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1.
Oxid Med Cell Longev ; 2019: 8219283, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089418

RESUMO

Chronic kidney disease (CKD) is accompanied by a disturbed redox homeostasis, especially in end-stage patients, which is associated with pathological complications such as anemia, atherosclerosis, and muscle atrophy. However, limited evidence exists about redox disturbances before the end stage of CKD. Moreover, the available redox literature has not yet provided clear associations between circulating and tissue-specific (muscle) oxidative stress levels. The aim of the study was to evaluate commonly used redox status indices in the blood and in two different types of skeletal muscle (psoas, soleus) in the predialysis stages of CKD, using an animal model of renal insufficiency, and to investigate whether blood redox status indices could be reflecting the skeletal muscle redox status. Indices evaluated included reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), catalase (CAT), total antioxidant capacity (TAC), protein carbonyls (PC), and thiobarbituric acid reactive substances (TBARS). Results showed that blood GSH was higher in the uremic group compared to the control (17.50 ± 1.73 vs. 12.43 ± 1.01, p = 0.033). In both muscle types, PC levels were higher in the uremic group compared to the control (psoas: 1.086 ± 0.294 vs. 0.596 ± 0.372, soleus: 2.52 ± 0.29 vs. 0.929 ± 0.41, p < 0.05). The soleus had higher levels of TBARS, PC, GSH, CAT, and GR and lower TAC compared to the psoas in both groups. No significant correlations in redox status indices between the blood and skeletal muscles were found. However, in the uremic group, significant correlations between the psoas and soleus muscles in PC, GSSG, and CAT levels emerged, not present in the control. Even in the early stages of CKD, a disturbance in redox homeostasis was observed, which seemed to be muscle type-specific, while blood levels of redox indices did not seem to reflect the intramuscular condition. The above results highlight the need for further research in order to identify the key mechanisms driving the onset and progression of oxidative stress and its detrimental effects on CKD patients.


Assuntos
Músculo Esquelético/metabolismo , Insuficiência Renal/sangue , Insuficiência Renal/metabolismo , Animais , Catalase/metabolismo , Modelos Animais de Doenças , Feminino , Dissulfeto de Glutationa/metabolismo , Oxirredução , Carbonilação Proteica , Coelhos , Uremia/sangue
2.
Eur J Pharmacol ; 854: 159-166, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30991047

RESUMO

After acute myocardial infarction (AMI), reactive oxygen species and oxidative stress have important roles in the progression to heart failure. As a therapeutic alternative, thyroid hormones (TH) revealed cardioprotective effects after AMI, including decreasing oxidative stress. Carvedilol beta-blocker, already used in the clinical treatment of AMI, also mitigate cardiac pathological remodelling. This study assessed the effects of post-AMI carvedilol and TH co-administration on oxidative stress and cardiac function as well as whether those effects were synergistic. Male Wistar rats were divided into five groups: sham-operated (SHAM), infarcted (MI), infarcted + TH (MI + TH), infarcted + carvedilol (MI + C) and infarcted + C + TH (MI + C + TH). Two days post-surgery, the SHAM and MI groups received saline, and treated groups received their respective treatments by gavage for 12 days. The animals were submitted to echocardiographic evaluation, ventricular catheterization and euthanized for heart collection to perform oxidative stress analysis. Treated groups improved for ejection fraction compared to the MI group. Carvedilol decreased the positive chronotropic TH effects in the MI + C + TH group. The MI and MI + C groups had increased reactive oxygen species and reduced sulfhydryl levels. Carvedilol and TH co-administration showed synergic effects in the MI + C + TH group, reducing reactive oxygen species levels and improving GSH/GSSG ratio. Moreover, co-treatment attenuated NADPH oxidase activity in the MI group. Therefore, this study showed for the first time that carvedilol and TH co-administration may improve redox balance and cardiac function after AMI. Such co-administration could represent a therapeutic strategy capable of preventing cardiac dysfunction and redox unbalance after AMI.


