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1.
Mol Med ; 28(1): 137, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36401163

RESUMO

BACKGROUND: Intestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism. METHODS: Cecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated. RESULTS: In vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6877002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1ß while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs. CONCLUSIONS: The inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.


Assuntos
Ligante de CD40 , Sepse , Humanos , Camundongos , Animais , Ocludina/metabolismo , Ligante de CD40/metabolismo , Células CACO-2 , S-Nitrosoglutationa/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , RNA Interferente Pequeno , Meios de Cultivo Condicionados/metabolismo , Ativação Plaquetária , Sepse/metabolismo , Neuroglia/metabolismo , Proteínas de Junções Íntimas/metabolismo
2.
Cell Death Dis ; 13(10): 859, 2022 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209194

RESUMO

Sustained oxidative stress in castration-resistant prostate cancer (CRPC) cells potentiates the overall tumor microenvironment (TME). Targeting the TME using colony-stimulating factor 1 receptor (CSF1R) inhibition is a promising therapy for CRPC. However, the therapeutic response to sustained CSF1R inhibition (CSF1Ri) is limited as a monotherapy. We hypothesized that one of the underlying causes for the reduced efficacy of CSF1Ri and increased oxidation in CRPC is the upregulation and uncoupling of endothelial nitric oxide synthase (NOS3). Here we show that in high-grade PCa human specimens, NOS3 abundance positively correlates with CSF1-CSF1R signaling and remains uncoupled. The uncoupling diminishes NOS3 generation of sufficient nitric oxide (NO) required for S-nitrosylation of CSF1R at specific cysteine sites (Cys 224, Cys 278, and Cys 830). Exogenous S-nitrosothiol administration (with S-nitrosoglutathione (GSNO)) induces S-nitrosylation of CSF1R and rescues the excess oxidation in tumor regions, in turn suppressing the tumor-promoting cytokines which are ineffectively suppressed by CSF1R blockade. Together these results suggest that NO administration could act as an effective combinatorial partner with CSF1R blockade against CRPC. In this context, we further show that exogenous NO treatment with GSNOR successfully augments the anti-tumor ability of CSF1Ri to effectively reduce the overall tumor burden, decreases the intratumoral percentage of anti-inflammatory macrophages, myeloid-derived progenitor cells and increases the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells, respectively. Together, these findings support the concept that the NO-CSF1Ri combination has the potential to act as a therapeutic agent that restores control over TME, which in turn could improve the outcomes of PCa patients.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Receptor de Fator Estimulador de Colônias de Macrófagos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Cisteína , Humanos , Fator Estimulador de Colônias de Macrófagos , Masculino , Óxido Nítrico , Óxido Nítrico Sintase Tipo III , S-Nitrosoglutationa , Microambiente Tumoral
3.
Planta ; 256(6): 101, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271196

RESUMO

MAIN CONCLUSION: NO enhances the resistance of tomato seedlings to salt stress through protein S-nitrosylation and transcriptional regulation, which involves the regulation of MAPK signaling and carbohydrate metabolism. Nitric oxide (NO) regulates various physiological and biochemical processes and stress responses in plants. We found that S-nitrosoglutathione (GSNO) treatment significantly promoted the growth of tomato seedling under NaCl stress, indicating that NO plays a positive role in salt stress resistance. Moreover, GSNO pretreatment resulted in an increase of endogenous NO level, S-nitrosothiol (SNO) content, S-nitrosoglutathione reductase (GSNOR) activity and GSNOR expression under salt stress, implicating that S-nitrosylation might be involved in NO-alleviating salt stress. To further explore whether S-nitrosylation is a key molecular mechanism of NO-alleviating salt stress, the biotin-switch technique and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were conducted. A total of 1054 putative S-nitrosylated proteins have been identified, which were mainly enriched in chloroplast, cytoplasm and mitochondrion. Among them, 15 and 22 S-nitrosylated proteins were involved in mitogen-activated protein kinase (MAPK) signal transduction and carbohydrate metabolism, respectively. In MAPK signaling, various S-nitrosylated proteins, SAM1, SAM3, SAM, PP2C and SnRK, were down-regulated and MAPK, MAPKK and MAPKK5 were up-regulated at the transcriptional level by GSNO treatment under salt stress compared to NaCl treatment alone. The GSNO pretreatment could reduce ethylene production and ABA content under NaCl stress. In addition, the activities of enzyme identified in carbohydrate metabolism, their expression at the transcriptional level and the metabolite content were up-regulated by GSNO supplication under salt stress, resulting in the activation of glycolysis and tricarboxylic acid cycle (TCA) cycles. Thus, these results demonstrated that NO might beneficially regulate MAPK signaling at transcriptional levels and activate carbohydrate metabolism at the post-translational and transcriptional level, protecting seedlings from energy deficiency and salinity, thereby alleviating salt stress-induced damage in tomato seedlings. It provides initial insights into the regulatory mechanisms of NO in response to salt stress.


