RESUMEN
Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.
Asunto(s)
Glutatión , Oxidación-Reducción , o-Ftalaldehído , o-Ftalaldehído/química , Glutatión/análisis , Glutatión/química , Glutatión/metabolismo , Humanos , Animales , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/química , Fosfinas/químicaRESUMEN
BACKGROUND: Periodontal diseases are the most frequently diagnosed problem in cats. It has been well-established that periodontal diseases could not only cause various oral health issues but could also contribute to systemic diseases. Oxidative stress is a possible link between systemic diseases and periodontitis. Our study aimed to illustrate the influence of periodontitis on oxidative stress development in cats. Furthermore, the changes in the bacterial flora of the gums were investigated. METHODS: Based on the clinical and laboratory examinations, fifty cats were divided into two groups normal (n = 25) and moderate to advanced periodontitis (n = 25). Serum total antioxidant capacity (TAC), total oxidant status (TOS), reduced (GSH) and oxidized glutathione (GSSG) were measured. In addition, samples were taken from the subgingival plaques of all cats for bacterial culture. RESULTS: Serum TOS, GSSG, GSSG to GSH ratio, and oxidative stress index (OSI), calculated as the ratio of TOS to TAC in cats with periodontal disease were significantly higher, and TAC was significantly lower (p < 0.05) compared with controls. The results of bacterial culture indicated that the number of isolated bacterial colonies is higher in patients than in the control group. Additionally, the analysis of these data showed a positive association between periodontal index and oxidative stress. CONCLUSIONS: Our results revealed that periodontitis in cats is related to a main oxidative stress. Furthermore, oxidant factors such as TOS and OSI, compared to antioxidant factors, may better indicate the presence of oxidative stress conditions in patients with periodontitis.
Asunto(s)
Antioxidantes , Enfermedades de los Gatos , Glutatión , Estrés Oxidativo , Periodontitis , Animales , Gatos , Enfermedades de los Gatos/microbiología , Enfermedades de los Gatos/sangre , Enfermedades de los Gatos/metabolismo , Estudios de Casos y Controles , Periodontitis/veterinaria , Periodontitis/microbiología , Femenino , Masculino , Antioxidantes/metabolismo , Glutatión/sangre , Glutatión/metabolismo , Disulfuro de Glutatión/sangre , Disulfuro de Glutatión/metabolismo , Oxidantes/metabolismo , Oxidantes/sangreRESUMEN
OBJECTIVE: The present systematic review explored the involvement of enzymatic and nonenzymatic antioxidants in periodontitis, drawing from established literature. MATERIALS AND METHODS: The research approach encompassed an extensive electronic search from 2000 to 2023 across databases such as PubMed, Science Direct, and Wiley Online Library and cross-referencing using specific keywords. RESULTS: The initial literature exploration generated a total of 766 articles. After thoroughly examining the abstracts, 693 articles were excluded from consideration due to duplication and lack of relevance to the central research inquiry. Following that, 73 articles were left for in-depth evaluation. Following a qualitative assessment, 35 studies that satisfied the inclusion criteria were chosen, while 38 were removed for not meeting the necessary standards. Within this selection, a meta-analysis was conducted on 11 articles that provided consistent data for quantitative synthesis. Specifically, the analysis of glutathione (GSH) levels in serum samples revealed a standardized mean difference (SMD) of -5.552 µg/mL (CI 95%: -9.078 to -2.026; P-0.002). In contrast, the analysis of glutathione peroxidase (GPx) enzymes in gingival crevicular fluid (GCF) samples displayed an overall SMD of 2.918 ng/µL (CI 95%: 0.372-5.465; P-0.025), while salivary samples exhibited an overall SMD value of 0.709 U/l (95% CI: -1.907-3.325; P-0.596) which is of insignificant. CONCLUSION: The systematic review findings suggest a notable decrease in antioxidant enzymes across various systemic biological samples among patients with periodontitis, contrasting with the results from gingival tissue samples meta-analysis of GPx enzyme.
Asunto(s)
Glutatión Peroxidasa , Glutatión Reductasa , Glutatión , Periodontitis , Humanos , Periodontitis/diagnóstico , Periodontitis/sangre , Periodontitis/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión/metabolismo , Glutatión Reductasa/metabolismo , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/sangre , Líquido del Surco Gingival/química , Antioxidantes/metabolismo , Antioxidantes/análisisRESUMEN
The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.