Assuntos
Carvedilol/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Infarto do Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Animais , Antioxidantes/metabolismo , Sinergismo Farmacológico , Eletrocardiografia/efeitos dos fármacos , Dissulfeto de Glutationa/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Infarto do Miocárdio/sangue , Infarto do Miocárdio/fisiopatologia , NADPH Oxidases/metabolismo , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Tireotropina/sangue
3.
PLoS One ; 14(4): e0215955, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022254

RESUMO

Oxidative stress plays a key role in steatohepatitis induced by both xenobiotic agents and high fat diet (HFD). The present study aimed to evaluate hepatic oxidative stress and anti-oxidant systems response in rats exposed to HFD and/or non-toxic dose of dichlorodiphenyldichloroethylene (DDE), the first metabolite of dichlorodiphenyltrichloroethane. Groups of 8 rats were so treated for 4 weeks: 1- standard diet (N group); 2- standard diet plus DDE (10 mg/kg b.w.) (N+DDE group); 3- HFD (D group); 4- HFD plus DDE (D+DDE group). Oxidative stress was analyzed by determining malondialdehyde as lipid peroxidation product, while the anti-oxidant systems were evaluating by measuring the levels of the principal cytosolic and mitochondrial antioxidant proteins and enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase 1 (GPx1) and uncoupling protein 2 (UCP2) involved in the control of hepatic reactive oxygens species (ROS) accumulation. The results showed malondialdehyde accumulation in livers of all groups, confirming the pro-oxidant effects of both HFD and DDE, but with a greater effect of DDE in absence of HFD. In addition, we found different levels of the analyzed anti-oxidant systems in the different groups. DDE mainly induced UCP2 and SOD2, while HFD mainly induced GPx1. Noteworthy, in the condition of simultaneous exposure to DDE and HFD, the anti-oxidant response was more similar to the one induced by HFD than to the response induced by DDE. Present findings confirmed that both HFD and xenobiotic exposure induced hepatic oxidative stress and showed that the anti-oxidant defense response was not the same in the diverse groups, suggesting that UCP2 induction could be an adaptive response to limit excessive ROS damage, mainly in condition of xenobiotic exposure.


Assuntos
Diclorodifenil Dicloroetileno/toxicidade , Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteína Desacopladora 2/metabolismo , Xenobióticos/toxicidade , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antioxidantes/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Fígado Gorduroso/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Dissulfeto de Glutationa/metabolismo , Macrófagos do Fígado/efeitos dos fármacos , Macrófagos do Fígado/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/análise , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Metaboloma/efeitos dos fármacos , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Proteína Desacopladora 2/genética , Ganho de Peso/efeitos dos fármacos
4.
Int J Mol Sci ; 20(6)2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893912

RESUMO

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.


Assuntos
Células Epiteliais/metabolismo , Deleção de Genes , Metabolômica , Receptores de GABA/genética , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Análise Discriminante , Células Epiteliais/efeitos dos fármacos , Glucose/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Metaboloma/efeitos dos fármacos , Nucleotídeos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Análise de Componente Principal , Receptores de GABA/metabolismo
5.
J Physiol Biochem ; 75(2): 185-197, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30868511

RESUMO

In this study, the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.


Assuntos
Glutationa/metabolismo , Melatonina/farmacologia , Células Estreladas do Pâncreas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dissulfeto de Glutationa/metabolismo , Humanos , Camundongos , Pâncreas/metabolismo , Células Estreladas do Pâncreas/citologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Ratos , Receptor MT1 de Melatonina/metabolismo
6.
Neurochem Res ; 44(7): 1613-1620, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919283

RESUMO

Ischemic stroke is a leading cause of mortality and morbidity worldwide, and oxidative stress plays a significant role in the ischemia stage and reperfusion stage. Previous studies have indicated that both calcium/calmodulin-dependent protein kinase II (CaMKII) and glucose 6-phosphate dehydrogenase (G6PD) are involved in the oxidative stress. Thus, the aim of this study was to investigate the roles of CaMKIIα, an important isoform of CaMKII, and G6PD in a rat model of middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of small interfering ribonucleic acid (siRNA) for CaMKIIα was performed at 48 h pre-MCAO surgery. Immunofluorescence Staining and western blot were performed to detect the expression of p-CaMKIIα and G6PD in the cortices. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was performed to investigate the infarct volume. In addition, neurological deficit, reactive oxygen species (ROS), ratio of reduced-to-oxidized glutathione (GSH/GSSG) and ratio of reduced-to-oxidized oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) were assessed. The results indicated that both p-CaMKIIα and G6PD were widely located in the neurons and astrocytes, and their expression was gradually increased in the cortices after MCAO, which was accompanied by increased level of ROS and decreased levels of GSH/GSSG and NADPH/NADP+. However, after treatment with siRNA for CaMKIIα, p-CaMKIIα expression was decreased and G6PD expression was increased. Moreover, inhibition of CaMKIIα improved the neurological deficit, reduced the infarct volume, decreased the level of ROS and increased the levels of GSH/GSSG and NADPH/NADP+. The results suggested that CaMKIIα inhibition exerted neuroprotective effects through regulating G6PD expression, which provides a new target for prevention and treatment of stroke.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Estresse Oxidativo/fisiologia , Animais , Astrócitos/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Inativação Gênica , Dissulfeto de Glutationa/metabolismo , Masculino , NADP/metabolismo , Neurônios/metabolismo , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
7.
Eur J Dermatol ; 29(2): 167-173, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30882351

