3-nitrotyrosine and Hemorrhage

Hemorrhagic shock may contribute to acute kidney injury (AKI) by profoundly altering renal mitochondrial function. Resveratrol (RSV), a naturally occurring sirtuin 1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related disease states. We hypothesized that RSV treatment during resuscitation would ameliorate kidney mitochondrial dysfunction and decrease oxidative damage following hemorrhagic shock.. Using a decompensated hemorrhagic shock model, male Long-Evans rats (n = 6 per group) were killed prior to hemorrhage (sham), at severe shock, and following either lactated Ringer's (LR) resuscitation or LR + RSV resuscitation (RSV: 30 mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, blood urea nitrogen, and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (complexes I, II, and IV) using high-resolution respirometry. Total mitochondria reactive oxygen species were measured using fluorometry, and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. Quantitative polymerase chain reaction was used quantify mRNA from peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α) SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis.. Resveratrol supplementation during resuscitation restored mitochondrial respiratory capacity and decreased mitochondrial reactive oxygen species and lipid peroxidation. Compared with standard LR resuscitation, RSV treatment significantly increased SIRT1 and PGC1-α expression and significantly increased both superoxide dismutase 2 and catalase expression. Although RSV was associated with decreased lactate production, pH, blood urea nitrogen, and serum creatinine values did not differ between resuscitation strategies.. Resuscitation with RSV significantly restored renal mitochondrial function and decreased oxidative damage following hemorrhagic shock.

Topics: Acute Kidney Injury; Aging; Aldehydes; Animals; Antioxidants; Citrate (si)-Synthase; Hemorrhage; Kidney; Male; Mitochondria; Oxidative Stress; Rats; Rats, Long-Evans; Reactive Oxygen Species; Resuscitation; Resveratrol; Shock, Hemorrhagic; Stilbenes; Tyrosine

Oxidative stress and inflammation contribute to hepatic injury after hemorrhage/resuscitation (H/R). Natural plant polyphenols, i.e., green tea extract (GTE) possess high anti-oxidant and anti-inflammatory activities in various models of acute inflammation. However, possible protective effects and feasible mechanisms by which plant polyphenols modulate pro-inflammatory, apoptotic, and oxidant signaling after H/R in the liver remain unknown. Therefore, we investigated the effects of GTE and its impact on the activation of NF-kappaB in the pathogenesis of hepatic injury induced by H/R.. Twenty-four female LEWIS rats (180-250 g) were fed a standard chow (ctrl) or a diet containing 0.1% polyphenolic extracts (GTE) from Camellia sinensis starting 5 days before H/R. Rats were hemorrhaged to a mean arterial pressure of 30 ± 2 mmHg for 60 min and resuscitated (H/R and GTE H/R groups). Control groups (sham, ctrl, and GTE) underwent surgical procedures without H/R. Two hours after resuscitation, tissues were harvested.. Plasma alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) increased 3.5-fold and fourfold, respectively, in vehicle-treated rats as compared to GTE-fed rats. Histopathological analysis revealed significantly decreased hepatic necrosis and apoptosis in GTE-fed rats after H/R. Real-time PCR showed that GTE diminished gene expression of pro-apoptotic caspase-8 and Bax, while anti-apoptotic Bcl-2 was increased after H/R. Hepatic oxidative (4-hydroxynonenal) and nitrosative (3-nitrotyrosine) stress as well as systemic IL-6 level and hepatic IL-6 mRNA were markedly reduced in GTE-fed rats compared with controls after H/R. Plant polyphenols also decreased the activation of both JNK and NFκB.. Taken together, GTE application blunts hepatic damage, apoptotic, oxidative, and pro-inflammatory changes after H/R. These results underline the important roles of JNK and NF-kappaB in inflammatory processes after H/R and the beneficial impact of plant polyphenols in preventing their activation.

