Red blood cells attenuate sinusoidal endothelial cell injury by scavenging xanthine oxidase-dependent hydrogen peroxide in hyperoxic perfused rat liver

AIMS/BACKGROUND: Rat liver perfused with an oxygenated buffered solution alone results in degenerative changes even when the perfusion flow is accelerated to give a sufficient oxygen supply. On the other hand, perfusion media supplemented with red blood cells (RBCs) preserve the viability of the liver. The present study was conducted to clarify how RBCs protect the isolated perfused liver. METHODS: The liver was perfused with and without RBCs in a perfusate equilibrated with supra-physiological oxygen tension at regulated inflow pressures, and controlled hepatic oxygen consumption. We examined alanine aminotransferase and purine nucleoside phosphorylase activity in the perfusate as specific markers of liver cells injury. Hydrogen peroxide (H2O2) production and morphological changes were determined using cerium electron microscopy. Apoptosis was detected by measuring CPP 32 protease activity and using TdT-mediated dUTP-digoxigenin nick end-labeling. RESULTS: When the liver was perfused with RBC-free buffer, H2O2 production and consequent injury progressing to apoptosis were initiated in the sinusoidal endothelial cells (SECs). After SECs were injured, H2O2 appeared in the hepatocytes. H2O2 production and associated degenerative changes were attenuated both morphologically and enzymatically by the addition of RBCs, a specific xanthine oxidase (XOD) inhibitor and the H2O2 radical scavenger, catalase. CONCLUSIONS: In the liver perfused with RBC-free buffer, H2O2 production and consequent injury were initiated in SECs. RBCs attenuate liver injury by scavenging XOD-dependent H2O2.     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short‐term ethanol perfusion in the rat

Abstract: Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O₂^·?) release into the hepatic sinusoids during short‐term exposure to ethanol. Methods: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert‐buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O₂^·? production, MCLA (2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one), a Cypridina luciferin analogue, was simultaneously infused and MCLA‐enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL₃) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine–threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4‐methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol‐induced chemiluminescence were also evaluated. Sites where O₂^·? could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Results: Both ethanol and tert‐buthanol rapidly caused O₂^·? release. GdCL₃ suppressed the ethanol‐induced O₂^·? release by 61%. Staurosporine and DPI, but neither IB nor 4‐MP, also significantly inhibited the ethanol‐induced O₂^·? release. In the histochemical examination, ethanol‐stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl₃‐pretreated liver had the precipitate only on SECs. Conclusions: This study shows that ethanol itself stimulates both SECs and KCs to release O₂^·? via activation of NADPH oxidase probably involving protein kinase C (PKC).     

Dense red blood cell and oxygen desaturation in sickle‐cell disease

Production of abnormal hemoglobin (HbS) in sickle‐cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled‐RBC formation. Dense RBCs (DRBCs), defined as density >1.11 and characterized by increased rigidity are absent in normal AA subjects, but present at percentages that vary of a patient to another remaining stable throughout adulthood for each patient. Polymerized HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen‐dissociation curve, leading to disturbances in oxygen transport. Ninety‐two SCD patients' total RBCs were separated into LightDRBC (LRBC) (d < 1.11 g/mL) and DRBC fractions. Venous blood partial oxygen pressure and RBC‐fraction–deoxygenation and –reoxygenation Hb–oxygen‐equilibrium curves were determined. All patients took a 6‐minute walking test (6MWT); 10 had results before and after >6 months on hydroxyurea. 6MWT time with SpO₂ < 88% (TSpO₂ < 88) assessed the physiological impact of exertion. Elevated mean corpuscular hemoglobin (Hb) concentrations, decreased %HbF, and 2,3‐bisphosphoglycerates in DRBCs modulated Hb–oxygen affinity. Deoxygenation and reoxygenation Hb–oxygen equilibrium curves differed between normal Hb AA and SS RBCs and between LRBCs and DRBCs, with rightward shifts confirming HbS‐polymerization's role in affinity loss. In bivariate analyses, 50% Hb saturation correlated positively with %DRBCs (P < 0.0001, r² = 0.34) and negatively with %HbF (P < 0.0001, r² = 0.25). The higher the %DRBCs, the longer the TSpO₂88 (P = 0.04). Hydroxyurea was associated with significantly shorter TSpO₂ < 88 (P = 0.01). We report that the %DRBCs directly affects SCD patients' SpO₂ during exertion; hydroxyurea improves oxygen affinity and lowers the %DRBCs. Am. J. Hematol. 91:1008–1013, 2016. © 2016 Wiley Periodicals, Inc.     