Assuntos
S-Nitrosotióis , Plântula/genética , Plântula/metabolismo , Óxido Nítrico/metabolismo , /metabolismo , S-Nitrosoglutationa/farmacologia , S-Nitrosoglutationa/metabolismo , Cromatografia Líquida , Biotina/metabolismo , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Aldeído Oxirredutases/metabolismo , Espectrometria de Massas em Tandem , S-Nitrosotióis/metabolismo , Estresse Salino , Processamento de Proteína Pós-Traducional , Etilenos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo
4.
Mol Med ; 28(1): 127, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36303116

RESUMO

BACKGROUND: Intestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism. METHODS: Cecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated. RESULTS: In vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6,877,002, a blocker of CD40L-CD40-TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1ß while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs. CONCLUSIONS: The inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.


Assuntos
Ligante de CD40 , Sepse , Humanos , Camundongos , Animais , Ligante de CD40/metabolismo , Células CACO-2 , Ocludina/metabolismo , S-Nitrosoglutationa/metabolismo , RNA Interferente Pequeno , Fator 6 Associado a Receptor de TNF/metabolismo , Meios de Cultivo Condicionados , Ativação Plaquetária , Sepse/metabolismo , Neuroglia/metabolismo , Proteínas de Junções Íntimas/metabolismo
5.
Int J Mol Sci ; 23(18)2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36142872

RESUMO

Brassinosteroids (BRs), a novel plant hormone, are widely involved in plant growth and stress response processes. Nitric oxide (NO), as an important gas signaling molecule, can regulate target protein activity, subcellular localization and function in response to various stresses through post-translational S-nitrosylation modifications. However, the relationship between BR and NO in alleviating low-temperature stress of mini Chinese cabbage remains unclear. The hydroponic experiment combined with the pharmacological and molecular biological method was conducted to study the alleviating mechanism of BR at low temperature in mini Chinese cabbage. The results showed that low temperature inhibited the growth of mini Chinese cabbage seedlings, as evidenced by dwarf plants and yellow leaves. Treatment with 0.05 mg/L BR and 50 µM NO donor S-nitrosoglutathione (GSNO) significantly increased the leaf area, stem diameter, chlorophyll content, dry and fresh weight and proline content. Meanwhile, the malondialdehyde (MDA) content in 0.05 mg/L BR- and 50 µM GSNO-treated leaves were significantly lower than those in other treated leaves under low-temperature conditions. In addition, BR and GSNO applications induced an increase in NO and S-nitrosothiol (SNO) levels in vivo under low-temperature stress. Similarly, spraying BR after the elimination of NO also increased the level of S-nitrosylation in vivo, while spraying GSNO after inhibiting BR biosynthesis decreased the level of NO and SNO in vivo. In contrast, the S-nitrosoglutathione reductase (BrGSNOR) relative expression level and GSNOR enzyme activity were downregulated and inhibited by BR treatment, GSNO treatment and spraying BR after NO clearance, while the relative expression level of BrGSNOR was upregulated and GSNOR enzyme activity was also increased when spraying GSNO after inhibiting BR synthesis. Meanwhile, the biotin switch assay showed that exogenous BR increased the level of total nitrosylated protein in vivo under low-temperature stress. These results suggested that BR might act as an upstream signal of NO, induced the increase of NO content in vivo and then induced the protein S-nitrosylation modification to alleviate the damage of mini Chinese cabbage seedlings under low-temperature stress.