Topics: Acute Disease; Alanine Transaminase; Aldehydes; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Female; Gene Expression Regulation; Hemorrhage; Inflammation; Interleukin-6; L-Lactate Dehydrogenase; Liver; NF-kappa B; Oxidative Stress; Plant Extracts; Polyphenols; Rats; Rats, Inbred Lew; Resuscitation; RNA; Tea; Tyrosine

Acute ethanol intoxication increases the production of reactive oxygen species (ROS). Hemorrhagic shock with subsequent resuscitation (H/R) also induces ROS resulting in cellular and hepatic damage in vivo. We examined the role of acute ethanol intoxication upon oxidative stress and subsequent hepatic cell death after H/R. 14 h before H/R, rats were gavaged with single dose of ethanol or saline (5 g/kg, EtOH and ctrl; H/R_EtOH or H/R_ctrl, resp.). Then, rats were hemorrhaged to a mean arterial blood pressure of 30 ± 2 mmHg for 60 min and resuscitated. Two control groups underwent surgical procedures without H/R (sham_ctrl and sham_EtOH, resp.). Liver tissues were harvested at 2, 24, and 72 h after resuscitation. EtOH-gavage induced histological picture of acute fatty liver. Hepatic oxidative (4-hydroxynonenal, 4-HNE) and nitrosative (3-nitrotyrosine, 3-NT) stress were significantly reduced in EtOH-gavaged rats compared to controls after H/R. Proapoptotic caspase-8 and Bax expressions were markedly diminished in EtOH-gavaged animals compared with controls 2 h after resuscitation. EtOH-gavage increased antiapoptotic Bcl-2 gene expression compared with controls 2 h after resuscitation. iNOS protein expression increased following H/R but was attenuated in EtOH-gavaged animals after H/R. Taken together, the data suggest that acute EtOH-gavage may attenuate H/R-induced oxidative stress thereby reducing cellular injury in rat liver.

Topics: Aldehydes; Animals; bcl-2-Associated X Protein; Caspase 8; Ethanol; Female; Hemorrhage; Liver; Nitric Oxide Synthase Type II; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Resuscitation; Tyrosine

Statins are established in the prevention and therapy of chronic cardiovascular diseases because of inhibition of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A), thus lowering blood cholesterol levels. However, their cholesterol-independent effects include regulation of Rho/Rho-kinases (ROCK) and eNOS, proteins centrally involved in various models of acute inflammation. Therefore, we tested the hypothesis that simvastatin confers protection after rat hemorrhage/resuscitation (H/R) and wanted to elucidate the mechanisms involved. Fifty-two female Lewis rats (180-250 g) were pretreated with simvastatin 5 mg/kg per day or vehicle for 6 days (i.p.). Then, rats were hemorrhaged to a mean arterial pressure of 30 +/- 2 mmHg for 60 min and resuscitated. Control group underwent surgical procedures without H/R. Two hours after resuscitation, tissues were harvested. Mortality was assessed 72 h after H/R. Simvastatin pretreatment increased survival after H/R from 20% to 80%. Serum alanine aminotransferase after H/R increased 2.2-fold in vehicle as compared with simvastatin-treated rats. Histopathological analysis revealed decreased hepatic necrosis in simvastatin-treated rats after H/R. Hepatic oxidative (4-hydroxynonenal) and nitrosative (3-nitrotyrosine) stress, inflammatory markers (serum IL-6 and hepatic infiltration with polymorphonuclear leukocytes), and actin cytoskeleton rearrangements were decreased after simvastatin pretreatment compared with vehicle-treated rats after H/R. Simvastatin increased eNOS and heme oxygenase 1 expression and eNOS activation. Expression of Rho/Rho-kinase and myosin phosphatase targeting subunit, Thr-MYPT1, a marker for Rho-kinase activity, decreased after simvastatin treatment compared with vehicle-treated rats after H/R. Simvastatin pretreatment exerts beneficial effects in this model of acute inflammation by supporting protective mechanisms that are important for hepatic microcirculation after H/R.

Topics: Actins; Aldehydes; Animals; Apoptosis; Cytoskeleton; DNA Fragmentation; Hemodynamics; Hemorrhage; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Lipid Peroxidation; Liver; Male; Neutrophils; Nitric Oxide Synthase Type III; Rats; Resuscitation; Simvastatin; Tyrosine

Hemorrhagic cystitis (HC) is a major dose-limiting side effect of cyclophosphamide (CP). The mechanism by which CP induces cystitis is not clear. Recent studies demonstrate that nitric oxide; (peroxynitrite) is involved in bladder damage caused by CP. However, the molecular targets of peroxynitrite are not known. The present study is aimed at investigating whether proteins and DNA are molecular targets of peroxynitrite using a rat model.. The experimental rats received a single i.p. injection of 150 mg kg(-1) body weight CP in saline and killed 6 or 16 h later. The control rats received saline. The bladders were used for histological and biochemical analysis. Nitrotyrosine and poly-(ADP-ribose) polymerase (PARP) were localized immunohistochemically as indicators of protein nitration and DNA damage, respectively. Nitrite, malondialdehyde, protein thiol and superoxide dismutase (SOD) activity were assayed in the bladder.. Hematuria and urinary bladder edema was observed in the CP-treated rats and histologically, moderate to severe damage to the urinary bladder was observed. The bladders of CP-treated rats stained strongly for nitrotyrosine as well as for PARP. Significant decrease in oxidized NAD levels was observed in the bladders of CP-treated rats 16 h following treatment with CP. Protein thiol was depleted and the activity of the peroxynitrite sensitive enzyme SOD was significantly reduced in the bladders of CP-treated rats.. The results of the present study reveal that protein nitration, PARP activation and NAD+ depletion may play a critical role in the pathogenesis of CP-induced hemorrhagic cystitis. Based on the results we propose a mechanism for CP-induced cystitis.