RESUMO

Dermatitis herpetiformis (DH) and celiac disease (CD) are considered to be autoimmune diseases that share a specific trigger (gluten) and a common genetic background (HLA-DQ2/DQ8). However, the pathogenesis of DH is not yet fully understood and no data are available regarding a possible role of fibroblasts in this disease. The aim of this study was to assess baseline DNA damage in fibroblasts in DH-diagnosed patients vs. fibroblasts of controls without DH or CD. Primary fibroblast cultures were derived from dermal biopsies from DH patients and controls (without DH or CD). In vitro genotoxic damage was investigated using the comet assay and ɣH2AX test after different treatments (with 33mer peptide and digested gliadin [DG]) in order to investigate a correlation between oxidative stress (evaluated by reactive oxygen species formation) and glutathione content. Our results demonstrate a difference in baseline DNA damage between cutaneous fibroblasts of controls and DH patients, moreover, DNA damage significantly increased after exposure to gluten (DG and 33mer peptide) in fibroblasts from DH patients. DNA damage in fibroblasts from patients under dapsone treatment was similar to that of the control group. Our data indicate that oxidative stress and DNA damage may be characteristics of fibroblasts from DH patients who are not treated with dapsone, particularly after exposure to gliadin peptides.


Assuntos
Doença Celíaca/genética , Dano ao DNA , Dermatite Herpetiforme/genética , Fibroblastos/citologia , Adulto , Idoso , Doença Celíaca/imunologia , Ensaio Cometa , Dermatite Herpetiforme/imunologia , Feminino , Gliadina/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo
8.
Neurochem Res ; 44(5): 1167-1181, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30806880

RESUMO

Menadione (2-methyl-1,4-naphthoquinone) is a synthetic derivative of vitamin K that allows rapid redox cycling in cells and thereby generates reactive oxygen species (ROS). To test for the consequences of a treatment of brain astrocytes with menadione, we incubated primary astrocyte cultures with this compound. Incubation with menadione in concentrations of up to 30 µM did not affect cell viability. In contrast, exposure of astrocytes to 100 µM menadione caused a time-dependent impairment of cellular metabolism and cell functions as demonstrated by impaired glycolytic lactate production and strong increases in the activity of extracellular lactate dehydrogenase and in the number of propidium iodide-positive cells within 4 h of incubation. In addition, already 5 min after exposure of astrocytes to menadione a concentration-dependent increase in the number of ROS-positive cells as well as a concentration-dependent and transient accumulation of cellular glutathione disulfide (GSSG) were observed. The rapid intracellular GSSG accumulation was followed by an export of GSSG that was prevented in the presence of MK571, an inhibitor of the multidrug resistance protein 1 (Mrp1). Menadione-induced glutathione (GSH) oxidation and ROS formation were found accelerated after glucose-deprivation, while the presence of dicoumarol, an inhibitor of the menadione-reducing enzyme NQO1, did not affect the menadione-dependent GSSG accumulation. Our study demonstrates that menadione rapidly depletes cultured astrocytes of GSH via ROS-induced oxidation to GSSG that is subsequently exported via Mrp1.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Dissulfeto de Glutationa/efeitos dos fármacos , Vitamina K 3/farmacologia , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicólise/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Ratos Wistar
9.
Redox Biol ; 21: 101050, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30654300

RESUMO

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.


