Zinc Dyshomeostasis in Cardiomyocytes after Acute Hypoxia/Reoxygenation

This study aimed to assess the protective roles of polysaccharides from Agaricus blazei Murill (ABP) against cadmium (Cd)-induced damage in chicken livers. A total of 80 Hy-Line laying chickens (7 days old) were randomly divided into four groups (n = 20). Group I (control) was fed with a basic diet and 0.2 ml saline per day, group II (Cd-treated group) was fed with a basic diet containing 140 mg/kg cadmium chloride (CdCl₂) and 0.2 ml saline per day, group III (Cd + ABP-treated group) was fed with a basic diet containing 140 mg/kg CdCl₂ and 0.2-ml ABP solution (30 mg/ml) per day via oral gavage, and group IV (ABP-treated group) was fed with 0.2-ml ABP solution (30 mg/ml) per day via oral gavage. The contents of Cd and malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), the messenger RNA (mRNA) levels of inflammatory cytokines and heat shock proteins (HSPs), the protein levels of HSPs, and the histopathological changes of livers were evaluated on days 20, 40, and 60. The results showed that Cd exposure resulted in Cd accumulating in livers and inhibiting the activities of antioxidant enzymes (SOD and GSH-PX). Cd exposure caused histopathological damage and increased the MDA content, the mRNA levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) and the protein levels of HSPs (HSP60, HSP70, and HSP90). ABP supplementation during dietary exposure to Cd reduced the histopathological damage and decreased the contents of Cd and MDA and the expression of inflammatory cytokines and HSPs and improved the activities of antioxidant enzymes. The results indicated that ABP could partly ameliorate the toxic effects of Cd on chicken livers.     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

番茄红素对大鼠乳鼠心肌细胞缺氧复氧的保护作用以及其机制

目的：探讨番茄红素对心肌细胞缺氧复氧的保护作用以及其分子机制。方法：采用原代培养心肌细胞建立缺氧/复氧损伤模型,实验分8组：正常对照组,H/R组,H/R＋番茄红素（1,2,4,8,16,32μmol/L）剂量组。观察各组细胞经H/R损伤后,细胞内天冬氨酸氨基转移酶（AST）、肌酸激酶（CK）、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量的变化情况,选择正常对照组,H/R组,最佳番茄红素剂量组做MTT分析细胞凋亡,Western检测TRL 4以及NF-κB的表达。结果：番茄红素（16,8,4,2μmol/L）剂量组可显著降低缺氧/复氧损伤心肌细胞内AST、CK、LDH释放量及MDA的生成,并能提高SOD活性。此外番茄红素可减少心肌细胞缺氧/复氧损伤后的心肌凋亡,减少TRL 4受体以及NF-κB的表达。结论：番茄红素具有抗缺氧/复氧损伤,保护心肌细胞的作用,其机制可能是通过抑制TRL 4通路来实现的。     

The Effects of Hypoxia/Reoxygenation on the Physiological Behaviour of U373-Mg Astrocytes

Nerve cells are very susceptible to hypoxia responsive for mitochondrial dysfunctions involved in the subsequent oxidative stress, apoptosis and necrosis. In this paper, we examined the effect of 12 h incubation of U-373 MG astrocytes in hypoxic environment (73% N₂: 2% O₂: 5% CO₂, v:v) by evaluating cell proliferation, modifications of NO and ATP production, intracellular Ca²⁺ concentration [Ca²⁺]_i, membrane potential, desferoxamine-chelatable free iron, esterified F2-isoprostanes levels and the production of phosphorylated ERK. The same parameters were evaluated also after a following re-oxygenation period of 24 h. Immediately after hypoxia the NO concentration increased significantly and returned to values similar to those of controls after the re-oxygenation period. At the same time, ATP levels remained similar to controls and the cell proliferation significantly decreased. This involved a significant increase of [Ca²⁺]_i immediately after hypoxia and the value remained significantly elevated after the following re-oxygenation period. Moreover, after hypoxia, astrocytes were slightly although not significantly depolarized. Indeed iron and F2-isoprostanes levels increased significantly after hypoxia. Finally ERK proteins increased slowly and not significantly after hypoxia and the same trend was observed after the re-oxygenation period. On the whole, our results indicate that 2% O₂ hypoxia induces a moderate oxidative stress, well tolerated by U-373 MG cells, remaining the ATP production, mitochondrial membrane potential and activated ERK proteins, similar to the values of controls.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Substrate Modulation of Fatty Acid Effects on Energization and Respiration of Kidney Proximal Tubules during Hypoxia/Reoxygenation

Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.     