Topics: Animals; Antineoplastic Agents, Alkylating; Antioxidants; Cyclophosphamide; Cystitis; Hemorrhage; Immunoenzyme Techniques; Male; Malondialdehyde; NAD; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Superoxide Dismutase; Tyrosine; Urinary Bladder

Although studies have shown that administration of testosterone receptor antagonist, flutamide, following trauma-hemorrhage, improves hepatic, cardiovascular, and immune functions, the precise cellular/molecular mechanisms responsible for producing these salutary effects remain largely unknown. To study this, male C3H/HeN mice were subjected to a midline laparotomy and hemorrhagic shock (35+/-5 mmHg for approximately 90 min), followed by resuscitation with Ringer lactate. Flutamide (25 mg/kg) or vehicle was administered subcutaneously at the onset of resuscitation, and animals were killed 2 h thereafter. Hepatic injury was assessed by plasma alpha-glutathione S-transferase concentration, liver myeloperoxidase activity, and nitrotyrosine formation. Hepatic malondialdehyde and 4-hydroxyalkenals (lipid peroxidation indicators), cellular DNA fragmentation, and the expression of inducible nitric oxide synthase and hypoxia-inducible factor-1alpha were also evaluated. Cytokines (TNF-alpha, IL-6) and chemokines (keratinocyte-derived chemokine and monocyte chemoattractant protein-1) levels were determined by cytometric bead array. The results indicate that flutamide administration after trauma-hemorrhage reduced liver injury, which was associated with decreased levels of alpha-glutathione S-transferase, myeloperoxidase activity, nitrotyrosine formation, lipid peroxidation, and cytokines/chemokines (systemic, liver tissue, and intracellular cytokines/chemokines). Cellular apoptosis, hepatocyte hypoxia-inducible factor-1alpha, and inducible nitric oxide synthase expression were also decreased under such conditions. Thus administration of flutamide following trauma-hemorrhage protects against liver injury via reduced inflammation, cellular oxidative stress, and apoptosis.

Topics: Androgen Antagonists; Animals; Apoptosis; Blotting, Western; Cell Separation; Chemokines; Cytokines; DNA Fragmentation; Flutamide; Glutathione Transferase; Hemorrhage; Hepatitis; Hepatocytes; Hypoxia-Inducible Factor 1, alpha Subunit; Kupffer Cells; Liver; Male; Mice; Mice, Inbred C3H; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxidase; Tyrosine

To monitor cascade of events following alveolar extravasation of blood due to exposure to shock wave (SW), we conducted spatiotemporal assessment of myeloperoxidase (MPO), heme oxygenase 1 (HO-1), Cu,Zn superoxide dismutase (SOD-1), transferrin (TRF), 3-nitrotyrosine (3NTyr), alveolar endothelial cadherin (VE-CDH), and the CD11b adhesion molecules on leukocytes using electron microscopy, electron paramagnetic resonance spectroscopy, immunofluorescence imaging, and immunoblotting. Accumulation of HO-1, MPO, 3NTyr, and SOD-1 in HIL at the first 12 h was associated with transmigration of inflammatory leucocytes (ILK) into hemorrhagic lesions (HLs). Biodegradation of extravasated hemoglobin (exvHb) and deposition of iron in alveoli occurred at 3-56 h post-exposure and was preceded by LKC degranulation and accumulation of MPO, HO-1, and SOD-1 in HLs. These alterations were accompanied by appearance of heme and non-heme iron complexes in HLs. A significant increase in TRF-bound [Fe(3+)] (i.e., 14.6 +/- 5.3 microM vs. 4.8 +/- 2.1 microM immediately after exposure) and non-TRF complexes of [Fe(3+)] (i.e., 4.5 +/- 1.8 microM vs. < 0.3 microM immediately after exposure) occurred at 24 h post-exposure. Transmigrations of ILK, nitroxidative stress, and iron deposition in endothelial and epithelial cells were accompanied by destruction of endothelial integrity at 3 h post-exposure, and alveolar capillary network and necrotic changes in the pulmonary epithelial cells at 24-56 h post-exposure.