Asunto(s)
Arabidopsis , Citosol , Glutatión , Peróxido de Hidrógeno , Oxidación-Reducción , Peróxido de Hidrógeno/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Glutatión/metabolismo , Citosol/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Peroxirredoxinas/metabolismo , Peroxirredoxinas/genética , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Disulfuro de Glutatión/metabolismo , NADP/metabolismoRESUMEN
Several studies have shown that berberine (BBR) is effective in protecting against myocardial ischemiareperfusion injury (MI/RI). However, the precise molecular mechanism remains elusive. The present study observed the mechanism and the safeguarding effect of BBR against hypoxia/reoxygenation (H/R) myocardial injury in H9c2 cells. BBR pretreatment significantly improved the decrease of cell viability, P62 protein, Rho Family GTPase 3 (RhoE) protein, ubiquinone subunit B8 protein, ubiquinolcytochrome c reductase core protein U, the Bcl2associated X protein/Bcell lymphoma 2 ratio, glutathione (GSH) and the GSH/glutathione disulphide (GSSG) ratio induced by H/R, while reducing the increase in lactate dehydrogenase, microtubuleassociated protein 1 light 3 protein, caspase3 activity, reactive oxygen species, GSSG and malonaldehyde caused by H/R. Transmission electron microscopy and LysoTracker Red DND99 staining results showed that BBR pretreatment inhibited H/Rinduced excessive autophagy by mediating RhoE. BBR also inhibited mitochondrial permeability transition, maintained the stability of the mitochondrial membrane potential, reduced the apoptotic rate, and increased the level of caspase3. However, the protective effects of BBR were attenuated by pAD/RhoEsmall hairpin RNA, rapamycin (an autophagy activator) and compound C (an AMPactivated protein kinase inhibitor). These new findings suggested that BBR protects the myocardium from MI/RI by inhibiting excessive autophagy, maintaining mitochondrial function, improving the energy supply and redox homeostasis, and attenuating apoptosis through the RhoE/AMPactivated protein kinase pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Autofagia , Berberina , Daño por Reperfusión Miocárdica , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis , Berberina/farmacología , Caspasa 3/metabolismo , Disulfuro de Glutatión/metabolismo , Isquemia/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/etiología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Animales , RatasRESUMEN
OBJECTIVE: To investigate the effects and possible mechanisms of negative air ions(NAIs) on blood pressure, oxidative stress, and inflammatory status in spontaneous hypertension rats(SHR). METHODS: A total of 60 SHR(half male and half female) were randomly divided into one-month and three-month groups, 30 rats per groups, based on the duration of the intervention. Each group was further randomized into three groups based on the daily intervention time: SHR control group, 2 h NAIs-SHR group, and 6 h NAIs-SHR group, 10 rats per groups. In addition, 20 Wistar Kyoto(WKY)(half male and half female), were randomized into one-month WKY group and three-month WKY group, 10 rats per groups, based on the intervention time. The 2 h NAIs-SHR group and 6 h NAIs-SHR group were exposed to an environment with NAIs concentrations of 4.5×10~4-5×10~4 cm~3 per day for 2 h and 6 h. The WKY group and SHR group were exposed to normal air on a daily basis. Blood pressure of rats in each group was measured every three days, while weight was measured once a week. After sacrificing the rats in the first month and the third month of rearing, wet weight of the organs was weighed. The enzyme linked immunosorbent assay(ELISA) was used to detect 8-hydroxylated deoxyguanosine(8-OHdG), interleukin-6(IL-6), interleukin-8(IL-8), tumor necrosis factor-α(TNF-α), nitric oxide(NO) and endothelin-1(ET-1) levels. Reactive oxygen species(ROS) detection kit was used to detect ROS level. Malondialdehyde(MDA) and superoxide dismutase(SOD), glutathione(GSH) and glutathione disulfide(GSSG) were measured by colorimetric analysis. HE staining was conducted to observe the histopathological morphological changes of the thoracic aorta in each group, and Western blot was conducted to detect the thoracic aortap38 mitogen-activated protein kinase(p38 MAPK), extracellular signal-regulated kinases(ERK), c-Jun n-terminal kinase(JNK), c-fos proteins, c-jun proteins and their phosphorylated proteins level. RESULTS: The weight of WKY male mice in the same week age group was higher than that of SHR control group, and