Hypoxia/Reoxygenation Stimulates Proliferation Through PKC-Dependent Activation of ERK and Akt in Mouse Neural Progenitor Cells

Cerebral ischemia increases neural progenitor cell proliferation and neurogenesis. However, the precise molecular mechanism is poorly understood. The present study was undertaken to determine roles of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt and their signaling pathways in neural progenitor cells exposed to hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion. Neural progenitor cells were isolated from postnatal mouse brain. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of MEK, but not by LY294002, a specific inhibitor of PI3K, whereas the Akt activation was blocked by LY294002, but not by U0126. Reoxygenation following 4-h hypoxia stimulated cell proliferation, which was dependent on ERK and Akt activation. Inhibitors of growth factor receptor (AG1478) and Src (PP2) and the antioxidant N-acetylcysteine did not affect activation of ERK and Akt, while the Ras and Raf inhibitors inhibited activation of ERK, but not Akt. PKC inhibitors inhibited both ERK and Akt activation. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt survival signaling pathways through a PKC-dependent mechanism. These pathways may be responsible for the repair process during ischemia/reperfusion.     

Insulin Protects Apoptotic Cardiomyocytes from Hypoxia/Reoxygenation Injury through the Sphingosine Kinase/Sphingosine 1-Phosphate Axis

Objective

Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated.

Methods and Results

Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P₁ receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes.

Conclusion

The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.     

Hypoxia/Reoxygenation induces nitric oxide and TNF-alpha release from cultured microglia but not astrocytes of the rat

Hypoxia/reoxygenation (H/R) elicits neuronal cell injury and glial cell activation within the central nervous system (CNS). Neuroinflammation is a process that primarily results from the acute or chronic activation of glial cells. This overactive state of glial cells results in the increased release of nitric oxide (NO) and/or tumor necrosis factor alpha (TNF-alpha), a process which can lead to neuronal damage or death. In this study, we found that hypoxia for eight or twelve hours (h) followed by 24 h reoxygenation (H8/ R24 or H12/R24) induced NO production and TNF-alpha release from cultures of enriched microglial or mixed glial cells. However, microglial cells could not survive longer periods of hypoxia (> or = 12 h) in microglia-enriched culture. While astrocytes retained a 95% viability following longer periods of H/R in astrocyte-enriched cultures, they did not produce any significant quantities of NO and TNF-alpha. Reoxygenation for prolonged periods (three and five days) following H24 resulted in progressively greater increases in NO production (about two-fold greater level in hypoxia as compared to normoxic conditions) accompanied by relatively less increases in TNF-alpha release in mixed glial cell cultures. Our data indicate that inflammatory mediators such as NO and TNF-alpha are released from glia-enriched mix culture in response to H/R. While microglial cells are more vulnerable than astrocytes during H/R, they survive longer in the presence of astrocyte and are the major cell type producing NO and TNF-alpha. Furthermore, the TNF-alpha release precedes NO production in response to a prolonged duration of reoxygenation following hypoxia for 24 h.     

Reversibility of Cisternal Stack Formation During Hypoxic Hypoxia and Subsequent Reoxygenation in Cerebellar Purkinje Cells

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca²⁺ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.     

Lin28a Protects against Hypoxia/Reoxygenation Induced Cardiomyocytes Apoptosis by Alleviating Mitochondrial Dysfunction under High Glucose/High Fat Conditions

Aim

The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions.

Methods

Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis.

Results

Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK.

Conclusions

The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.     

Moderate Hypoxia Followed by Reoxygenation Results in Blood-Brain Barrier Breakdown via Oxidative Stress-Dependent Tight-Junction Protein Disruption

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O₂, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.     