Assuntos
Brassica rapa , Brassica , S-Nitrosotióis , Biotina/metabolismo , Brassica/metabolismo , Brassica rapa/metabolismo , Brassinosteroides/metabolismo , China , Clorofila/metabolismo , Malondialdeído/metabolismo , Óxido Nítrico/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Prolina/metabolismo , S-Nitrosoglutationa/metabolismo , S-Nitrosotióis/metabolismo , Plântula/metabolismo , Temperatura
6.
J Colloid Interface Sci ; 628(Pt B): 911-921, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36030716

RESUMO

HYPOTHESIS: Alginate is widely used in biomedical applications due to its high biocompatibility as well as structural and mechanical similarities to human tissue. Further, simple ionic crosslinking of alginate allows for the formation of alginate beads capable of drug delivery. S-nitrosoglutathione is a water-soluble molecule that releases nitric oxide in physiological conditions, where it acts as a potent antimicrobial gas, among other functions. As macrophages and endothelial cells endogenously produce nitric oxide, incorporating nitric oxide donors into polymers and hydrogels introduces a biomimetic approach to mitigate clinical infections, including those caused by antibiotic-resistant microorganisms. The incorporation of S-nitrosoglutathione into macro-scale spherical alginate beads is reported for the first time and shows exciting potential for biomedical applications. EXPERIMENTS: Herein, nitric oxide-releasing crosslinked alginate beads were fabricated and characterized for surface and cross-sectional morphology, water uptake, size distribution, and storage stability. In addition, the NO release was quantified by chemiluminescence and its biological effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were investigated. The biocompatibility of the alginate beads was tested against 3T3 mouse fibroblast cells. FINDINGS: Overall, nitric oxide-releasing alginate beads demonstrate biologically relevant activities without eliciting a cytotoxic response, revealing their potential use as an antimicrobial material with multiple mechanisms of bacterial killing.


Assuntos
Anti-Infecciosos , Gasotransmissores , Camundongos , Animais , Humanos , Alginatos/química , Doadores de Óxido Nítrico/química , Óxido Nítrico/metabolismo , S-Nitrosoglutationa , Biomimética , Células Endoteliais , Estudos Transversais , Antibacterianos/farmacologia , Antibacterianos/química , Hidrogéis/química , Polímeros/química , Água
7.
Redox Biol ; 56: 102439, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35995009

RESUMO

Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase. In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•-) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40-70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•- dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP+-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•- indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•- or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•- appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•- could represent a mechanism to regain reducing equivalents in situations of excess O2•- formation, e.g. in the reperfusion phase after ischemia.


Assuntos
Compostos de Sulfidrila , Superóxidos , Ditiotreitol , Glutationa/metabolismo , Isocitrato Desidrogenase , NADP , Óxido Nítrico , Nitrosação , Ácido Peroxinitroso , S-Nitrosoglutationa/metabolismo , Tiorredoxinas
8.
Chemistry ; 28(58): e202201494, 2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-35851725

RESUMO

S-Nitrosylation has been found to play an important role in regulating mitochondrial function. However, probes for detection of protein S-nitrosylation in mitochondria remain unexplored. Herein, a novel 4-(pyridin-4-yl)vinyl-substituted indole was designed, exhibiting a long-wavelength emission and a high fluorescent quantum yield. Functionalization of the 7-position of the indole ring with an arylphosphine ester resulted with probes with efficient mitochondria-targeting ability. Furthermore, the indole-arylphosphine displayed a significant fluorescence enhancement upon exposure to S-nitrosoglutathione (GSNO) at low micromolar concentrations in A431 cells. Taken together, this study provides a new indole-based fluorescent probe with a unique long-wavelength emission for direct detection of S-nitrosylation in mitochondria, which may represent a powerful tool for understanding the critical roles of S-nitrosylation within mitochondria of living organisms.


