Plasma Nitrotyrosine Research Articles

Glycyrrhizin Prevents Hemorrhagic Transformation and Improves Neurological Outcome in Ischemic Stroke with Delayed Thrombolysis Through Targeting Peroxynitrite-Mediated HMGB1 Signaling.

Peroxynitrite (ONOO-) and high mobility group box 1 protein (HMGB1) are important cytotoxic factors contributing to cerebral ischemia-reperfusion injury. However, the roles of ONOO- in mediating HMGB1 expression and its impacts on hemorrhagic transformation (HT) in ischemic brain injury with delayed t-PA treatment remain unclear. In the present study, we tested the hypothesis that ONOO- could directly mediate the activation and release of HMGB1 in ischemic brains with delayed t-PA treatment. With clinical studies, we found that plasma nitrotyrosine (NT, a surrogate marker of ONOO-) was positively correlated with HMGB1 level in acute ischemic stroke patients. Hemorrhagic transformation and t-PA-treated ischemic stroke patients had increased levels of nitrotyrosine and HMGB1 in plasma. In animal experiments, we found that FeTmPyP, a representative ONOO- decomposition catalyst (PDC), significantly reduced the expression of HMGB1 and its receptor TLR2, and inhibited MMP-9 activation, preserved collagen IV and tight junction claudin-5 in ischemic rat brains with delayed t-PA treatment. ONOO- donor SIN-1 directly induced expression of HMGB1 and its receptor TLR2 in naive rat brains in vivo and induced HMGB1 in brain microvascular endothelial b.End3 cells in vitro. Those results suggest that ONOO- could activate HMGB1/TLR2/MMP-9 signaling. We then addressed whether glycyrrhizin, a natural HMGB1 inhibitor, could inhibit ONOO- production and the antioxidant properties of glycyrrhizin contribute to the inhibition of HMGB1 and the neuroprotective effects on attenuating hemorrhagic transformation in ischemic stroke with delayed t-PA treatment. Glycyrrhizin treatment downregulated the expressions of NADPH oxidase p47 phox and p67 phox and iNOS, inhibited superoxide and ONOO- production, reduced the expression of HMGB1, TLR2, MMP-9, preserved type IV collagen and claudin-5 in ischemic brains. Furthermore, glycyrrhizin significantly decreased the mortality rate, attenuated hemorrhagic transformation, brain swelling, blood-brain barrier damage, neuronal apoptosis, and improved neurological outcomes in the ischemic stroke rat model with delayed t-PA treatment. In conclusion, peroxynitrite-mediated HMGB1/TLR2 signaling contributes to hemorrhagic transformation, and glycyrrhizin could be a potential adjuvant therapy to attenuate hemorrhagic transformation, possibly through inhibiting the ONOO-/HMGB1/TLR2 signaling cascades.

The features of the nitric oxide system in the left ventricle myocardium in the rats with experimental intermittent hypoxia of different duration

The aim was to determine the features of the NO system status in the left ventricular myocardium in the rats with intermittent hypoxic hypobaric hypoxia during 15 and 60 days.Material and methods. The study was conducted on the 30 Wistar male rats, which were divided into 3 experimental groups: the 1st – control, the 2nd – rats exposed to intermittent hypoxia during 15 days and the 3rd group – the rats exposed to intermittent hypoxia during 60 days. Blood pressure was measured in all the rats. The objects and methods of the study were blood plasma (nitrotyrosine detection with immunoassay) and the myocardium of the left ventricle (concentration of nitrites by Griess nitrite test and content of immunoreactive material to the nitric oxide synthase isoforms by immunofluorescence method). mRNA expression of NOS isoforms was determined by PCR-RT method in the myocardium.Results. In the rats with 15-day hypoxia there was an increase in systolic pressure compared to control, and in 60-day hypoxia, there was also an increase in diastolic pressure although this changes were within the normotensive range. mRNA to all the myocardial NOS isoforms was characterized by an increase in both hypoxia groups. At the same time, the indices of the immunoreactive material content to the NOS isoforms were dependent on the hypoxia term. The concentration of the nitrotyrosine increased in both hypoxic groups, but in the long term it occurred along with a decrease in the level of nitrite, which indicates the possibility of nitro-oxidative stress.Conclusions. The 15-day intermittent hypoxia changes the myocardial NO system: an increase in the expression of all 3 isoforms, an increase in the nitrite and the nitrotyrosine content. iNOS becomes the predominant form of the enzyme in the myocardium with the increasing its mRNA. In 60-day hypoxia, the profile of the NOS enzyme is characterized by increased expression of the constitutive isoforms and decreased inducible NOS expression that was accompanied with significant increase in mRNA of all three forms. The level of terminal metabolites of NO was characterized by a decrease in the nitrite while the content of the nitrotyrosine increased.