Assuntos
Glutationa Redutase/química , Glutationa Redutase/metabolismo , Glutationa/biossíntese , Algoritmos , Alquilação , Sequência de Aminoácidos , Animais , Catálise , Domínio Catalítico , Dissulfeto de Glutationa/metabolismo , Espaço Intracelular , Cinética , Camundongos , Modelos Teóricos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo , Células RAW 264.7 , Espécies Reativas de Oxigênio , Compostos de Sulfidrila
10.
Ecotoxicol Environ Saf ; 171: 373-381, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30616154

RESUMO

Considerable evidence exists that microorganisms play a significant role in the remediation of soil contaminated with heavy metals. Aspergillus aculeatus (A. aculeatus) isolated from Cd-polluted soil has been shown to increase the tolerance of turfgrasses to Cd stress. In this study, we assessed the tolerance, biosorption capacity for Cd and surface characteristics of this fungus and investigated the effect of plant inoculation with A. aculeatus on the lipid peroxidation, antioxidant activities and photosynthetic rates in rice cultivated in Cd-contaminated soil. The results indicated that the removal efficiency of A. aculeatus was 46.8% at a Cd concentration of 10 mg L-1. The A. aculeatus strains had the capacity to produce indole acetic acid, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase and to solubilize phosphate. The O2- accumulation and the amount of MDA in rice roots inoculated with A. aculeatus were significantly lower than those in uninoculated plants. Nevertheless, no decrease in leaf ROS accumulation and photosynthetic activity was observed between the inoculated and uninoculated plants. Inoculation with A. aculeatus contained more of the ROS-scavenging metabolite GSH, a higher GSH/GSSG ratio, and higher antioxidative enzyme (SOD, POD, and CAT) activities, possibly explaining the lower ROS concentrations observed in inoculated roots in the presence of Cd. These results suggest that application of A. aculeatus has the potential to protect crops against Cd stress.


Assuntos
Antioxidantes/metabolismo , Aspergillus/efeitos dos fármacos , Cádmio/análise , Oryza/microbiologia , Fotossíntese , Aspergillus/metabolismo , Dissulfeto de Glutationa/metabolismo , Ácidos Indolacéticos , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Oryza/efeitos dos fármacos , Fosfatos/metabolismo , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismo , Solo/química , Microbiologia do Solo , Poluentes do Solo/análise
11.
Redox Biol ; 21: 101071, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576927

RESUMO

Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H2O2 production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.


Assuntos
Técnicas Biossensoriais , Glutarredoxinas/genética , Proteínas Luminescentes/genética , Oxirredução , Proteínas Recombinantes de Fusão , Animais , Expressão Gênica , Genes Reporter , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicólise , Células HEK293 , Células HeLa , Humanos , Hipóxia/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Estresse Oxidativo , Peixe-Zebra
12.
Artigo em Inglês | MEDLINE | ID: mdl-30508739

RESUMO

Angiotensin converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1) plays an important role in the regulation of blood pressure. In this study, ACE was purified from human plasma by affinity chromatography in single step. The enzyme purified in 5367-fold from human plasma and specific activity was found to be 1208 EU/mg protein. The purity and molecular weight of ACE were determined by SDS-PAGE, which indicated two bands at around 60 kDa and 70 kDa on the gel. Effect of oxidized glutathione (GSSG) peptide and reduced glutathione (GSH) peptide on purified ACE activity were also investigated in which lisinopril was used as reference inhibitor. GSSG showed activation effect on ACE activity whereas GSH provided inhibition effect. In the lights of activity (%) versus activator graph for GSSG and activity (%) versus inhibitor graphs for GSH and lisinopril; IC50 values for GSH and lisinopril were determined to be 16.2 µM and 0.781 nM, respectively. Type of inhibition for GSH and lisinopril from graph Lineweaver-Burk was found to be reversible non-competitive inhibition and Ki constants for GSH and lisinopril were calculated as 11.7 µM and 0.662 nM, respectively.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/química , Cromatografia de Afinidade , Glutationa/química , Dissulfeto de Glutationa/química , Humanos , Modelos Lineares , Fragmentos de Peptídeos/química , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/isolamento & purificação , Peptidil Dipeptidase A/metabolismo
13.
Animal ; 13(8): 1641-1650, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30458891