Assuntos
Corantes Fluorescentes , S-Nitrosoglutationa , Corantes Fluorescentes/metabolismo , S-Nitrosoglutationa/metabolismo , Proteína S/metabolismo , Mitocôndrias/metabolismo , Indóis/metabolismo , Ésteres/metabolismo
9.
Talanta ; 250: 123736, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-35858531

RESUMO

The identification and quantitation of S-nitrosothiols (RSNO) has aroused enormous levels of attention, due to RSNO have many roles in vivo. Here, we synthesized the nanocomposites of ultrafine Cu2O/layered double hydroxide (u-Cu2O/LDH) by the in situ topotactic reduction of a Cu2+-containing LDH with ascorbic acid under gentle conditions and applied these u-Cu2O/LDH to detect and monitor RSNO. Electrochemical signals of u-Cu2O/LDH exhibited a wide N-acetyl-S-nitrosopenicillamine detection range from 5.0 nM-4.0 µM and 4.0 µM-400 µM, with a low detection limit of 1.58 nM. The sensor also exhibited good performance for other RSNO, such as S-nitrosoglutathione, S-nitrosocysteine, and S-nitrosohomocysteine with corresponding limits of detection at 1.94 nM, 1.23 nM and 1.62 nM, respectively. The high levels of selectivity and sensitivity to RSNO in complex biological environments can be attributed to the abundance of exposed active sites, and the underlying support structure. In addition, u-Cu2O/LDH also exhibited dynamic nitric oxide (NO) monitoring ability from living cells. Collectively, these results reveal that u-Cu2O/LDH exhibit a remarkable ability to quantify RSNO levels in complex samples, and could therefore provide new tools for exploring ultrafine nanomaterials as a potential biosensor to investigate biological events.


Assuntos
Nanocompostos , Óxido Nítrico , Ácido Ascórbico , Hidróxidos/química , Óxido Nítrico/química , S-Nitroso-N-Acetilpenicilamina , S-Nitrosoglutationa
10.
Redox Biol ; 54: 102387, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35793584

RESUMO

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , S-Nitrosoglutationa , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Fotossíntese , S-Nitrosoglutationa/metabolismo , Compostos de Sulfidrila/metabolismo
11.
Bull Exp Biol Med ; 173(1): 28-32, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35624350

RESUMO

We studied the effect of nitric oxide (NO) on the functioning of P-glycoprotein transporter (Pgp) in Caco-2 cells. NO donor S-nitrosoglutathione (GSNO) was used in concentrations of 1, 10, 50, 100, and 500 µM; the duration of exposure was 24 h. The content of Pgp was analyzed by the Western blotting, activity of the transport protein was analyzed by the transport of its substrate fexofenadine. It was shown that GSNO in concentrations of 10 and 50 µM increased the content and activity of Pgp. Increasing the GSNO concentration to 500 µM led to the development of nitrosative stress and a decrease in the content and activity of the transporter protein.