Effects of deferoxamine on H2O2-induced oxidative stress in isolated rat heart

During myocardial reperfusion injury, iron has been implicated in the Fenton based generation of hydroxyl radical, ·OH, leading to further organ injury. Although previous studies have investigated the protective effect of iron chelators including deferoxamine (DFX) in myocardial reperfusion injury, there is little information regarding the role of iron chelation during oxidative stress produced by H₂O₂ on the heart. Isolated hearts from male Sprague-Dawley rats were retrograde-perfused with Krebs-Henseleit solution at 5 ml/min. After a 60-min equilibration, oxyradical challenge was instituted by the addition of H₂O₂ (200–600 M) to the perfusate for 60 min. A subgroup of animals received DFX (400 M) in the perfusate prior to challenge with 400 M H₂O₂. Contractility was continuously monitored; perfusate samples for glutathione (GSH) and lactate dehydrogenase (LDH) estimations were collected at 30-min intervals. Headspace ethane, an indicator of lipid peroxidation, was estimated at 30-min intervals by gas chromatography. Control hearts maintained contractility during the perfusion period. H₂O₂ perfusion caused a dose dependent decrease in myocardial contractility; DFX pretreatment was partialy protective. Headspace ethane slowly accumulated in control hearts; perfusion with H₂O₂ caused dose dependent increase in ethane accumulation indicative of enhanced lipid peroxidation. GSH and LDH in the perfusate remained low in control hearts. In contrast, H₂O₂ treated hearts had a dose dependent inclease in the efflux of GSH and LDH which was markedly increased by perfusion with 600 M H₂O₂. Pretreatment with DFX did not significantly reduce GSH or LDH efflux from hearts perfused with peroxide. While H₂O₂ perfused with peroxide. While H₂O₂ perfusion causes a dose dependent decrease in myocardial contractility with a corresponding increase in headspace ethane release with GSH & LDH efflux indicative of oxidative stress, concurrent treatment with DFX reduces myocardial dysfunction and ethane generation. However, sublethal damage of plasma membrane still continues as reflected by continuous enhancement of LDH efflux, possibly indicating involvement of other reactive species besides hydroxyl radical.     

Sinusoidal endothelial cell injury by superoxide anion and iron in the Propionibacterium acnes‐pretreated and lipopolysaccharide‐stimulated rat liver

Abstract: Aims/Background: We attempted to measure the generation of superoxide anion, examine its site of release and determine its pathological role in Propionibacterium acnes‐lipopolysaccharide‐induced liver injury in the rat. Methods: The P. acnes‐pretreated (16 mg/kg i.v.) rat liver was perfused with buffer containing lipopolysaccharide (2.5 μg/ml). Chemiluminescence enhanced with Cypridina luciferin analog, MCLA, and reduction of nitro blue tetrazolium were used for detecting superoxide anion. Leakage of enzymes and release of cytokines into the perfusate, and histological specimens were also examined. Results: Superoxide dismutase‐inhibitable chemiluminescence peaked at 30 min of lipopolysaccharide infusion and blue formazan precipitate was histochemically deposited mainly on hepatic macrophages. Purine nucleoside phosphorylase (PNP) activity in the perfusate, as a marker of sinusoidal endothelial cell injury, reached its maximum at 50 min and aspartate aminotransferase (AST) activity, as a marker of hepatocyte injury, reached a plateau at 90 min. Simultaneous treatment with superoxide dismutase and deferoxamine mesylate significantly suppressed the leakage of PNP and AST. Release of tumor necrosis factor‐α and growth‐related oncogene/cytokine‐induced neutrophil chemoattractant‐1 lagged behind PNP leakage. Light microscopy showed destruction of the sinusoids followed by hepatocyte necrosis. Electron microscopy revealed adherence of hepatic macrophages to sinusoidal endothelial cells. Conclusion: These results indicate that superoxide anion released from hepatic macrophages may induce sinusoidal endothelial cell injury via interaction with iron in the P. acnes‐lipopolysaccharide‐treated liver.     