RESUMO

Glutathione (GSH) is considered to play an important role in maintaining the integrity of the small intestine. In piglets, altered mucosal GSH levels might therefore be involved in weaning-induced changes of the small intestinal morphology and barrier function. To test this hypothesis, we aimed to challenge the mucosal GSH redox status during the first 28 days after weaning, by feeding diets containing 5% fresh linseed oil (CON), or 2.5% (OF1) or 5% (OF2) peroxidized linseed oil (peroxide value 225 mEq O2/kg oil) and exploring the effects on gut integrity. Piglets were pair-fed and had a total daily feed allowance of 32 g/kg BW. A fourth treatment included animals that were fed the control diet ad libitum (ACON). Animals were sampled at days 5 and 28 post-weaning. The malondialdehyde (MDA) concentration and GSH redox status (GSH/GSSG Eh) were determined in blood, liver and small intestinal mucosa. Histomorphology of the duodenal and jejunal mucosa was determined, and Ussing chambers were used to assess fluorescein isothiocyanate dextran (FD4) and horseradish peroxidase (HRP) fluxes across the mucosa. Results show that peroxidized linseed oil imposed an oxidative challenge at day 28, but not at day 5 post-weaning. At day 28, increasing levels of dietary peroxides to pair-fed pigs linearly increased MDA levels in duodenal and jejunal mucosa. Moreover, FD4 fluxes were significantly increased in OF1 (+75%) and OF2 (+64%) in the duodenum, and HRP fluxes tended (P=0.099) to show similar differences, as compared to CON. This co-occurred with a significant 11 mV increase of the hepatic GSH/GSSG Eh, potentiated by a significantly increased GSH peroxidase activity for treatments OF1 (+47%) and OF2 (+63%) in liver as compared to CON. Furthermore; duodenal HRP flux significantly correlated with the hepatic glutathione disulphide (GSSG) level (r=0.650), as also observed in the jejunum for hepatic GSSG (r=0.627) and GSH/GSSG Eh (r=0.547). The jejunal permeability was not affected, but FD4 and HRP fluxes significantly correlated with the local GSH (r=0.619; r=0.733) and GSSG (r=0.635; r=0586) levels. Small intestinal histomorphology was not affected by dietary lipid peroxides, nor were there any correlations found with the GSH redox system. To conclude, under oxidative stress conditions, jejunal barrier function is related to the local and hepatic GSH redox system. It is suggested that the hepatic GSH system participates in the elimination of luminal peroxides, and thereby impacts on duodenal barrier function.


Assuntos
Glutationa/metabolismo , Intestino Delgado/efeitos dos fármacos , Óleo de Semente do Linho/farmacologia , Suínos/metabolismo , Ração Animal , Animais , Dieta/veterinária , Suplementos Nutricionais , Dissulfeto de Glutationa/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/fisiologia , Óleo de Semente do Linho/administração & dosagem , Óleo de Semente do Linho/química , Malondialdeído/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Desmame
14.
Mol Neurobiol ; 56(4): 2760-2773, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30058022

RESUMO

S-Adenosylmethionine (AdoMet) concentrations are highly elevated in tissues and biological fluids of patients affected by S-adenosylhomocysteine hydrolase deficiency. This disorder is clinically characterized by severe neurological symptoms, whose pathophysiology is not yet established. Therefore, we investigated the effects of intracerebroventricular administration of AdoMet on redox homeostasis, microglia activation, synaptophysin levels, and TAU phosphorylation in cerebral cortex and striatum of young rats. AdoMet provoked significant lipid and protein oxidation, decreased glutathione concentrations, and altered the activity of important antioxidant enzymes in cerebral cortex and striatum. AdoMet also increased reactive oxygen (2',7'-dichlorofluorescein oxidation increase) and nitrogen (nitrate and nitrite levels increase) species generation in cerebral cortex. Furthermore, the antioxidants N-acetylcysteine and melatonin prevented most of AdoMet-induced pro-oxidant effects in both cerebral structures. Finally, we verified that AdoMet produced microglia activation by increasing Iba1 staining and TAU phosphorylation, as well as reduced synaptophysin levels in cerebral cortex. Taken together, it is presumed that impairment of redox homeostasis possibly associated with microglia activation and neuronal dysfunction caused by AdoMet may represent deleterious pathomechanisms involved in the pathophysiology of brain damage in S-adenosylhomocysteine hydrolase deficiency.


Assuntos
Encéfalo/patologia , Homeostase , Microglia/patologia , Neurônios/patologia , S-Adenosilmetionina/administração & dosagem , S-Adenosilmetionina/farmacologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Dissulfeto de Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Homeostase/efeitos dos fármacos , Injeções Intraventriculares , Lipídeos/química , Malondialdeído/metabolismo , Melatonina/farmacologia , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinaptofisina/metabolismo , Proteínas tau/metabolismo
15.
Fish Shellfish Immunol ; 86: 1044-1052, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30590160

RESUMO

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.


