenal and Ischemia

Chlorogenic acid (CGA), an ester of caffeic acid and quinic acid, is among the phenolic acid compounds which can be naturally found in green coffee extract and tea. CGA has been studied since it displays significant pharmacological properties. The aim of this study was to investigate the effects of CGA on cognitive function and neuroprotection including its mechanisms in the hippocampus following transient forebrain ischemia in gerbils. Memory and learning following the ischemia was investigated by eight-arm radial maze and passive avoidance tests. Neuroprotection was examined by immunohistochemistry for neuronal nuclei-specific protein and Fluoro-Jade B histofluorescence staining. For mechanisms of the neuroprotection, alterations in copper, zinc-superoxide dismutase (SOD1), SOD2 as antioxidant enzymes, dihydroethidium and 4-hydroxy-2-nonenal as indicators for oxidative stress, and anti-inflammatory cytokines (interleukin (IL)-4 and IL-13) and pro-inflammatory cytokines (tumor necrosis factor α (TNF-α) and IL-2) were examined by Western blotting and/or immunohistochemistry. As a result, pretreatment with 30 mg/kg CGA attenuated cognitive impairment and displayed a neuroprotective effect against transient forebrain ischemia (TFI). In Western blotting, the expression levels of SOD2 and IL-4 were increased due to pretreatment with CGA and, furthermore, 4-HNE production and IL-4 expressions were inhibited by CGA pretreatment. Additionally, pretreated CGA enhanced antioxidant enzymes and anti-inflammatory cytokines and, in contrast, attenuated oxidative stress and pro-inflammatory cytokine expression. Based on these results, we suggest that CGA can be a useful neuroprotective material against ischemia-reperfusion injury due to its antioxidant and anti-inflammatory efficacies.

Topics: Aldehydes; Animals; Anti-Inflammatory Agents; Antioxidants; Chlorogenic Acid; Cognition; Hippocampus; Interleukin-2; Interleukin-4; Ischemia; Mice; Neurons; Neuroprotective Agents; Superoxide Dismutase

Renal ischemia-reperfusion (I/R) is a major cause of acute renal failure. The mechanisms of I/R injury include endoplasmic reticulum (ER) stress, inflammatory responses, hypoxia, and generation of reactive oxygen species (ROS). CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is involved in the ER stress signaling pathways. CHOP is a transcription factor and a major mediator of ER stress-induced apoptosis. However, the role of CHOP in renal I/R injury is still undefined. Here, we investigated whether CHOP could regulate I/R-induced renal injury using CHOP-knockout mice and cultured renal tubular cells as models.. In CHOP-knockout mice, loss of renal function induced by I/R was prevented. Renal proximal tubule damage was induced by I/R in wild-type mice; however, the degree of alteration was significantly less in CHOP-knockout mice. CHOP deficiency also decreased the I/R-induced activation of caspase-3 and -8, apoptosis, and lipid peroxidation, whereas the activity of endogenous antioxidants increased. In an in vitro I/R model, small interfering RNA targeting CHOP significantly reversed increases in H2O2 formation, inflammatory signals, and apoptotic signals, while enhancing the activity of endogenous antioxidants in renal tubular cells.. To the best of our knowledge, this is the first study which demonstrates that CHOP deficiency attenuates oxidative stress and I/R-induced acute renal injury both in vitro and in vivo.. These findings suggest that CHOP regulates not only apoptosis-related signaling but also ROS formation and inflammation in renal tubular cells during I/R. CHOP may play an important role in the pathophysiology of I/R-induced renal injury.

Topics: Aldehydes; Animals; Antioxidants; Apoptosis; Cell Hypoxia; Cell Line; Gene Knockdown Techniques; Glutathione; Ischemia; Kidney; Lipid Peroxidation; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; RNA, Small Interfering; Transcription Factor CHOP

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.

Topics: Aldehydes; Animals; ATP Synthetase Complexes; Chronic Disease; Citrate (si)-Synthase; Female; Gene Expression Regulation; Hindlimb; Humans; Ischemia; Mice; Mice, Inbred C57BL; Mitochondria, Muscle; Muscle, Skeletal; Muscular Diseases; Oxidative Stress; Superoxide Dismutase

The pathogenesis of ischemia-reperfusion involves generation of reactive oxygen and resulting lipid peroxidation. However, investigation that ischemia-reperfusion following tourniquet release enhances lipid peroxidation is insufficient.. Tourniquet was applied to a unilateral hind limb of mice for 3h followed by 5-, 15-, 30- and 60-min release. To examine superoxide production immunohistochemically in ischemia-reperfusion muscles, a primary antibody directed to 4-hydroxy-nonenal (HNE) was used. Furthermore, we analyzed 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol, 7alpha- and 7beta-hydroxycholesterol, and 7-ketocholesterol by HPLC in the gastrocnemius muscles, kidneys, liver, heart and lungs of mice after 1-h reperfusion.. Increased HNE immunoreactivitiy was observed in the tourniquet-applied side of gastrocnemius muscles of hind limb particularly after 5-min reperfusion. All the oxysterols were significantly higher in the gastrocnemius muscles of the tourniquet-applied side than of the contralateral muscles. Oxysterols were elevated in the kidneys and the liver. Together with the presence of high blood urea nitrogen, these data indicate that the kidney is vulnerable to ischemia-reperfusion.. The enhanced oxidative stress due to ischemia-reperfusion appears to increase HNE in muscle and oxysterols by peroxidation not only in the gastrocnemius muscles but also in the kidneys and liver.