there was no significant difference in the weight between the other groups. The coefficient of heart in SHR control group(4.66±0.48) was higher than that in WKY group(3.73±0.15)(P<0.05), while there were no significant differences in the coefficients of brain, kidney, liver and spleen among the groups. Blood pressure in WKY group at the same age was lower than that in SHR group, and blood pressure in SHR control group at 2-5 and 8-11 weeks was higher than that in 2 h NAIs-SHR and 6 h NAIs-SHR groups(P<0.05). HE staining showed that the internal, middle and external membranes of thoracic aorta in 2 h NAIs-SHR group and 6 h NAIs-SHR group were improved to varying degrees compared with those in SHR control group, including disordered internal membrane structure, thickened middle membrane and broken external membrane. In terms of oxidative stress levels, compared with the SHR control group, the ROS(0.66%±0.17%, 0.49%±0.32%) and 8-OHdG((48.29±8.00) ng/mL, (33.13±14.67)ng/mL) levels were lower in the 6 h NAIs-SHR group(P<0.05), while the GSH/GSSG ratio was higher in the one-month 6 h NAIs-SHR group(10.08±4.93). Compared with the 2 h NAIs-SHR group, the ROS level(0.99%±0.19%) was lower in the 6 h NAIs-SHR group(P<0.05). In terms of inflammatory factor levels, compared with the SHR control group, the IL-8 levels((160.44±56.54) ng/L, (145.77±38.39) ng/L) were lower in the 6 h NAIs-SHR group(P<0.05), while the ET-1 level((249.55±16.98) ng/L) was higher in the one-month WKY group. There was no significant difference in NO levels among the groups. The relative expression of p-p38 protein in the thoracic aorta of rats in the one-month SHR control group was lower than that in the WKY group(P<0.05). The relative expression of p-p38 and p-c-fos proteins in the thoracic aorta of rats at three-months was higher in the SHR control group than in the 2 h NAIs-SHR and 6 h NAIs-SHR groups(P<0.05). CONCLUSION: The intervention of NAIs at a concentration of 4.5×10~4-5×10~4/cm~3 may regulate the partial oxidation and inflammatory state of SHR rats through the ROS/MAPK/AP1 signaling pathway, thereby reducing their blood pressure level.
Asunto(s)
Hipertensión , Interleucina-8 , Femenino , Ratas , Masculino , Ratones , Animales , Ratas Endogámicas SHR , Presión Sanguínea , Ratas Endogámicas WKY , Interleucina-8/metabolismo , Interleucina-8/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/farmacología , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/farmacología , Especies Reactivas de Oxígeno , Estrés Oxidativo , InflamaciónRESUMEN
The parenteral nutrition (PN) received by premature newborns is contaminated with peroxides that induce global DNA hypermethylation via oxidative stress. Exposure to peroxides could be an important factor in the induction of chronic diseases such as those observed in adults who were born preterm. As endogenous H2O2 is a major regulator of glucose-lipid metabolism, our hypothesis was that early exposure to PN induces permanent epigenetic changes in H2O2 metabolism. Three-day-old guinea pigs were fed orally (ON), PN or glutathione-enriched PN (PN+GSSG). GSSG promotes endogenous peroxide detoxification. After 4 days, half the animals were sacrificed, and the other half were fed ON until 16 weeks of age. The liver was harvested. DNA methylation and mRNA levels were determined for the SOD2, GPx1, GCLC, GSase, Nrf2 and Keap1 genes. PN induced GPx1 hypermethylation and decreased GPx1, GCLC and GSase mRNA. These findings were not observed in PN+GSSG. PN+GSSG induced Nrf2 hypomethylation and increased Nrf2 and SOD2 mRNA. These observations were independent of age. In conclusion, in neonatal guinea pigs, PN induces epigenetic changes, affecting the expression of H2O2 metabolism genes. These changes persist for at least 15 weeks after PN. This disruption may signify a permanent reduction in the capacity to detoxify peroxides.
Asunto(s)
Peróxido de Hidrógeno , Factor 2 Relacionado con NF-E2 , Animales , Cobayas , Peróxido de Hidrógeno/metabolismo , Disulfuro de Glutatión/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Animales Recién Nacidos , Nutrición Parenteral/efectos adversos , Glutatión/metabolismo , Peróxidos/metabolismo , Suplementos Dietéticos , Epigénesis Genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. METHODS: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. RESULTS: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. CONCLUSIONS: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. GENERAL SIGNIFICANCE: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.