Assuntos
Autofagia , Glutamina/metabolismo , Dissulfeto de Glutationa/metabolismo , Perciformes/virologia , Vesiculovirus/fisiologia , Animais , Butionina Sulfoximina , Linhagem Celular , Glutationa , Perciformes/fisiologia , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/veterinária , Replicação Viral
16.
Biomed Res Int ; 2018: 7210783, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30533439

RESUMO

Chronic exposure to stress factors contributes to the development of depression by generating excess of reactive oxygen species which leads to oxidative stress and inflammatory processes. The aim of the study was to assess the potential protective properties of α-tocopherol supplementation on the rats exposed to chronic variable stress (CVS). Male Wistar rats (50-55 days old, weighing 200-250 g) were divided into three groups (n=10): control, stressed, and stressed and receiving (+)-α-tocopherol solution in a dose of 100 mg/kg/day. Rats in the stressed groups were exposed to CVS for 40 days. Markers of redox disorders (glutathione reduced and oxidized levels, GSH/GSSG ratio, glutathione peroxidase, glutathione reductase activities, total antioxidant status, and lipid peroxidation) and inflammatory response (IL-1ß, IL6, and TNF-α) were determined in the blood. Additionally, molecular biomarkers of depression (expression of Fkbp5 and Tph2) were studied in hippocampus. The biochemical analysis was inconclusive about the presence of oxidative stress in the blood of rats exposed to CVS. However, changes in all parameters suggest presence of redox equilibrium disorders. Similarly, activation of inflammatory processes was observed as a result of CVS. Molecular effects of environmental stress in hippocampus were also observed. Generally, α-tocopherol ameliorated redox equilibrium disorders, tempered inflammatory response, and protected from changes in determined molecular markers of depression.


Assuntos
Inflamação/patologia , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/patologia , alfa-Tocoferol/uso terapêutico , Animais , Antioxidantes/metabolismo , Doença Crônica , Citocinas/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Inflamação/sangue , Masculino , Malondialdeído/metabolismo , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Estresse Psicológico/sangue , Estresse Psicológico/genética , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , alfa-Tocoferol/farmacologia
17.
J Agric Food Chem ; 66(48): 12651-12656, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30417643

RESUMO

Glutathione, a tripeptide antioxidant, has recently been shown to be either utilized or synthesized by Gram-positive bacteria, such as lactic acid bacteria. Glutathione plays an important role in countering environmental stress, such as oxidative stress. In this study, cellular activity regarding glutathione in Lactobacillus fermentum CECT 5716 is characterized. We demonstrate that L. fermentum CECT 5716 has a better survival rate in the presence of glutathione under both oxidative and metal stress. As L. fermentum CECT 5716 does not possess the ability to synthesize glutathione under the conditions tested, it shows the ability to uptake both reduced and oxidized glutathione from the environment, regenerate reduced glutathione from oxidized glutathione, and perform secretion of glutathione to the environment.


Assuntos
Glutationa/metabolismo , Lactobacillus fermentum/metabolismo , Antioxidantes/metabolismo , Cobre/metabolismo , Dissulfeto de Glutationa/metabolismo , Lactobacillus fermentum/crescimento & desenvolvimento , Estresse Oxidativo
18.
Biochem J ; 475(23): 3725-3743, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401685

RESUMO

Fast and uniform germination is key to agricultural production and can be achieved by seed 'priming' techniques. Here, we characterised the responses of bread wheat (Triticum aestivum L.) seeds to a hot steam treatment ('BioFlash'), which accelerated water uptake, resulting in faster germination and seedling growth, typical traits of primed seed. Before the completion of germination, metabolite profiling of seeds revealed advanced accumulation of several amino acids (especially cysteine and serine), sugars (ribose, glucose), and organic acids (glycerate, succinate) in hot steam-treated seeds, whereas sugar alcohols (e.g. arabitol, mannitol) and trehalose decreased in all seeds. Tocochromanols (the 'vitamin E family') rose independently of the hot steam treatment. We further assessed shifts in the half-cell reduction potentials of low-molecular-weight (LMW) thiol-disulfide redox couples [i.e. glutathione disulfide (GSSG)/glutathione (GSH) and cystine/cysteine], alongside the activities of the reactive oxygen species (ROS)-processing enzyme superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase. Upon the first 4 h of imbibition, a rapid conversion of LMW disulfides to thiols occurred. Completion of germination was associated with a re-oxidation of the LMW thiol-disulfide cellular redox environment, before more reducing conditions were re-established during seedling growth, accompanied by an increase in all ROS-processing enzyme activities. Furthermore, changes in the thiol-disulfide cellular redox state were associated to specific stages of wheat seed germination. In conclusion, the priming effect of the hot steam treatment advanced the onset of seed metabolism, including redox shifts associated with germination and seedling growth.