Topics: Aldehydes; Animals; Cholesterol; Heart; Hindlimb; Ischemia; Kidney; Lipid Peroxidation; Liver; Lung; Male; Mice; Mice, Inbred C57BL; Muscles; Reperfusion Injury; Superoxides; Time Factors; Tourniquets

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.

Topics: Aldehydes; Animals; Glutathione Peroxidase; Ischemia; Lipid Peroxidation; Liver; Liver Function Tests; Male; Malondialdehyde; Oxidative Stress; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Hydroperoxide Glutathione Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Survival Rate

Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2',7'-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and alpha-phenyl-N-tert-butylnitrone reduced levels of oxidative stress after reoxygenation. These changes are consistent with an ischemia-reperfusion injury, and mirror those seen in preeclampsia. Consequently, in vitro hypoxia/reoxygenation may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy.

Topics: Aldehydes; Cyclic N-Oxides; Deferoxamine; Female; Fluorescent Antibody Technique; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Immunohistochemistry; Ischemia; Nitrogen Oxides; Oxidative Stress; Oxygen; Placenta; Pregnancy; Pregnancy Complications; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tissue Distribution; Tyrosine

Ischemia-reperfusion (IR) injury is an intractable process associated not only with therapeutic recanalization of vessels, but also with partial resection or transplantation of solid organs including liver. To develop methods for predicting the degree of hepatic IR injury and further to identify injured cells, we studied the formation of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE)-modified proteins in the normothermic hepatic IR model of rats using immunohistochemistry, high-performance liquid chromatography (HPLC) determination and Western blot. The Pringle maneuver for either 15 or 30 min duration produced reversible or lethal damage, respectively. The levels of both products were significantly increased in proportion to ischemia duration 40 min after reperfusion, suggesting the involvement of hydroxyl radicals. Increased immunoreactivity of 8-OHdG was observed not only in the nuclei of hepatocytes but also in those of bile canalicular and endothelial cells. However, immunoreactivity of HNE-modified proteins was detected in the cytoplasm of hepatocytes, which was confirmed by Western blot, and in addition, in the nuclei of hepatocytes after severe injury. Thus, localization of the two oxidatively modified products was not identical. Our data suggest that these two products could be used for the assessment of hepatic IR injury in tissue, but that the biological significance of the two products might be different.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Blotting, Western; Body Temperature; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Adducts; DNA Damage; Hepatocytes; Hydroxyl Radical; Immunoenzyme Techniques; Ischemia; Liver; Liver Function Tests; Male; Molecular Weight; Nuclear Proteins; Oxidation-Reduction; Oxidative Stress; Proteins; Rats; Rats, Wistar; Reperfusion Injury; Specific Pathogen-Free Organisms; Time Factors

4-Hydroxy-2-nonenal (HNE)-modified proteins and 3-nitro-L-tyrosine were evaluated as a specific marker of reactive oxygen species (ROS)- and nitric oxide (NO)-mediated peroxynitrite-induced tissue injuries in ischemic and reperfused skin flap by Western blot analysis. Specimens were taken from island skin flaps of rats during the following three conditions: ischemia only, 5 hours of ischemia and reperfusion, and 10 hours of ischemia and reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine increased with ischemic time (3, 6, and 10 hours postischemia). In the reperfused skin flap after both 5 and 10 hours of ischemia, HNE-modified proteins and 3-nitro-L-tyrosine were increased 3 hours postreperfusion, and they reached a maximum 6 hours after reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine 1 hour postreperfusion were higher with 10 hours ischemia-reperfusion than with 5 hours ischemia-reperfusion. These results indicate (1) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity in ischemic flaps is dependent on the ischemic period and (2) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity occurs during an early stage of reperfusion if the ischemic period is long.