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Citosol , Glutatión , Mitocondrias , Oxidación-Reducción , Pez Cebra , Animales , Pez Cebra/metabolismo , Pez Cebra/embriología , Glutatión/metabolismo , Mitocondrias/metabolismo , Citosol/metabolismo , Desarrollo Embrionario , Disulfuro de Glutatión/metabolismo , Embrión no Mamífero/metabolismoRESUMEN
BACKGROUND: Reduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies. METHODS: Here we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined. RESULTS: Twelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state. CONCLUSION: The described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.
Asunto(s)
Cromatografía Líquida con Espectrometría de Masas , Espectrometría de Masas en Tándem , Humanos , Etilmaleimida , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Especies Reactivas de Oxígeno , Reproducibilidad de los Resultados , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Oxidación-ReducciónRESUMEN
Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.
Asunto(s)
Glutarredoxinas , Glutatión , Proteínas Fluorescentes Verdes/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Oxidación-Reducción , Disulfuros/metabolismo , Catálisis , Disulfuro de Glutatión/metabolismoRESUMEN
BACKGROUND: Lipid peroxidation is a characteristic metabolic manifestation of diabetic retinopathy (DR) that causes inflammation, eventually leading to severe retinal vascular abnormalities. Selenium (Se) can directly or indirectly scavenge intracellular free radicals. Due to the narrow distinction between Se's effective and toxic doses, porous Se@SiO2 nanospheres have been developed to control the release of Se. They exert strong antioxidant and anti-inflammatory effects. METHODS: The effect of anti-lipid peroxidation and anti-inflammatory effects of porous Se@SiO2 nanospheres on diabetic mice were assessed by detecting the level of Malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), decreased reduced/oxidized glutathione (GSH/GSSG) ratio, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL) -1ß of the retina. To further examine the protective effect of porous Se@SiO2 nanospheres on the retinal vasculopathy of diabetic mice, retinal acellular capillary, the expression of tight junction proteins, and blood-retinal barrier destruction was observed. Finally, we validated the GPX4 as the target of porous Se@SiO2 nanospheres via decreased expression of GPX4 and detected the level of MDA, GSH/GSSG, TNF-α, IFN-γ, IL -1ß, wound healing assay, and tube formation in high glucose (HG) cultured Human retinal microvascular endothelial cells (HRMECs). RESULTS: The porous Se@SiO2 nanospheres reduced the level of MDA, TNF-α, IFN-γ, and IL -1ß, while increasing the level of GPX4 and GSH/GSSG in diabetic mice. Therefore, porous Se@SiO2 nanospheres reduced the number of retinal acellular capillaries, depletion of tight junction proteins, and vascular leakage in diabetic mice. Further, we identified GPX4 as the target of porous Se@SiO2 nanospheres as GPX4 inhibition reduced the repression effect of anti-lipid peroxidation, anti-inflammatory, and protective effects of endothelial cell dysfunction of porous Se@SiO2 nanospheres in HG-cultured HRMECs. CONCLUSION: Porous Se@SiO2 nanospheres effectively attenuated retinal vasculopathy in diabetic mice via inhibiting excess lipid peroxidation and inflammation by target GPX4, suggesting their potential as therapeutic agents for DR.