The use of the mechanical fragility test in evaluating sublethal RBC injury during storage

Background The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion, thus the MFI was determined serially during routine RBC storage. Methods Leucoreduced AS‐5‐ and SAGM‐preserved RBCs were stored under routine blood bank conditions. The mechanical fragility (MF) of each unit was serially measured during storage. Results For both AS‐5 and SAGM units, male and female RBCs demonstrated statistically significant increases in the MFI during storage. The MFI was significantly lower in AS‐5 units compared to SAGM units throughout storage. Female RBCs had significantly lower MFI vs. male RBCs in both AS‐5 and SAGM units at all times. No significant differences in MFI were observed between ABO groups for both genders for AS‐5 RBCs. Conclusions The MF of RBCs increases during storage. Both gender and preservation solution influenced the MFI; however, the male:female MFI ratios were similar at all time‐points and remained stable, suggesting that gender‐based biological differences exist independent of storage solution. The MF could be a useful test for evaluating the effect of novel interventions intended to mitigate the susceptibility of RBCs to sublethal injury during storage.     

The effects of asthma medications on reactive oxygen species production in human monocytes

Objective: Asthma is a chronic inflammatory airway disease induced by many environmental factors. The inhalation of allergens and pollutants promotes the production of reactive oxygen species (ROS) leading to airway inflammation, hyper-responsiveness, and remodeling in allergic asthma. The effects of asthma medications on ROS production are unclear. The present study investigated the anti-ROS effects of current asthma medications including inhaled corticosteroid (ICS; budesonide and fluticasone), leukotriene receptor antagonist (LTRA; montelukast), long-acting β2 agonists (LABAs; salmeterol and formoterol), and a new extra-LABA (indacaterol). Methods: The human monocyte cell line THP-1 cells were pre-treated with different concentrations of the asthma medications at different time points after hydrogen peroxide (H₂O₂) stimulation. H₂O₂ production was measured with DCFH-DA by flow cytometry. Results: Montelukast, fluticasone, and salmeterol suppressed H₂O₂-induced ROS production. Indacaterol enhanced H₂O₂-induced ROS production. Budesonide and formoterol alone had no anti-ROS effects, but the combination of these two drugs significantly suppressed H₂O₂-induced ROS production. Conclusions: Different asthma medications have different anti-ROS effects on monocytes. The combination therapy with LABA and ICS seemed not to be the only choice for asthma control. Montelukast may also be a good supplemental treatment for the poorly controlled asthma because of its powerful anti-ROS effects. Our findings provide a novel therapeutic view in asthma.     

Physiological and pathological functions of NADPH oxidases during myocardial ischemia–reperfusion

Oxidative stress, the presence of reactive oxygen species (ROS) in excess of the antioxidant capacity in the heart induces myocardial damage, accumulation of which leads to ischemic heart disease and heart failure. NADPH oxidase (Nox) 2 and 4 are the major sources of O₂^? and H₂O₂ in the heart and play a crucial role in the regulation of growth and death in cardiomyocytes. Both Nox2 and Nox4 are upregulated in response to ischemia–reperfusion (I/R), thereby contributing to ROS production and consequent myocardial injury. Suppression of either one of them can reduce ROS and I/R injury in the heart. Importantly, however, a minimum level of ROS production by either Nox2 or Nox4 is essential for the activation of HIF-1α and inhibition of PPARα during I/R, such that combined suppression of both Nox2 and Nox4 exacerbates myocardial I/R injury. Thus, either excessive activation or suppression of Noxs below physiological levels can induce cardiac injury. Here we discuss both detrimental and salutary functions of Nox isoforms during myocardial I/R.     