P717Glucagon-like peptide 1 (GLP-1) improves endothelial dysfunction and vascular inflammation in polymicrobial sepsis induced by cecal ligation and puncture (CLP)

Abstract Objective Sepsis causes severe hypotension, accompanied by high mortality in the setting of septic shock. LEADER, SUSTAIN-6 and other clinical trials revealed cardioprotective and anti-inflammatory properties of GLP-1 analogs like Liraglutide (Lira). We already demonstrated improved survival by amelioration of disseminated intravasal coagulation (DIC) in lipopolysaccharide (LPS)-induced endotoxemia by inhibition of the GLP-1 degrading enzyme dipeptidylpeptidase-4 (DPP-4). With the present study we aim to investigate the mechanism of protective effects of the GLP-1 analog Lira and the DPP4 inhibitor Linagliptin (Lina) in the clinically relevant sepsis model cecal ligation and puncture (CLP). Methods C57/BL6j and endothelial cell-specific GLP-1 receptor knockout mice (Cdh5crexGLP-1rfl/flmice) were used and sepsis was induced by cecal ligation and puncture (CLP). DPP4 inhibitor (Lina, 5mg/kg/d; 3 days) and GLP-1 analog (Lira, 200μg/kg/d; 3 days) were applied subcutaneously. Aortic vascular function was tested by isometric tension recording. Aorta and heart tissue was used for Western blotting, dot blot and qRT-PCR. Endogenous GLP-1 (7–36 and 9–36) and insulin was determined by ELISA. Blood samples were collected for examination of cell count, oxidative stress and glucose levels. Results Body temperature was increased by CLP and normalized by Lina and Lira. Sham- and Lira- but not Lina-treated septic mice showed low blood glucose levels compared to healthy controls. Acetylcholine-induced (endothelium-dependent) vascular relaxation in aorta was impaired by CLP. This was accompanied by vascular inflammation and elevation of IL-6, iNOS, ICAM-1, and TNF-alpha mRNA levels in aortic tissue. Vascular, cardiac and whole blood oxidative stress were increased by CLP. Furthermore, we detected higher levels of IL-6, 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-NHE) in plasma of CLP animals. Lina and Lira reduced oxidative stress and vascular inflammation, which was accompanied by improved endothelial function. In addition, CLP treatment in endothelial specific knockout mice of the GLP-1r strongly induced mortality compared to WT mice, with the effect being strongest in the Lira-treated group. Conclusion The present study demonstrates that Lina (DPP4 inhibitor) and the GLP-1 analog Lira ameliorate sepsis-induced endothelial dysfunction by reduction of vascular inflammation and oxidative stress. Clinical trials like LEADER and SUSTAIN-6 proved that GLP-1 analogs like Lira have cardioprotective effects in T2DM patients. The present study, performed in a clinically relevant model of polymicrobial sepsis, reveals that the known cardioprotective effects of GLP-1 might be translated to other diseases which affect the cardiovascular system like sepsis, underlining the potent anti-inflammatory effects of GLP-1 analogs.

1166-P: Bromocriptine-QR (BQR) Therapy Reduces Elevated Sympathetic Nervous System Activity (SNSA) and Oxidative Stress (OS) in Type 2 Diabetes (T2D) Subjects whose Dysglycemia Is Poorly Controlled on GLP-1 Receptor Agonist (GLP-1RA)