Topics: Aldehydes; Animals; Blotting, Western; Free Radicals; Ischemia; Male; Proteins; Rats; Rats, Wistar; Reperfusion Injury; Skin; Specific Pathogen-Free Organisms; Surgical Flaps; Tyrosine

Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes. In renal transplantation, this mechanism is triggered by ischemia/reperfusion and may be of relevance in graft failure.. Using specific antibodies directed against malondialdehyde (MDA) and 4-hydroxynonenal (HNE) adducts, major aldehydic metabolites of lipid peroxidation, we investigated, in situ, by means of an immunohistochemical procedure, the occurrence of lipid peroxidation during different warm ischemic periods of 0, 15, 30, 45 and 60 minutes in rat kidneys prior to reperfusion. The same experiments included followup of the rats after nephrectomy and reperfusion for 10 days.. We observed superficial and deep cortex immunostaining with both antibodies against MDA and HNE after 30 minutes of warm ischemia. This immunostaining was observed in the absence of any histological lesions, as assessed by routine staining. After 45 and 60 minutes of warm ischemia, lipid peroxidation byproducts were detected both in the cortex and in the medulla, which is associated with 33% and 66% of rat deaths respectively.. This study confirms the involvement of the lipid peroxidation process in kidney damage during anoxia before reperfusion, and its extension to the whole organ. Lipid peroxidation byproducts were detectable in warm ischemic kidney, and the presence of medulla immunostaining was associated with the animals' death. Lipid peroxidation immunostaining might thus be useful as a sensitive tool to detect ischemic damage after warm ischemia prior to reperfusion, as well as in the decision to carry out kidney transplantation in humans.

Topics: Aldehydes; Animals; Biomarkers; Ischemia; Kidney; Lipid Peroxidation; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley

Topics: Aldehydes; Animals; Immunohistochemistry; Ischemia; Kidney; Lipid Metabolism; Lipid Peroxidation; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley

To assess the role of superoxide (O2-) and nitric oxide (NO) in ischaemic-reperfusion-induced acute renal failure, we investigated whether an activation of the L-arginine-NO pathway contributes to ischaemia-reperfusion-induced kidney membrane peroxidation by measurement of 4-hydroxynonenal (HNE) content in anaesthetized rats submitted to acute renal ischaemia. Following ischaemia-reperfusion injury, renal blood flow (RBF) was significantly reduced, while renal vascular resistance was significantly increased. Infusion of neither L-arginine nor D-arginine led to a recovery of RBF. L-Arginine, but not D-arginine, caused a significant increase in HNE accumulation in the ischaemic kidney. L-Arginine infusion enhanced the degree of lipid peroxidation afforded by ischaemia-reperfusion injury in the kidney suggesting that products of the endogenous L-arginine-NO pathway may react with O2- to initiate lipid peroxidation.

Topics: Aldehydes; Animals; Arginine; Cricetinae; Ischemia; Kidney; Lipid Peroxidation; Male; Nitric Oxide; Rats; Rats, Wistar; Reactive Oxygen Species; Renal Circulation; Reperfusion Injury; Superoxides; Vascular Resistance

The metabolism and the washout of 4-hydroxynonenal (HNE), a major lipid peroxidation product, was determined in jejunal intestine of rats under normoxic, ischemic and postischemic conditions. The HNE utilization was by several orders higher than the HNE washout. Under ischemic conditions a drastic decrease of HNE utilization was found, compared with normoxic and postischemic utilization rates. The real flux rate of intracellular HNE formation during ischemia and reperfusion of small intestine was for the first time estimated on the basis of tissue HNE concentration, HNE utilization rate at defined HNE tissue concentration and HNE washout by perfusate circulation. Such estimation led to intestinal HNE formation rates of about 20 nmoles/g/min at normoxia, 20 to 40 nmoles/g/min at ischemia, 100 nmoles/g/min at 10 min of reperfusion and 20 nmoles/g/min at 60 min of reperfusion. Therefore, one can conclude that the tissue concentration of HNE only partly represents the real HNE formation rate and that the formation rate is of particular pathophysiological importance.

Topics: Aldehydes; Animals; Intestine, Small; Ischemia; Male; Rats; Rats, Wistar; Reperfusion

4-Hydroxynonenal (HNE) as an indicator of lipid peroxidation was determined in rat jejunal mucosa. HNE was extracted as the dinitrophenylhydrazone derivative from the tissue, partially separated from other carbonyl compounds by thin-layer chromatography and measured by HPLC. During reperfusion of the small intestine following an ischemic period of 60 minutes a marked increase of the tissue concentration of HNE was observed. The mucosal HNE level passed a maximum value of 3.0 +/- 0.5 microM 10 min after the onset of reperfusion in comparison with 0.7 +/- 0.2 microM as initial value. The increased tissue level of the highly cytotoxic 4-hydroxyalkenal is suggested to be involved in the reperfusion induced morphological and biochemical changes of the small intestine.

Topics: Aldehydes; Animals; Chromatography, High Pressure Liquid; Glutathione; Glutathione Disulfide; Hypoxanthine; Hypoxanthines; Intestine, Small; Ischemia; Jejunum; Lipid Peroxidation; Male; Phenylhydrazines; Rats; Rats, Wistar; Reperfusion; Thiobarbituric Acid Reactive Substances