Impaired lymphocyte mitochondrial antioxidant defences in variegate porphyria are accompanied by more inducible reactive oxygen species production and DNA damage

This study aimed to analyse lymphocyte reactive oxygen species (ROS) production and detoxification mechanisms and the appearance of oxidative damage in variegate porphyria (VP) patients. Twelve women affected by VP and 12 pair‐matched healthy control women participated in the study. VP women presented impaired expression of the mitochondrial proteins protoporphyrinogen oxidase, uncoupling protein‐3, Bcl‐2 and sirtuin 3. Lymphocytes from VP women presented higher H₂O₂ production than controls after stimulation with phorbol myristate acetate. The inhibition of H₂O₂ production after in vitro lymphocyte treatment with myxothiazol pointed towards complex III of the mitochondrial respiratory chain as the main contributor of the higher ROS production in porphyric subjects. No differences were observed between VP and control subjects in the levels of DNA damage, assessed by the comet assay method in un‐treated lymphocytes. However, DNA damage, expressed both as a percentage of DNA in tail and as the tail moment, was greater in VP women than controls after lymphocyte treatment with H₂O₂. In conclusion, lymphocytes from VP women showed impaired expression of mitochondrial antioxidant defences but no significant signs of oxidative stress were evidenced in basal, non‐stressing conditions; however, lymphocytes of VP women were more susceptible to producing mitochondrial ROS and to suffering oxidative damage when submitted to stressful situations.     

Epimorphin protects hepatocytes from oxidative stress by inhibiting mitochondrial injury

Background and Aims: Many investigations have demonstrated that cell injuries caused by generation of reactive oxygen species (ROS) is a common mechanism of various hepatic disorders. Recently, we have demonstrated that epimorphin, originally cloned as a mesenchymal protein, protects cultured intestinal epithelial cells from ROS. We therefore examine whether epimorphin protects primary cultured hepatocytes from ROS‐induced cell injury. Methods: We explored the cell viability and the intracellular ROS levels of purified murine hepatocytes after exposure to 0.5 mM H₂O₂ with or without pretreatment of epimorphin. Then, we observed mitochondrial permeability transition (MPT) and depolarization using confocal microscopy to make clear the mechanism that epimorphin inhibited cell injuries after exposure to H₂O₂. In addition, to clarify the signaling pathways related to cell survival, we carried out Western blotting analysis with phosphorylated stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) polyclonal antibody to evaluate the inhibition of JNK by epimorphin. Finally, we evaluated the cell viability in hepatocytes administered JNK inhibitor. Results: Epimorphin protected primary cultured hepatocytes from H₂O₂‐induced cell injuries independent of intracellular ROS levels. Epimorphin also inhibited onset of MPT, depolarization of the mitochondrial membrane potential, and eventually cell killing. The cell protective function of epimorphin after exposure to H₂O₂ was not dependent on Akt signaling but on JNK signaling. Conclusion: Epimorphin can protect hepatocytes from MPT‐dependent cell injury induced by ROS. Since hepatic disorders could be caused by MPT‐dependent cell injuries with excessive ROS, epimorphin might open a new therapeutic avenue for hepatic disorders.     

Relationship between coronary flow and adenosine production and release

Hearts from 5 guinea pigs were excised and simultaneously perfused for 30 min with Krebs-Henseleit solution at 37°C and equilibrated with 95% O₂ and 5% CO₂. The coronary flow at the end of the equilibration period was taken as the control flow (100%). Thereafter the hearts were perfused for 10 min with solutions equilibrated with various oxygen mixtures (95, 60, 30, 15, 10 and 5% O₂). Experiments were performed on two groups of 5 hearts at each oxygen level. Coronary flow was measured and the perfusates were collected for adenosine analysis. At the end of 10 min the hearts were instantaneously frozen and myocardial adenosine and adenine nucleotides were determined. As the PO₂ of the gas phase was reduced, per cent change in coronary flow, tissue adenosine and the rate of adenosine release into perfusates were the same for 95 and 60% PO₂ but increased in a parallel fashion at the lower PO₂ values. However, oxygen consumption remained relatively constant and the estimated energy charge (E = 1/2ADP + ATP/ATP + ADP + AMP) decreased slightly. The striking parallelism between coronary flow and adenosine production suggests that the decrease in coronary resistance associated with reduction of oxygen tension in the perfusing fluid may be attributed to the release of adenosine by myocardial cells. Since 5′-nucleotidase is activated in vitro by a decrease in E, it is possible that the small change in the balance of the myocardial adenine nucleotides may control adenosine production.     