Assuntos
Óxido Nítrico , S-Nitrosoglutationa , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Células CACO-2 , Humanos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , S-Nitrosoglutationa/farmacologia
12.
Biochemistry (Mosc) ; 87(4): 366-379, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35527375

RESUMO

Mechanisms of regulation of the P-glycoprotein (Pgp) transporter under the action of nitric oxide (NO) were studied in Caco-2 cells. S-Nitrosoglutathione (GSNO) was used as a NO donor, which was added to the cells at concentrations 1, 10, 50, 100, and 500 µM and incubated for 3, 24, or 72 h. The amount of Pgp was analyzed using Western blotting, activity was determined by monitoring transport of its substrate, fexofenadine. The study showed that a short-term exposure to GSNO for 3 h at 500 µM concentration caused increase in the concentration of peroxynitrite in Caco-2 cells, which reduced the activity, but not the amount of Pgp. Increase in the duration of exposure to 24 h increased the amount and activity of Pgp at GSNO concentrations of 10 and 50 µM, increased the amount without increasing activity at 100 µM concentration, and decreased the amount of the transporter protein at 500 µM. Duration of exposure to GSNO of 72 h at concentration of 10 µM resulted in the increase of the amount and activity of Pgp, while at concentration of 100 and 500 µM it decreased the amount of the transport protein. At the same time, it was shown using specific inhibitors that the increase in the amount of Pgp under the influence of low concentrations of GSNO was realized through the NO-cGMP signaling pathway, and the effect of the higher concentration of GSNO and the respective development of nitrosative stress was realized through Nrf2 and the constitutive androstane receptor.


Assuntos
Óxido Nítrico , S-Nitrosoglutationa , Subfamília B de Transportador de Cassetes de Ligação de ATP , Células CACO-2 , Humanos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , S-Nitrosoglutationa/metabolismo , S-Nitrosoglutationa/farmacologia
13.
Nitric Oxide ; 122-123: 35-44, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35257853

RESUMO

The present study evaluated the protective role of S-nitrosoglutathione (GSNO) in preventing hyperglycemia-induced nitro-oxidative stress and alterations in monoaminergic system associated with neurobehavioral deficits in mice. Mice were subjected to diabetes by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days, whereas GSNO (100 µg/kg body weight) was administered daily via oral route for 8 weeks. Diabetic mice showed deficits in neurobehavioral functions associated with memory, learning, anxiety and motor coordination. These neurobehavioral deficits observed in diabetic mice may be attributed to decrease in norepinephrine (NE), dopamine (DA), serotonin (5-HT) and increased monoamine oxidase (MAO) activity in cortex and hippocampus. Further, a significant increase in reactive oxygen species (ROS), protein carbonyls, nitrotyrosine (NT) and lipid peroxidation were observed in brain regions of diabetic animals suggesting increased nitro-oxidative stress. Hyperglycemia induced nitro-oxidative stress appears to involve reduction in redox ratio (GSH/GSSG) and enzymatic antioxidants; catalase (CAT) and superoxide dismutase (SOD) in cortex and hippocampus. However, GSNO supplementation was able to ameliorate alterations in monoaminergic system and nitro-oxidative stress in the brain regions thereby restoring neurobehavioural functions. These findings suggest GSNO as potential therapeutic molecule to prevent diabetic encephalopathy.


Assuntos
Diabetes Mellitus Experimental , Hiperglicemia , Animais , Antioxidantes/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Hiperglicemia/induzido quimicamente , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Peroxidação de Lipídeos , Camundongos , Estresse Oxidativo , S-Nitrosoglutationa/metabolismo , S-Nitrosoglutationa/farmacologia , Superóxido Dismutase/metabolismo
14.
Brain Res Bull ; 183: 184-200, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35304287