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Diabetes Mellitus Experimental , Retinopatía Diabética , Nanosferas , Selenio , Humanos , Ratones , Animales , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Selenio/metabolismo , Selenio/farmacología , Selenio/uso terapéutico , Dióxido de Silicio/metabolismo , Dióxido de Silicio/farmacología , Dióxido de Silicio/uso terapéutico , Diabetes Mellitus Experimental/metabolismo , Células Endoteliales/metabolismo , Peroxidación de Lípido , Porosidad , Factor de Necrosis Tumoral alfa/metabolismo , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/farmacología , Disulfuro de Glutatión/uso terapéutico , Inflamación/metabolismo , Antiinflamatorios/uso terapéutico , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Rapid restoration of perfusion in ischemic myocardium is the most direct and effective treatment for coronary heart disease but may cause myocardial ischemia/reperfusion injury (MIRI). Cinnamaldehyde (CA, C9H8O), a key component in the well-known Chinese medicine cinnamomum cassia, has cardioprotective effects against MIRI. This study aimed to observe the therapeutic effect of CA on MIRI and to elucidate its potential mechanism. H9C2 rat cardiomyocytes were pretreated with CA solution at 0, 10, and 100 µM, respectively and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Then the cell viability, the NF-κB and caspase3 gene levels, the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, superoxide dismutase (SOD) level, reactive oxygen species (ROS) generation, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) were detected. The severity of DNA damage was assessed by tail moment (TM) values using alkaline comet assay. Besides, the DNA damage-related proteins and the key proteins of the Nrf2 pathway were detected by western blot. CA treatment increased the cell viability, GHS/GSSG ratio, SOD level, PARP1, Nrf2, PPAR-γ, and HO-1 protein levels of H9C2 cardiomyocytes, while reducing NF-κB, caspase3, ROS level, 4-HNE and MDA content, γ-H2AX protein level, and TM values. Inhibition of the Nrf2 pathway reversed the effect of CA on cell viability and apoptosis of OGD/R induced H9C2 cardiomyocytes. Besides, 100 µM CA was more effective than 10 µM CA. In the OGD/R-induced H9C2 cardiomyocyte model, CA can protect cardiomyocytes from MIRI by attenuating lipid peroxidation and repairing DNA damage. The mechanism may be related to the activation of the Nrf2 pathway.
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Acroleína , Miocitos Cardíacos , Factor 2 Relacionado con NF-E2 , Oxígeno , Animales , Ratas , Acroleína/análogos & derivados , Acroleína/farmacología , Apoptosis , Daño del ADN , Glucosa/farmacología , Disulfuro de Glutatión/genética , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/farmacología , Peroxidación de Lípido , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
The parameters of oxidative damage of DNA, proteins, as well as the parameters of the thiol-disulfide system and C-reactive protein in adolescent girls and boys with exogenous constitutional obesity (ECO) were evaluated. In girls and boys with obesity, the levels of 8-hydroxy-2'-deoxyguanosine (marker of DNA destruction) were higher than in controls. Evaluation of the activity of the thiol-disulfide system revealed increased levels of oxidized glutathione (GSSG) and decreased levels of the reduced glutathione (GSH) and GSSG ratio (GSH/GSSG) in adolescents with ECO regardless of the sex in comparison with the control. C-reactive protein was also higher in the ECO groups regardless of the sex. The levels of glutathione peroxidase in obese boys were higher than in girls. In view of the revealed shifts, corrective measures with the prescription of drugs with antioxidant properties are recommended in adolescents with ECO to stabilize the indices.
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Proteína C-Reactiva , Estrés Oxidativo , Masculino , Adolescente , Femenino , Humanos , Disulfuro de Glutatión/metabolismo , Proteína C-Reactiva/metabolismo , Glutatión/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Compuestos de Sulfhidrilo , Obesidad , ADN/metabolismo , Disulfuros , Oxidación-ReducciónRESUMEN
2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals.
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Contaminantes Ambientales , Oryza , Antioxidantes/metabolismo , Plantones , Oryza/metabolismo , Pentanoles/metabolismo , Pentanoles/farmacología , 1-Butanol/metabolismo , 1-Butanol/farmacología , Contaminantes Ambientales/metabolismo , Disulfuro de Glutatión/metabolismo , Estrés Oxidativo , Glutatión/metabolismo , Raíces de Plantas/metabolismoRESUMEN
Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.