The Physiological Behaviour of IMR-32 Neuroblastoma Cells is Affected by a 12-h Hypoxia/24-h Reoxygenation Period

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N₂: 2% O₂: 5% CO₂, v:v) by evaluating cell viability, modifications of NO, intracellular Ca²⁺ concentration [Ca²⁺]_i and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca²⁺]_i immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O₂ hypoxia induces variations of NO and [Ca²⁺]_i with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.     

Lack of Correlation Between the Effects of Transient Exposure to Glutamate and Those of Hypoxia/Reoxygenation in Immature Neurons In Vitro

Abstract: To assess the influence of brain immaturity on the effects of oxygen deprivation and the participation of excitotoxicity, the consequences of a 6-h exposure to either hypoxia (95% N₂/5% CO₂) or 100 µ M glutamate were studied in cultured fetal rat forebrain neurons taken at two maturational stages, i.e., 6 and 13 days in vitro. Cells were examined for their morphology, viability, energy metabolism reflected by 2- d -[³H]deoxyglucose uptake, and protein synthesis assessed by [³H]leucine incorporation. Apoptosis and necrosis were scored using the fluorescent dye 4,6-diamidino-2-phenylindole. Whereas 6-day-old neurons responded to a 6-h hypoxia by transient hypermetabolism, biphasic increase in protein synthesis, and cycloheximide-sensitive apoptotic death within 72 h postexposure, glutamate did not affect cell characteristics by the same time. In 13-day-old neurons, hypoxia induced both apoptosis (8.2%) and necrosis (22.3%). At this age, glutamate definitely reduced energy metabolism (26%) and protein synthesis (17%) by the end of exposure. The percentage of necrotic neurons reached 40.7%, but the rate of apoptosis was unchanged compared with controls. Therefore, excitotoxicity cannot account for hypoxia-induced injury in immature neurons, but its participation is suggested in older cells by the suppression of the necrotic component of hypoxia by glutamate receptor antagonists at 13 days.     

The Glutathione Status of Perfused Rat Hearts Subjected to Hypoxia and Reoxygenation: The Oxygen Paradox

Langendorff perfused rat hearts subjected to 30min hypoxia followed by 20min reoxygenation and the levels of the oxidised and reduced forms of glutathione measured. No change in the concentration of oxidised glutathione was detected in reoxygenated hearts when compared to normoxic controls. In contrast hearts exposed to oxidative stress in the form of H₂O₂ showed elevated levels of both oxidised glutathione (GSSG) and the glutathione-protein mixed disulphide. These results suggest that if oxidants do contribute to cell damage on reoxygenation of the hypoxic myocardium then their action is local and not through overwhelming of the cells antioxidant defences.     

The Glutathione Status of Perfused Rat Hearts Subjected to Hypoxia and Reoxygenation: The Oxygen Paradox

Langendorff perfused rat hearts subjected to 30min hypoxia followed by 20min reoxygenation and the levels of the oxidised and reduced forms of glutathione measured. No change in the concentration of oxidised glutathione was detected in reoxygenated hearts when compared to normoxic controls. In contrast hearts exposed to oxidative stress in the form of H₂O₂ showed elevated levels of both oxidised glutathione (GSSG) and the glutathione-protein mixed disulphide. These results suggest that if oxidants do contribute to cell damage on reoxygenation of the hypoxic myocardium then their action is local and not through overwhelming of the cells antioxidant defences.     

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

Objective

To investigate whether lipoxin A₄ (LXA₄) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA₄-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA₄-induced HO-1 induction.

Methods

Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA₄ or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay.

Results

Pretreatment of the cells undergoing H/R lesion with LXA₄ significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA₄ on the cells undergoing H/R lesion. LXA₄ increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure.

Conclusion

The protection role of LXA₄ against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway.     

Exposure of Astrocytes to Hypoxia/Reoxygenation Enhances Expression of Glucose-Regulated Protein 78 Facilitating Astrocyte Release of the Neuroprotective Cytokine Interleukin 6

Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78-kDa polypeptide that demonstrated sequence identity with glucose-regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high-molecular-weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti-sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R-induced astrocyte release of IL-6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL-6 under the stress of H/R.