Elevated SNSA induces dysglycemia by enhancing adipose lipolysis and stimulating hepatic glucose production. It also promotes cardiovascular disease (CVD) by multiple mechanisms including augmenting systemic and vascular reactive oxygen and nitrogen species (ROS, RNS) generation. Elevated plasma prolactin (subclinical hyperprolactinemia) has also been associated with systemic inflammation, which can potentiate dysglycemia and CVD. BQR, the only dopamine agonist approved for treatment of T2D, improves postprandial dysglycemia and reduces CVD events. To evaluate mechanisms for the glucose lowering and CVD protective effects of BQR, we evaluated BQR’s effect on plasma markers of SNSA and ROS/RNS and plasma prolactin and leptin from a mechanistic study in which BQR added to 15 T2D subjects (HbA1c 8.3%) treated with a GLP-1 RA significantly improved HbA1c (-0.6%), postprandial hyperglycemia (by 22%) and endothelial dysfunction (by 36%). Plasma norepinephrine (NE), normetanephrine (NM), TBAR (a marker of systemic OS), nitrotyrosine (NT, a marker of systemic oxidative/nitrosative stress), prolactin and leptin were measured at baseline and after 4 months of BQR add-on therapy (3.2 mg/day). Plasma NE, NM, TBAR, NT and prolactin levels were elevated at baseline and decreased significantly with BQR [NE by 33% (2.95 to 1.95 ng/mL, p <0.0005); NM by 22% (56.5 to 44.3 pg/ml, p<0.018); TBAR by 10% (10.2 to 9.2 μM, p=0.04); NT by 13% (214 to 187 nM, p<0.02); prolactin by 42% (13.0 to 7.6 ng/ml, p<0.012)]. Plasma leptin, unchanged in the whole group, decreased 23% (35.7 to 27.5 ng/mL, p=0.038) in subjects with elevated leptin (>20 ng/dl, N=7) at baseline. Conclusion: Bromocriptine-QR is the only T2D therapy known to simultaneously reduce elevated plasma NE, NM, prolactin and OS and such actions may be important for the drug’s glucose lowering and CVD protective effects. Disclosure B. Chamarthi: Employee; Self; VeroScience LLC. E. Cersosimo: None. J.M. Adams: None. M. Alatrach: None. C. Agyin: None. C.L. Triplitt: Consultant; Self; Abbott. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Janssen Pharmaceuticals, Inc. M. Ezrokhi: Employee; Self; VeroScience LLC. S. Luo: Employee; Self; VeroScience LLC. E. Duvallet: Employee; Self; VeroScience LLC. A.H. Cincotta: Employee; Self; VeroScience LLC. R.A. DeFronzo: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Elcelyx Therapeutics, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk Inc. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Speaker's Bureau; Self; AstraZeneca, Novo Nordisk Inc.

Effects of the Dietary Inclusion of Partially Defatted Black Soldier Fly (Hermetia illucens) Meal on the Blood Chemistry and Tissue (Spleen, Liver, Thymus, and Bursa of Fabricius) Histology of Muscovy Ducks (Cairina moschata domestica).

Simple SummaryInsects represent a promising feed ingredient for poultry diets, as an alternative to conventional feedstuffs. Black soldier fly (BSF; Hermetia illucens) larvae are processed to obtain two main products: the protein and fat fractions. The possible utilization of BSF defatted meal in Muscovy duck (Cairina moschata domestica) diets has been poorly investigated. However, its effect on in vivo and post-mortem traits, which are extremely important for animal welfare, has not yet been investigated. Therefore, the present study has evaluated the effect of 0%, 3%, 6%, and 9% dietary BSF meal replacement on the in vivo haematological parameters and on the post-mortem organ traits. Overall, the obtained results are encouraging as increasing dietary BSF meal did not impair the growth performance or the haematological traits. Furthermore, both the liver and renal function were unaffected or even improved. The antioxidant picture appeared improved and the histological traits were not influenced by the dietary inclusion of BSF meal. From a productive and biological point of view, the dietary replacement up to 9% of BSF meal in Muscovy duck diet is feasible and BSF meal could represent a promising feed ingredient.The present study has evaluated the effects of dietary partially-defatted black soldier fly (BSF; Hermetia illucens L.) larva meal on the blood parameters, antioxidant status, and histological features of the organs of broiler ducks. A total of 192 female 3-days of age Muscovy ducklings (Cairina moschata domestica) were divided into four dietary treatments (0%, 3%, 6%, and 9% BSF meal inclusion; 6 pens/treatment, 8 birds/pen). A total of 12 ducks/treatment (2 birds/pen) were slaughtered at 51 days of age and blood samples were collected to evaluate the haematological traits, serum protein, lipid and minerals, liver and renal function serum enzymes, plasma oxidative enzymes, and metabolites. Liver, spleen, thymus, and bursa of Fabricius samples were submitted to histopathological investigations. Between the serum and plasma traits, triglycerides, cholesterol, creatinine, alkaline phosphatase, magnesium, malondialdehyde, and nitrotyrosine showed a linear decrease for increasing amounts of dietary BSF meal (p <0.01); in contrast, the serum iron concentration showed a linear increase (p <0.01). Moreover, the histopathological findings were not significantly affected by the dietary BSF larva meal inclusion. The results showed that the inclusion of up to 9% BSF larva meal represents a promising feed ingredient for Muscovy duck nutrition, and improved blood traits were observed.

High-intensity interval training modulates retinal microvascular phenotype and DNA methylation of p66Shc gene: a randomized controlled trial (EXAMIN AGE)