Effects of oxidative stress on red blood cell rheology in sickle cell patients

Sickle cell anaemia (SS) and sickle cell‐haemoglobin C disease (SC) patients exhibit severe red blood cell (RBC) rheological alterations involved in the development of several complications. The contribution of oxidative stress in these haemorheological abnormalities is still unknown. We compared RBC reactive oxygen species (ROS) and glutathione (GSH) content, and the haemorheological profile of SS (n = 11), SC (n = 11) and healthy subjects (n = 12) at baseline and after in‐vitro treatment with t‐butyl hydroperoxide (TBHP). We showed: (i) higher RBC ROS content in SS and SC patients, with the highest level observed in SS patients; (ii) lower RBC GSH content in sickle syndrome patients, especially in SS patients; (iii) TBHP increased RBC ROS production and decreased RBC GSH content in all groups; (iv) TBHP decreased RBC aggregation and increased the strength of RBC aggregates in all groups but the increase in RBC aggregates strength was greater in sickle cell patients; (v) TBHP decreased RBC deformability in the three groups but with a higher magnitude in sickle cell patients. These data suggest that RBCs from sickle cell patients have an exaggerated response to oxidative stress, which is accompanied by a profound abnormal haemorheological profile, with greater alterations in SS than in SC patients.     

Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat

Abstract: Aims/Background: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. Methods: Sinusoidal endothelial cells were isolated from rat liver. After 2–3 days of primary culture, the cells were exposed to hypoxia (N₂/CO₂ 95/5) for 120 min and reoxygenation (O₂/CO₂ 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O₂ alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondria dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. Results: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. Conclusions: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.     

Effects of Chronic Ethanol Administration on 02 Consumption in Whole Body and Perfused Liver of the Rat

The effects of chronic ethanol consumption on thyroid hormone levels and the rates of whole animal and perfused liver oxygen consumption were determined to test the hypothesis that alcoholic liver damage is a result of thyroid mediated liver hypermetabolism (L. Videla, J. Bernstein, and Y. Israel: Biochem. J, 134: 507–514, 1973). Whole animal minimal oxygen consumption, a sensitive indicator of the effects of thyroid hormone (W. D. Denckla: J. Clin. Invest, 53:572–581, 1974) was unchanged in rats maintained 3 wk on a liquid diet containing 34% of the calories as ethanol (2.49 ± 0.06 ml of O₂/min/1O0 g of fat-free body weight) when compared to animals fed an equicaloric sucrose containing liquid diet (2.61 ± 0.20 ml of 0₂/min/100 g of fat-free body weight) or Purina chow (2.50 ± 0.12 ml of O₂/min/100 g of fat-free body weight). Ethanol treatment lowered serum thyroxine (5.09 ± 0.20 pg/100 ml) compared to sucrose-fed control rats (7.66 ± 0.40 pg/100 ml), while serum triiodothyronine was unaffected (59.3 ± 4.0 compared to 66.9 ± 3.1 ng/100 ml for controls). Measurement of O₂ consumption in the isolated perfused rat liver showed no significant difference after chronic treatment with the ethanol diet compared to either the sucrose or chow control diets. Infusion of 10^-7 M norepinephrine into the perfusion medium resulted in an approximately 22% increase in O₂ consumption in ethanol-fed animal and sucrose controls, while a 31 % increase was observed for sucrose-treated animals given 10 μg of T₃/kg of body weight/day for 3 wk. These data indicate that T₃ potentiates the ability of norepinephrine to increase O₂ consumption. The data presented give no support to the concept that chronic ethanol ingestion results in hyperthyroidism or liver hypermetabolism and, consequently, the rationale for treatment of alcoholic hepatitis with the antithyroid drug, propylthiouracil, is incorrect (H. Orrego, H. Ka-lant, Y. Israel, et al.: Gastroenterology, 76:105–115, 1979).     

Long‐chain fatty acid uptake and oxidation in the perfused liver of Walker‐256 tumour‐bearing rats