Assuntos
Temperatura Alta , Plântula/metabolismo , Sementes/metabolismo , Vapor , Triticum/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Germinação , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Sementes/crescimento & desenvolvimento , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Triticum/crescimento & desenvolvimento
19.
Food Funct ; 9(11): 5891-5902, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30375606

RESUMO

Acetaminophen (APAP) is commonly used to relieve pain and fever in a clinical setting, but its excessive use can lead to serious hepatotoxicity. Our previous study demonstrated that polydatin (PD) can effectively attenuate d-galactose- and alcohol-induced hepatotoxicity, however, its effect on APAP-induced hepatotoxicity is still unknown. In this study, we explore the protective effect and potential mechanism of PD against APAP-induced hepatotoxicity in mice. The results indicate that PD effectively improves the survival of mice with APAP-induced hepatotoxicity, significantly alleviating histopathologic alterations in the liver, and decreasing the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). PD significantly and dose-dependently reduces oxidative stress by lowering the content of oxidized glutathione (GSSG), reactive oxygen species (ROS), nitric oxide (NO) and malonaldehyde (MDA), while enhancing the hepatic activities of glutathione (GSH), glutathione peroxidase (GSH-Px) and the GSH/GSSG ratio. Meanwhile, PD also substantially inhibits the levels and mRNA expressions of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (NOX2). Additionally, PD markedly arrests apoptosis by assuaging TUNEL-positive hepatocytes and the apoptotic index, decreasing the levels and expression of cytochrome c (CytC), cleaved-caspase-9, apoptotic protease activating factor 1 (Apaf-1), cleaved-caspase-3, and Bax and increasing the level and expression of Bcl-2. Overall, PD pretreatment shows a potent protective effect against APAP-induced hepatotoxicity by relieving oxidative stress and inhibiting apoptosis.


Assuntos
Acetaminofen/toxicidade , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Glucosídeos/farmacologia , Substâncias Protetoras/farmacologia , Estilbenos/farmacologia , Alanina Transaminase/sangue , Animais , Antioxidantes/metabolismo , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aspartato Aminotransferases/sangue , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/sangue , Glutationa Peroxidase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Camundongos , Camundongos Endogâmicos ICR , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
20.
Sci Rep ; 8(1): 15213, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30315269

RESUMO

As the first fungal quorum sensing molecule, farnesol-induced morphological transition is usually studied in dimorphic fungi, but in basidiomycetes the morphological changes regulated by farnesol are rarely investigated. In this study, we found that farnesol made the basidiomycete Coriolus versicolor develop into a hyperbranched morphology with short hyphae and bulbous tips. Farnesol treatment resulted in a significant increase of intracellular oxidative stress level, which influenced the expression of several morphogenesis-related genes, and thereby led to the morphological changes. High oxidative stress level significantly stimulated the expression of laccase genes for improving intracellular laccase biosynthesis. The resulted hyperbranched morphology further accelerated the secretion of intracellular laccase into culture medium. As a result, extracellular laccase production reached a maximum of 2189.2 ± 54.7 U/L in farnesol-induced cultures, which was 6.8-fold greater than that of control cultures. SDS-PAGE and native-PAGE showed that farnesol increased laccase production by promoting the biosynthesis of three laccase isoforms. Together these results provide new opportunities in not only understanding the farnesol-regulated mycelial morphology in basidiomycetes, but also developing novel strategies for enhancing the production of secreted enzymes of biotechnological interest.


Assuntos
Agaricales/fisiologia , Farneseno Álcool/farmacologia , Hifas/fisiologia , Agaricales/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Dissulfeto de Glutationa/metabolismo , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Lacase/metabolismo , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
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