Impairments of retinal vessel diameter are associated with major adverse cardiovascular (CV) events. Promoter DNA methylation is a repressor of the mitochondrial adaptor p66Shc gene transcription, a key driver of ageing-induced reactive oxygen species. The study aimed to investigate whether high-intensity interval training (HIIT) affects retinal microvascular phenotype as well as p66Shc expression and oxidative stress in ageing subjects with increased CV risk from the EXAMIN AGE cohort. Eighty-four sedentary subjects (mean age 59.4 ± 7.0 years) with ≥2 CV risk factors were randomized into either a 12-week HIIT or standard physical activity recommendations. Retinal arteriolar and venular diameters were measured by use of a retinal vessel analyser. As a marker of oxidative stress plasma 3-nitrotyrosine (3-NT) level was determined by ELISA. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by RT-qPCR and methylated-DNA capture (MethylMiner Enrichment Kit) coupled with qPCR, respectively. High-intensity interval training reduced body mass index, fat mass, low-density lipoprotein and increased muscle mass, as well as maximal oxygen uptake (VO2max). Moreover, HIIT restored microvascular phenotype by inducing retinal arteriolar widening (pre: 175 ± 14 µm vs. post: 181 ± 13 µm, P = 0.001) and venular narrowing (pre: 222 ± 14 µm vs. post: 220 ± 14 µm, P = 0.007). After HIIT, restoration of p66Shc promoter methylation (P = 0.034) reduced p66Shc gene expression (P = 0.037) and, in turn, blunted 3-NT plasma levels (P = 0.002). High-intensity interval training rescues microvascular dysfunction in ageing subjects at increased CV risk. Exercise-induced reprogramming of DNA methylation of p66Shc gene may represent a putative mechanistic link whereby exercise protects against age-related oxidative stress. ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

Rectus Sheath Block (RSB) Analgesia Could Enhance Significantly the Patient Satisfaction Following Midline Laparotomy in Benign Disease and in Cancer: A Prospective Study With Special Reference to Nitrosative Stress Marker Nitrotyrosine (NT) Plasma Concentrations.

Our hypothesis was that rectus sheath block (RSB) analgesia could enhance satisfaction following midline laparotomy in patients with benign disease and cancer patients. Initially, 56 patients were randomized into four groups; control group (n=12), single-dose (n=16), repeated-dose (n=12) and continuous infusion (n=16) RSB analgesia groups. The plasma concentrations of the NT marker were measured just before, immediately after and 24 h after operation. Patient satisfaction at 24 h postoperatively was filed on a 11-point numeric rating scale (SFS24; 0=fully unsatisfied; 10=fully satisfied). The RSB analgesia significantly enhanced the SFS24 scores in the study groups (p=0.001). The median plasma NT concentrations (pg/ml) following surgery (POP1) were significantly lower in patients with cancer versus patients with benign disease (5.3 vs. 7.6, p=0.008). Jitter plots of the individual SFS24 values versus plasma NT concentrations were significantly correlated in benign and cancer patients (r=-0.284, p=0.028). The RSB analgesia could significantly enhance patient satisfaction following midline laparotomy. Plasma NT concentrations versus patient satisfaction following surgery are significantly correlated in benign disease and cancer.

Nitrotyrosine (NT), a Nitrosative Stress Biomarker, Plasma Concentrations in Gallstone Disease and Cancer Patients.

The aim of this study was to assess the plasma concentration of the nitrosative stress biomarker nitrotyrosine (NT) in gallstone disease and cancer patients. Initially, 114 patients with symptomatic gallstone disease were randomized into the laparoscopic cholecystectomy (LC) (n=54) and the minicholecystectomy (MC) (n=60) groups. The plasma concentrations of NT were measured just before, immediately after (POP1) and 6 h after operation (POP2). The cancer patients of this study included ten patients with gastrointestinal cancer and 19 patients with gynecological cancer. There was a statistically significant correlation in the median plasma NT concentrations versus plasma catalase (CAT) concentrations in cholecystectomy patients (r=0.169, p=0.001). Interestingly, there was a statistically significant inverse correlation between the individual values of the pain assessed and filed using a 11-point numeric rating scale 8 h postoperatively (NAD8) and plasma NT median values in cholecystectomy patients (r=-0.337, p=0.004). Patients with high plasma concentrations of NT appeared to have significantly lower pain scores 8 h postoperatively.

Mg-supplementation attenuated lipogenic and oxidative/nitrosative gene expression caused by Combination Antiretroviral Therapy (cART) in HIV-1-transgenic rats.