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Listeria monocytogenes , Animales , Cisteína/metabolismo , Disulfuro de Glutatión/genética , Disulfuro de Glutatión/metabolismo , Compuestos de Azufre/metabolismo , Glutatión , Azufre/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , MamíferosRESUMEN
ABSTRACT: Chronic stress induces a group of unrecognized cardiovascular impairments, including elevated hemodynamic variables and vascular dysfunction. Moreover, hydrogen sulfide (H 2 S), a gasotransmitter that regulates the cardiovascular system decreases under chronic stress. Thus, this study assessed the impact of sodium hydrosulfide (NaHS) (H 2 S donor) on chronic restraint stress (CRS)-induced cardiovascular changes. For that purpose, male Wistar rats were restrained for 2 hours a day in a transparent acrylic tube over 8 weeks. Then, body weight, relative adrenal gland weight, serum corticosterone, H 2 S-synthesizing enzymes, endothelial nitric oxide synthetize expression, reactive oxygen species levels, lipid peroxidation, and reduced glutathione-to-oxidized glutathione (GSH 2 :GSSG) ratio were determined in the thoracic aorta. The hemodynamic variables were measured in vivo by the plethysmograph method. The vascular function was evaluated in vitro as vasorelaxant responses induced by carbachol or sodium nitroprusside, and norepinephrine (NE)-mediated vasocontractile responses in the thoracic aorta. CRS increased (1) relative adrenal gland weight; (2) hemodynamic variables; (3) vasoconstrictor responses induced by NE, (4) reactive oxygen species levels, and (5) lipid peroxidation in the thoracic aorta. In addition, CRS decreased (1) body weight; (2) vasorelaxant responses induced by carbachol; (3) GSH content, and (4) GSH 2 :GSSG ratio. Notably, NaHS administration (5.6 mg/kg) restored hemodynamic variables and lipid peroxidation and attenuated the vasoconstrictor responses induced by NE in the thoracic aorta. In addition, NaHS treatment increased relative adrenal gland weight and the GSH 2 :GSSG ratio. Taken together, our results demonstrate that NaHS alleviates CRS-induced hypertension by reducing oxidative stress and restoring vascular function in the thoracic aorta.
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Sulfuro de Hidrógeno , Sulfuros , Ratas , Animales , Masculino , Especies Reactivas de Oxígeno/metabolismo , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/farmacología , Carbacol/farmacología , Ratas Wistar , Sulfuro de Hidrógeno/metabolismo , Estrés Oxidativo , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Peso CorporalRESUMEN
BACKGROUND: HucMSCs had shown promising efficacy in treating childhood diseases, but oxidative stress induced by the poor microenvironment at the site of damage resulted in low cell survival after transplantation, thus preventing the cells from maximizing therapeutic efficacy. Therefore, this study aimed to investigate the role and mechanism of keap1 in oxidative stress injury of human umbilical cord mesenchymal stem cells (hucMSCs), and to provide theoretical support for improving the efficacy of stem cell therapy. METHODS: The hucMSCs were treated with hypoxic low-sugar-free serum (GSDH) to mimic the damaged site microenvironment after implantation. Adenoviral overexpression of keap1 gene of hucMSCs was performed in vitro, and cell proliferation ability was detected by CCK8 assay, crystal violet staining assay, and cell cycle assay. Cellular redox level was assessed by Amplex Red, MDA, and GSH/GSSG kit. Mitochondrial morphology was evaluated by mitotracker Red staining. ATP production was estimated by ATP detection kit. The mRNA and protein expression levels were tested by western blotting and RT-qPCR. RESULTS: GSDH treatment substantially upregulated keap1 expression. Subsequently, we found that overexpression of keap1 notably inhibited cell proliferation and caused cells to stagnate in G1 phase. At the same time, overexpression of keap1 induced the production of large amounts of H2O2 and the accumulation of MDA, but suppressed the GSH/GSSG ratio and the expression of antioxidant proteins NQO1 and SOD1, which caused oxidative stress damage. Overexpression of keap1 induced cells to produce a large number of dysfunctional mitochondria resulting in reduced ATP production. Moreover, Overexpression of keap1 significantly decreased the IKKß protein level, while upregulating IkB mRNA levels and downregulating P50 mRNA levels. CONCLUSIONS: Overexpression of keap1 may induce oxidative stress injury in hucMSCs by down-regulating IKKß expression and inhibiting NF-κB pathway activation. This implies the importance of keap1 in hucMSCs and it may be a potential gene for genetic modification of hucMSCs.