Abstract: Aims/Background: The effect of the Walker‐256 tumour on uptake and oxidation of long‐chain fatty acids was investigated in perfused livers of rats. Methods: Isolated livers were perfused in a non‐recirculating system. Fatty acid uptake, ketogenesis, oxygen uptake and ¹⁴CO₂‐production were measured as well as the activities of the acyl carnitine transferases I and II (CAT I and CAT II). Results: Basal oxygen uptake of livers from tumour‐bearing rats was lower. Ketone bodies production derived from the long‐chain fatty acids in livers from starved tumour‐bearing rats was lower relative to the controls, but ¹⁴CO₂ production was similar in both groups. The oxygen uptake increment and the mitochondrial NADH/NAD⁺ redox ratio were also decreased in tumour‐bearing rats. The extent of these differences was dependent on the chain length and structure of the fatty acid, the following decreasing sequence of differences between control and tumour‐bearing animals being valid: palmitate > oleate > stearate. The CAT I activity of the enzyme isolated from livers of tumour‐bearing rats was half that from normal rats when palmitoyl‐CoA and oleoyl‐CoA were the substrates. Conclusions: Ketogenesis from exogenous fatty acids is decreased in the livers of Walker‐256 tumour‐bearing rats in consequence of the diminished activity of the mitochondrial CAT I. The lower rates of oxygen uptake indicate a decreased ATP synthesis, which is consistent with the in vivo lower phosphorylation potential.     

N-acetylcysteine attenuates reactive-oxygen-species-mediated endoplasmic reticulum stress during liver ischemia-reperfusion injury

AIM: To investigate the effects of N-acetylcysteine (NAC) on endoplasmic reticulum (ER) stress and tissue injury during liver ischemia reperfusion injury (IRI).METHODS: Mice were injected with NAC (300 mg/kg) intraperitoneally 2 h before ischemia. Real-time polymerase chain reaction and western blotting determined ER stress molecules (GRP78, ATF4 and CHOP). To analyze the role of NAC in reactive oxygen species (ROS)-mediated ER stress and apoptosis, lactate dehydrogenase (LDH) was examined in cultured hepatocytes treated by H₂O₂ or thapsigargin (TG).RESULTS: NAC treatment significantly reduced the level of ROS and attenuated ROS-induced liver injury after IRI, based on glutathione, malondialdehyde, serum alanine aminotransferase levels, and histopathology. ROS-mediated ER stress was significantly inhibited in NAC-treated mice. In addition, NAC treatment significantly reduced caspase-3 activity and apoptosis after reperfusion, which correlated with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment significantly inhibited LDH release from hepatocytes treated by H₂O₂ or TG.CONCLUSION: This study provides new evidence for the protective effects of NAC treatment on hepatocytes during IRI. Through inhibition of ROS-mediated ER stress, NAC may be critical to inhibit the ER-stress-related apoptosis pathway.     

Glutathione‐dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.)

Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonin‐treated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H₂O₂ and GSH also induced tolerance to photooxidative stress, while inhibition of H₂O₂ accumulation and GSH synthesis compromised melatonin‐induced local and systemic tolerance to photooxidative stress. H₂O₂ treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H₂O₂ accumulation prevented a melatonin‐induced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H₂O₂‐induced photooxidative stress tolerance, whereas scavenging or inhibition of H₂O₂ production attenuated but did not abolish GSH‐induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin‐enhanced GSH/GSSG ratio in a H₂O₂‐dependent manner is critical in the induction of tolerance.     

Red blood cells induce hypoxic lung inflammation

Hypoxia, which commonly associates with respiratory and cardiovascular diseases, provokes an acute inflammatory response. However, underlying mechanisms are not well understood. Here we report that red blood cells (RBCs) induce hypoxic inflammation by producing reactive oxygen species (ROS) that diffuse to endothelial cells of adjoining blood vessels. Real-time fluorescence imaging of rat and mouse lungs revealed that in the presence of RBC-containing vascular perfusion, hypoxia increased microvascular ROS, and cytosolic Ca²⁺, leading to P-selectin–dependent leukocyte recruitment. However, in the presence of RBC-free perfusion, all hypoxia-induced responses were completely inhibited. Because hemoglobin (Hb) autoxidation causes RBC superoxide formation that readily dismutates to H₂O₂, hypoxia-induced responses were lost when we inhibited Hb autoxidation with CO or nitrite, or when the H₂O₂ inhibitor, catalase was added to the infusion to neutralize the RBC-derived ROS. By contrast, perfusion with RBCs from BERK-trait mice that are more susceptible to Hb autoxidation and to hypoxia-induced superoxide production enhanced the hypoxia-induced responses. We conclude that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H₂O₂ that diffuses to the lung microvascular endothelium, thereby initiating Ca²⁺-dependent leukocyte recruitment. These findings are the first evidence that RBCs contribute to hypoxia-induced inflammation.