We determined if HIV-1 expression in transgenic (HIV-1-Tg) rats enhanced hepatic genomic changes related to oxidative/nitrosative stress and lipogenesis during cART-treatment, and assessed effects of Mg-supplementation. A clinically used cART (atazanavir-ritonavir+Truvada) was given orally to control and HIV-1-Tg rats (18 weeks) with normal or 6-fold dietary-Mg. Oxidative/nitrosative and lipogenic genes were determined by real-time RT-PCR. cART induced a 4-fold upregulation of sterol regulatory element-binding protein-1 (SREBP-1) in HIV-1-Tg-rats, but not in controls; Tg rats displayed a 2.5-fold higher expression. Both were completely prevented by Mg-supplementation. Nrf2 (Nuclear erythroid-derived factor 2), a master transcription factor controlling redox homeostasis, was down-regulated 50% in HIV-Tg rats, and reduced further to 25% in Tg+cART-rats. Two downstream antioxidant genes, heme oxygenase-1(HmOX1) and Glutathione-S-transferase(GST), were elevated in HIV-Tg alone but were suppressed by cART treatment. Decreased Nrf2 in Tg±cART were normalized by Mg-supplementation along with the reversal of altered HmOX1 and GST expression. Concomitantly, iNOS (inducible nitric oxide synthase) was upregulated 2-fold in Tg+cART rats, which was reversed by Mg-supplementation. In parallel, cART-treatment led to substantial increases in plasma 8-isoprostane, nitrotyrosine, and RBC-GSSG (oxidized glutathione) levels in HIV-1-Tg rats; all indices of oxidative/nitrosative stress were suppressed by Mg-supplementation. Both plasma triglyceride and cholesterol levels were elevated in Tg+cART rats, but were lowered by Mg-supplementation. Thus, the synergistic effects of cART and HIV-1 expression on lipogenic and oxidative/nitrosative effects were revealed at the genomic and biochemical levels. Down-regulation of Nrf2 in the Tg+cART rats suggested their antioxidant response was severely compromised; these abnormal metabolic and oxidative stress effects were effectively attenuated by Mg-supplementation at the genomic level.

Combination ART-Induced Oxidative/Nitrosative Stress, Neurogenic Inflammation and Cardiac Dysfunction in HIV-1 Transgenic (Tg) Rats: Protection by Mg.

Chronic effects of a combination antiretroviral therapy (cART = tenofovir/emtricitatine + atazanavir/ritonavir) on systemic and cardiac oxidative stress/injury in HIV-1 transgenic (Tg) rats and protection by Mg-supplementation were assessed. cART (low doses) elicited no significant effects in normal rats, but induced time-dependent oxidative/nitrosative stresses: 2.64-fold increased plasma 8-isoprostane, 2.0-fold higher RBC oxidized glutathione (GSSG), 3.2-fold increased plasma 3-nitrotyrosine (NT), and 3-fold elevated basal neutrophil superoxide activity in Tg rats. Increased NT staining occurred within cART-treated HIV-Tg hearts, and significant decreases in cardiac systolic and diastolic contractile function occurred at 12 and 18 weeks. HIV-1 expression alone caused modest levels of oxidative stress and cardiac dysfunction. Significantly, cART caused up to 24% decreases in circulating Mg in HIV-1-Tg rats, associated with elevated renal NT staining, increased creatinine and urea levels, and elevated plasma substance P levels. Strikingly, Mg-supplementation (6-fold) suppressed all oxidative/nitrosative stress indices in the blood, heart and kidney and substantially attenuated contractile dysfunction (>75%) of cART-treated Tg rats. In conclusion, cART caused significant renal and cardiac oxidative/nitrosative stress/injury in Tg-rats, leading to renal Mg wasting and hypomagnesemia, triggering substance P-dependent neurogenic inflammation and cardiac dysfunction. These events were effectively attenuated by Mg-supplementation likely due to its substance P-suppressing and Mg’s intrinsic anti-peroxidative/anti-calcium properties.

Level of NO/nitrite and 3-nitrotyrosine in seminal plasma of infertile men: Correlation with sperm number, motility and morphology

Nitric oxide (NO) is a signaling molecule responsible for initiation of molecular events that influence sperm functionality in a concentration-dependent manner. It is still not fully understood how seminal plasma NO contributes to sperm pathologies. The aim of this study was to elucidate whether and how NO is implicated in etiology of different sperm abnormalities.To this end we determine NO, nitrite and 3-nitrotyrosine (3-NT) content in seminal plasma of infertile men with specific pathologies (terato-, oligoterato- and oligoasthenoteratospermia) and relate it to infertile normospermic samples. To gain further understanding of NO metabolism in seminal plasma we determine protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS).Here we show that NO, nitrite and 3-NT levels in seminal plasma of men with suboptimal semen parameters are significantly lower compared to normospermic men. An increase in protein expression of eNOS and no change in protein expression of iNOS is observed in men with sperm pathologies. Association of seminal plasma 3-NT level with functional sperm parameters is observed - positive correlation with sperm count, motility and morphology in normospermia, teratospermia and oligoteratospermia, as well as negative correlation with sperm morphology and motility in oligoasthenoteratospermia.Present study revealed that suboptimal seminal plasma NO content is found in all examined sperm pathologies. This result unequivocally shows the importance of NO for sperm function and involvement of suboptimal NO level in etiology of sperm abnormalities. Lower seminal plasma 3-NT level and its significant association with sperm parameters, found in pathologies, strongly indicates that protein nitration is important for spermatozoa function and that failure to establish this post-translational protein modification might be involved in etiology of sperm abnormalities. According to our results, NO measurement can discriminate infertile men with sperm pathologies from infertile normospermic men but is not indicative of a specific type of sperm pathology.