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Peróxido de Hidrógeno , Células Madre Mesenquimatosas , Niño , Humanos , Adenosina Trifosfato , Disulfuro de Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Quinasa I-kappa B/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , ARN Mensajero/metabolismo , Cordón UmbilicalRESUMEN
Yellow fever (YF) presents a wide spectrum of severity, with clinical manifestations in humans ranging from febrile and self-limited to fatal cases. Although YF is an old disease for which an effective and safe vaccine exists, little is known about the viral- and host-specific mechanisms that contribute to liver pathology. Several studies have demonstrated that oxidative stress triggered by viral infections contributes to pathogenesis. We evaluated whether yellow fever virus (YFV), when infecting human hepatocytes cells, could trigger an imbalance in redox homeostasis, culminating in oxidative stress. YFV infection resulted in a significant increase in reactive oxygen species (ROS) levels from 2 to 4 days post infection (dpi). When measuring oxidative parameters at 4 dpi, YFV infection caused oxidative damage to lipids, proteins, and DNA, evidenced by an increase in lipid peroxidation/8-isoprostane, carbonyl protein, and 8-hydroxy-2'-deoxyguanosine, respectively. Furthermore, there was a significant reduction in the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), in addition to a reduction in the ratio of reduced to oxidized glutathione (GSH/GSSG), indicating a pro-oxidant environment. However, no changes were observed in the enzymatic activity of the enzyme catalase (CAT) or in the gene expression of SOD isoforms (1/2/3), CAT, or GPx. Therefore, our results show that YFV infection generates an imbalance in redox homeostasis, with the overproduction of ROS and depletion of antioxidant enzymes, which induces oxidative damage to cellular constituents. Moreover, as it has been demonstrated that oxidative stress is a conspicuous event in YFV infection, therapeutic strategies based on antioxidant biopharmaceuticals may be new targets for the treatment of YF.
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Antioxidantes , Fiebre Amarilla , Humanos , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Virus de la Fiebre Amarilla/metabolismo , Glutatión/metabolismo , Estrés Oxidativo , Oxidación-Reducción , Catalasa/genética , Catalasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Disulfuro de Glutatión/metabolismo , Hepatocitos/metabolismo , Peroxidación de Lípido , Glutatión Peroxidasa/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina/metabolismoRESUMEN
TMAO, a gut microbiota derived byproduct, has been associated with various cardiometabolic diseases by promoting oxidative stress and inflammation. The liver is the main organ for TMAO production and chronic exposure to high doses of TMAO could alter its function. In this study, we evaluated the effect of chronic exposure of high TMAO doses on liver oxidative stress, inflammation, and fibrosis. TMAO was administered daily via gastric gavage to laboratory rats for 3 months. Blood was drawn for the quantification of TMAO, and liver tissues were harvested for the assessment of oxidative stress (MDA, GSH, GSSG, GPx, CAT, and 8-oxo-dG) and inflammation by quantification of IL-1α, TNF-α, IL-10, TGF-ß, NOS and COX-2 expression. The evaluation of fibrosis was made by Western blot analysis of α-SMA and Collagen-3 protein expression. Histological investigation and immunohistochemical staining of iNOS were performed in order to assess the liver damage. After 3 months of TMAO exposure, TMAO serum levels enhanced in parallel with increases in MDA and GSSG levels in liver tissue and lower values of GSH and GSH/GSSG ratio as well as a decrease in GPx and CAT activities. Inflammation was also highlighted, with enhanced iNOS, COX-2, and IL-10 expression, without structural changes and without induction of liver fibrosis.
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Interleucina-10 , Hígado , Metilaminas , Ratas , Animales , Interleucina-10/metabolismo , Ciclooxigenasa 2/metabolismo , Disulfuro de Glutatión/metabolismo , Fibrosis , Inflamación/inducido químicamente , Inflamación/metabolismo , Estrés OxidativoRESUMEN
Dose-dependent heart failure is a major complication of the clinical use of doxorubicin (Dox), one of the most potent chemotherapeutic agents. Effective adjuvant therapy is required to prevent Dox-induced cardiotoxicity. Currently, plant-derived exosome-like nanovesicle (PELNV) has revealed their salubrious antioxidant and immunological regulating actions in various disease models. In this study, we isolated, purified and characterized Beta vulgaris-derived exosome-like nanovesicle (BELNV). Dox or normal saline was given to HL-1 cells (3 µM) and 8-week C57BL/6N mice (5 mg/kg bodyweight per week for 4 weeks) to establish the in vitro and in vivo model of Dox-induced cardiotoxicity. Administration of BELNV significantly alleviated chronic Dox-induced cardiotoxicity in terms of echocardiographic and histological results. A reduced malondialdehyde (MDA), increased ratio of glutathione (GSH) to oxidized glutathione (GSSG) and levels of system xc- and glutathione peroxidase 4 were observed, indicating that DOX-stimulated ferroptosis was reversed by BELNV. Besides, the safety of BELNV was also validated since no liver, spleen, and kidney toxicity induced by BELNV was observed. These findings provide evidence that BELNV may act as a novel therapeutic biomaterial for patients undergoing adverse effects of Dox, at least partly mediated by inhibiting Dox-induced ferroptosis.