Topics: Animals; Antigens, CD; Blast Injuries; Cadherins; Cell Movement; Endothelial Cells; Epithelial Cells; Heme Oxygenase-1; Hemoglobins; Hemorrhage; Inflammation; Iron; Leukocytes; Male; Oxidative Stress; Peroxidase; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Superoxide Dismutase; Superoxide Dismutase-1; Transferrin; Tyrosine

NO in vivo has both beneficial and nonbeneficial effects depending on site and concentration. Peroxynitrite, resulting from the reaction of NO with superoxide radical, causes cellular damage. Nitrotyrosine, end product of NO's toxic effects on cellular proteins, is a stable compound that can be used to detect evidence of harmful quantities of NO. We sought to detect nitrotyrosine in coronary arterioles of DBA/2 mice injected intraperitoneally with Lactobacillus casei cell wall. The inflammatory response induced occurred in perivascular fashion and involved mainly macrophages. It was variable according to time points, being severe on days 10 and 14 and mild to moderate on days 3 and 7. Few basal inflammatory cells appeared in controls injected with phosphate-buffered saline. Western immunoblots of homogenized hearts on days 10 and 14 demonstrated specific nitrated proteins. Immunohistochemistry of frozen sections of diseased hearts showed positive immunoreactivity for nitrotyrosine in coronary arterioles at the same time points. These findings were absent in the controls. We also determined the expression of inducible nitric oxide synthase (iNOS) in controls on days 10 and 14. iNOS colocalized with nitrotyrosine in perivascular macrophages and coronary arterioles of treated mice. Additionally, aneurysms were found on day 10 and intracardiac hemorrhage with consequent death on day 14. These observations supply evidence that NO through its reactive product, peroxynitrite, and its antigen/tissue marker, nitrotyrosine, is directly involved in coronary arteritis and aneurysm development in mice models of Kawasaki disease (KD). This article shows that macrophages are central to this and bolsters the likelihood of L. casei being the cause of KD.

Topics: Aneurysm; Animals; Blotting, Western; Cell Wall; Coronary Vessels; Disease Models, Animal; Hemorrhage; Immunohistochemistry; Inflammation; Lacticaseibacillus casei; Macrophages; Male; Mice; Mucocutaneous Lymph Node Syndrome; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Thrombosis; Time Factors; Tyrosine; Vasculitis

Several studies have suggested that exposure to hyperoxia causes lung injury through increased generation of reactive oxygen and nitrogen species. The present study was aimed to investigate the effects of hyperoxia exposure on protein nitration in lungs. Rats were exposed to hyperoxia (>95%) for 48, 60, and 72 h. Histopathological analysis showed a dramatic change in the severity of lung injury in terms of edema and hemorrhage between 48- and 60-h exposure times. Western blot for nitrotyrosine showed that several proteins with molecular masses of 29-66 kDa were nitrated in hyperoxic lung tissues. Immunohistochemical analyses indicate nitrotyrosine staining of alveolar epithelial and interstitial regions. Furthermore, immunoprecipitation followed by Western blot revealed the nitration of surfactant protein A and t1alpha, proteins specific for alveolar epithelial type II and type I cells, respectively. The increased myeloperoxidase (MPO) activity and total nitrite levels in bronchoalveolar lavage and lung tissue homogenates were observed in hyperoxic lungs. Neutrophils and macrophages isolated from the hyperoxia-exposed rats, when cocultured with a rat lung epithelial L2 cell line, caused a significant protein nitration in L2 cells. Inclusion of nitrite further increased the protein nitration. These studies suggest that protein nitration during hyperoxia may be mediated in part by MPO generated from activated phagocytic cells, and such protein modifications may contribute to hyperoxia-mediated lung injury.

Topics: Amino Acid Sequence; Animals; Bronchoalveolar Lavage Fluid; Cell Line; Edema; Hemorrhage; Hyperoxia; Lung; Macrophages, Alveolar; Neutrophils; Nitrates; Nitrites; Peptide Fragments; Peroxidase; Proteins; Pulmonary Surfactant-Associated Protein A; Rats; Respiratory Mucosa; Time Factors; Tyrosine