Chronic aerobic exercise training alleviates myocardial fibrosis in aged rats through restoring bioavailability of hydrogen sulfide.

Age-related fibrosis is attenuated by aerobic exercise; however, little is known concerning the underlying molecular mechanism. To address this question, aged rats were given moderate-intensity exercise for 12 weeks. After exercise in aged rats, hydrogen sulfide levels in plasma and heart increased 39.8% and 90.9%, respectively. Exercise upregulated expression of cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase in heart of aged rats. Furthermore, aged rats were given moderate-intensity exercise for 12 weeks or treated with NaHS (intraperitoneal injection of 0.1 mL/kg per day of 0.28 mol/L NaHS). After exercise in aged rats, Masson-trichrome staining area decreased 34.8% and myocardial hydroxyproline levels decreased 29.6%. Exercise downregulated expression of collagen-I and α- smooth muscle actin in heart of aged rats. Exercise in aged rats reduced malondialdehyde levels in plasma and heart and 3-nitrotyrosine in heart. Exercise in aged rats reduced mRNA and protein expression of C/EBP homologous protein, glucose regulated protein 78, and X-box protein 1. Exercise also reduced mRNA and protein expression of interleukin 6 and monocyte chemotactic protein 1and suppressed activation of c-Jun N-terminal kinase in aging heart. Similar effects were demonstrated in aged rats treated with NaHS. Collectively, exercise restored bioavailability of hydrogen sulfide in the heart of aged rats, which partly explained the benefits of exercise against myocardial fibrosis of aged population.

Pregestational diabetes increases fetoplacental vascular resistance in rats

IntroductionDiabetes is a well-known risk factor in pregnancy. Because maternal diabetes involves oxidative stress that is also induced by chronic hypoxia and can alter vascular function, we sought to determine the effects of chronic maternal hyperglycemia on the fetoplacental vasculature in rats and to compare it with the effects of chronic hypoxia. MethodsDiabetes was induced in female rats by a streptozotocin injection at a neonatal age. When these animals reached adulthood, their hyperglycemia was confirmed and they were inseminated. Half of them were exposed to hypoxia (10% O2) for the last week before the delivery. One day before the expected date of delivery, one of their placentae was isolated and perfused. ResultsFetoplacental vascular resistance was increased equally by experimental diabetes, chronic hypoxia, and their combination. Fetoplacental perfusion pressure-flow analysis suggested increased resistance in the small vessels in chronic hypoxia and in larger vessels in diabetes. Fetal plasma nitrotyrosine levels, measured as a marker of peroxynitrite (reaction product of superoxide and nitric oxide), mirrored the differences in fetoplacental resistance, suggesting a causative role. Fetoplacental vasoconstrictor reactivity to acute hypoxic stimuli was reduced similarly in all groups. Fasudil, a strong vasodilator agent, reduced fetoplacental vascular resistance similarly in all groups, suggesting that for the observed differences among the groups, the changes in vascular morphology were more important than variances in vascular tone. DiscussionMaternal diabetes increases fetoplacental vascular resistance to a similar extent as chronic hypoxia. These stimuli are not additive. Changes in vascular tone are not responsible for these effects.

Allopurinol attenuates acute kidney injury following Bothrops jararaca envenomation.

Snakebites have been recognized as a neglected public health problem in several tropical and subtropical countries. Bothrops snakebites frequently complicate with acute kidney injury (AKI) with relevant morbidity and mortality. To date, the only treatment available for Bothrops envenomation is the intravenous administration of antivenom despite its several limitations. Therefore, the study of novel therapies in Bothrops envenomation is compelling. The aim of this study was to evaluate the protective effect of Allopurinol (Allo) in an experimental model of Bothrops jararaca venom (BJ)-associated AKI. Five groups of Wistar rats were studied: Sham, Allo, BJ, BJ+Allo, BJ+ipAllo. BJ (0.25 mg/kg) was intravenously injected during 40’. Saline at same dose and infusion rate was administered to Sham and Allo groups. Allo and BJ+Allo groups received Allo (300 mg/L) in the drinking water 7 days prior to Saline or BJ infusion respectively. BJ+ipAllo rats received intraperitoneal Allo (25 mg/Kg) 40’ after BJ infusion. BJ rats showed markedly reduced glomerular filtration rate (GFR, inulin clearance) associated with intense renal vasoconstriction, hemolysis, hemoglobinuria, reduced glutathione and increased systemic and renal markers of nitro-oxidative stress (Nitrotyrosine). Allo ameliorated GFR, renal blood flow (RBF), renal vascular resistance and arterial lactate levels. In addition, Allo was associated with increased serum glutathione as well as reduced levels of plasma and renal Nitrotyrosine. Our data show that Allo attenuated BJ-associated AKI, reduced oxidative stress, improved renal hemodynamics and organ perfusion. It might represent a novel adjuvant approach for Bothrops envenomation, a new use for an old and widely available drug.

162 - Combination Antiretroviral Therapy (cART) Altered Redox/Nitrosative genes, Enhanced Systemic Stress and Cardiac dysfunction in HIV-1 Transgenic (HIV-Tg) Rats: Effects of Magnesium Supplementation (Mg-Supp)

We determined if clinically used cART promoted redox/nitrosative stress and cardiac toxicity in control and HIV-Tg rats and the effects of Mg. Treatment with cART (Truvada+atazanavir/ritonavir) up to 18 weeks, induced significant hypomagnesemia (-25%), elevated plasma 8-isoprostane, and RBC GSSG 2-3 folds (at 6 wks); plasma 3-nitrotyrosine (NT) increased 3.5-fold in the HIV-Tg rats (at 18 wks). In association, liver Nrf2 mRNA (by real time RT-PCR) was down-regulated 75% along with altered expression of HmOX-1 and GST; however iNOS and TNFα mRNAs were increased 2- & 4-fold. Prominent ventricular NT staining was observed in HIV-Tg+cART rats; echocardiography detected time-dependent moderate, but significant decreases in cardiac systolic (Fractional Shortening. -17%, 18 wks) and diastolic (E/A, -26%) functions. Mg-Supp (6X normal) in diet, substantially prevented rises in levels of 8-isoprostane, GSSG, and plasma NT. Most strikingly, Mg-Supp restored expressions of Nrf2 and HmOX-1, GST close to controls in HIV-Tg rats; both iNOS and TNFα up-regulations were blunted. In association, cardiac NT staining was substantially reduced; both cardiac systolic and diastolic functional decreases were attenuated 60-70% by Mg-supp. Conclusions cART treatment synergized with HIV-1 expression in down-regulating Nrf2 pathway, but up-regulating iNOS and TNFα expression; elevation of systemic redox/nitrosative stress contributed to cardiac dysfunction. The cardiac protective effects of Mg-Supp might be mediated by its antioxidant actions at the genomic and systemic levels through the Nrf2 pathway

Zinc-Induced Metallothionein in Centenarian Offspring From a Large European Population: The MARK-AGE Project.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35-75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan.

Nitrotyrosine and its role in assessing the severity of oxidative stress and predicting the incidence of early postoperative complications

Background. Currently, the incidence of complications and perioperative mortality in cardiac surgery is still higher than in general surgery. This is partly associated with the development of oxidative stress, which is regarded as excessive accumulation of reactive oxygen species and nitrogen species. There are nowadays few studies demonstrating an increase of nitrotyrosine plasma level in patients during cardiac surgery. The relationship of these changes with post-operative complications and adverse outcomes has not been studied yet. Aim. Investigation the dynamics of the nitrotyrosine plasma level in patients after cardiac surgery and the assessment of its prognostic significance in terms of the development of early postoperative complications.Methods. The prospective observational cohort study involved 28 adult patients admitted at the cardiac intensive care unit of Moscow Regional Research and Clinical Institute (Moscow, Russian Federation). The relationship of the oxidative stress severity measured by intraoperative nitrotyrosine plasma levels and the development of acute kidney injury, cardiac failure and systemic inflammatory response syndrome was studied. Results. There was no significant perioperative dynamics of nitrotyrosine plasma levels when studying this oxidative stress marker in patients undergoing cardiac surgery. The level of nitrotyrosine plasma by the end of surgery was 12.1 [9.9; 13.0] nmol/mg of protein, which was 5% higher (p&gt;0.5) than the initial level of 11.6 [9.3; 12.2] nmol/mg of protein, and returned to 11.5 [10.9; 12.4] after the 1st postoperative day. The same perioperative dynamics occurred in the subgroups with different surgery techniques: valve surgery, сoronary artery bypass grafting with cardiopulmonary bypass and off-pump сoronary artery bypass grafting.Conclusion. The negative result achieved in the given study might suggest that there are no significant changes of the perioperative nitrotyrosine plasma level in patients undergoing cardiac surgery, as well as that there is no significant correlation between the nitrotyrosine plasma level of and the incidence of postoperative complications.Received 26 December 2016. Accepted 4 May 2017.Funding: The study was carried out within the state-funded research project “Oxidant stress in cardiac surgery: new markers-predictors of complication development and pathogenetically substantiated therapy” granted to Moscow Regional Research and Clinical Institute.Conflict of interest: The authors declare no conflict of interest.Author contributionsConceptualization, study design: V.V. Likhvantsev.Data collection and analysis: T.S. Zabelina, Yu.V. Skripkin, O.N. Ulitkina. Drafting the article: O.A. Grebenchikov, Zh.S. Philippovskaya.Critical revision of the article: O.A. Grebenchikov.Final approval of the version to be published: V.V. Likhvantsev.

Plasma Nitrotyrosine Level does not Differentiate between Patients with Minimal Hepatic Encephalopathy and without Minimal Hepatic Encephalopathy in Liver Cirrhosis

Plasma Nitrotyrosine Level does not Differentiate between Patients with Minimal Hepatic Encephalopathy and without Minimal Hepatic Encephalopathy in Liver Cirrhosis

Inorganic Nitrate Supplementation in Young and Old Obese Adults Does Not Affect Acute Glucose and Insulin Responses but Lowers Oxidative Stress

Background: Aging and obesity are associated with raised oxidative stress and a reduction of nitric oxide (NO) bioavailability, with subsequent decline in insulin sensitivity and endothelial function. Inorganic nitrate is converted into NO via a 2-step reduction process and may be an effective nutritional intervention to modify vascular and metabolic functions.Objectives: This study tested whether inorganic nitrate supplementation improved glucose disposal and attenuated the acute effects of hyperglycemia on oxidative stress, inflammation, and vascular function in young and old obese participants.Methods: Ten young (aged 18–44 y) and 10 old (aged 55–70 y) obese participants consumed 75 g glucose followed by either potassium nitrate (7 mg/kg body weight) or potassium chloride (placebo) in a randomized, double-blind crossover design. Resting blood pressure (BP), endothelial function, and blood biomarkers were measured for 3 h postintervention. Biomarkers included plasma nitrate/nitrite (NOx), glucose, insulin, cyclic GMP, interleukin 6, 3-nitrotyrosine, E- and P-selectins, intercellular adhesion molecule 3 (ICAM-3), and thrombomodulin, as well as superoxide in freshly isolated peripheral blood mononuclear cells (PBMCs).Results: Inorganic nitrate supplementation did not affect plasma glucose (P = 0.18) or insulin (P = 0.26) responses. The increase in plasma NOx concentrations 3 h after the administration of inorganic nitrate was significantly higher in young than in old participants (234% increase compared with 149% increase, respectively, P < 0.001). Plasma 3-nitrotyrosine concentrations declined significantly after inorganic nitrate supplementation compared with placebo (3 h postdose, 46% decrease compared with 27% increase, respectively, P = 0.04), and a similar nonsignificant trend was observed for superoxide concentrations (3 h postdose, 16% decrease compared with 23% increase, respectively, P = 0.06). Plasma cyclic GMP, ICAM-3, and thrombomodulin concentrations differed between young and old participants (P < 0.01). Inorganic nitrate supplementation did not improve BP or endothelial function.Conclusions: Oral supplementation with inorganic nitrate did not improve glucose and insulin responses but reduced oxidative stress in old individuals during acute hyperglycemia. This trial was registered at http://www.controlled-trials.com as ISRCTN42776917.

Gradually increased oxygen administration promoted survival after hemorrhagic shock.

Gradually increased oxygen administration (GIOA) seems promising in hemorrhagic shock. However, the effects of GIOA on survival remain unclear, and details of GIOA are to be identified. After the induction of hemorrhagic shock, the rats were randomized into five groups (n = 9): normoxic group (Normo), hyperoxic group (Hypero), normoxic to hyperoxic group (GIOA1), long-time hypoxemic to hyperoxic group (GIOA2), and short-time hypoxemic to hyperoxic group (GIOA3). Survival was recorded for 96 h, plasma alanine transaminase, oxidative stress, hemodynamics, and blood gas were measured. The mean survival time of the GIOA3 was significantly longer than that of the Normo, Hypero, and GIOA2. Plasma alanine transaminase levels were significantly lower in the Normo, GIOA1, and GIOA3 compared to the Hypero and GIOA2 at 2 h post-resuscitation (PR). Plasma 3-nitrotyrosine levels at 2 h PR were significantly lower in the GIOA2 and GIOA3 compared to the Normo and Hypero. Central venous oxygen saturation at 2 h PR in the GIOA3 was significantly higher than the Normo; however, no significant difference was observed between GIOA1 and Normo. Besides, at 2 h PR, mean arterial pressure in the GIOA3 was significantly higher than the GIOA2; however, no significant difference was observed between GIOA1 and GIOA2. (1) GIOA could significantly prolong survival time compared to normoxemic resuscitation and hyperoxic resuscitation; (2) early moments of GIOA are critical to the benefits; and (3) hypoxemia at onset of resuscitation may be imperative, more works are needed to determine the optimal initial oxygen concentration of GIOA.