RESUMO

Subarachnoid hemorrhage (SAH) is a hemorrhagic stroke with a high mortality and disability rate. Nitric oxide (NO) can promote blood supply through vasodilation, leading to protein S-nitrosylation. However, the function of S-nitrosylation in neurons after SAH remains unclear. Excessive NO in the pathological state is converted into S-nitrosoglutathione (GSNO) and stored in cells, which leads to high S-nitrosylation of intracellular proteins and causes nitrosative stress. S-nitrosoglutathione reductase (GSNOR) promotes GSNO degradation and protects cells from excessive S-nitrosylation. We conducted an in vivo rat carotid puncture model and an in vitro neuron hemoglobin intervention. The results showed that SAH induction increased NO, GSNO, neuron protein S-nitrosylation, and neuronal apoptosis, while decreasing the level and activity of GSNOR. GSNOR overexpression by lentivirus decreased GSNO but had little effect on NO. GSNOR overexpression also improved short- and long-term neurobehavioral outcomes in rats and alleviated nitrosative stress. Furthermore, GSNOR reduced neuronal apoptosis and played a neuroprotective role by alleviating Drp1 S-nitrosylation, reducing mitochondrial division. Thus, the regulation of GSNOR in early brain injury and neuronal denitrosylation may play an important role in neuroprotection.


Assuntos
Oxirredutases , Hemorragia Subaracnóidea , Aldeído Oxirredutases/metabolismo , Animais , Apoptose , Óxido Nítrico/metabolismo , Ratos , S-Nitrosoglutationa/farmacologia
15.
ACS Appl Mater Interfaces ; 14(9): 11116-11123, 2022 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-35225600

RESUMO

Blood-contacting medical devices (BCMDs) are inevitably challenged by thrombi formation, leading to occlusion of flow and device failure. Ideal BCMDs seek to mimic the intrinsic antithrombotic properties of the human vasculature to locally prevent thrombotic complications, negating the need for systemic anticoagulation. An emerging category of BCMD technology utilizes nitric oxide (NO) as a hemocompatible agent, as the vasculature's endothelial layer naturally releases NO to inhibit platelet activation and consumption. In this paper, we report for the first time the novel impregnation of S-nitrosoglutathione (GSNO) into polymeric poly(vinyl chloride) (PVC) tubing via an optimized solvent-swelling method. Material testing revealed an optimized GSNO-PVC material that had adequate GSNO loading to achieve NO flux values within the physiological endothelial NO flux range for a 4 h period. Through in vitro hemocompatibility testing, the optimized material was deemed nonhemolytic (hemolytic index <2%) and capable of reducing platelet activation, suggesting that the material is suitable for contact with whole blood. Furthermore, an in vivo 4 h extracorporeal circulation (ECC) rabbit thrombogenicity model confirmed the blood biocompatibility of the optimized GSNO-PVC. Platelet count remained near 100% for the novel GSNO-impregnated PVC loops (1 h, 91.08 ± 6.27%; 2 h, 95.68 ± 0.61%; 3 h, 97.56 ± 8.59%; 4 h, 95.11 ± 8.30%). In contrast, unmodified PVC ECC loops occluded shortly after the 2 h time point and viable platelet counts quickly diminished (1 h, 85.67 ± 12.62%; 2 h, 54.46 ± 10.53%; 3 h, n/a; 4 h, n/a). The blood clots for GSNO-PVC loops (190.73 ± 72.46 mg) compared to those of unmodified PVC loops (866.50 ± 197.98 mg) were significantly smaller (p < 0.01). The results presented in this paper recommend further investigation in long-term animal models and suggest that GSNO-PVC has the potential to serve as an alternative to systemic anticoagulation in BCMD applications.


Assuntos
Polímeros/farmacologia , S-Nitrosoglutationa/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Circulação Extracorpórea/métodos , Hemólise/efeitos dos fármacos , Masculino , Teste de Materiais , Modelos Animais , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Polímeros/uso terapêutico , Cloreto de Polivinila/química , Coelhos , S-Nitrosoglutationa/química , S-Nitrosoglutationa/uso terapêutico , Propriedades de Superfície , Suínos , Trombose/prevenção & controle
16.
Small ; 18(11): e2105762, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35060323

RESUMO

Ceria nanoparticles (NPs) are widely reported to scavenge nitric oxide (NO) radicals. This study reveals evidence that an opposite effect of ceria NPs exists, that is, to induce NO generation. Herein, S-nitrosoglutathione (GSNO), one of the most biologically abundant NO donors, is catalytically decomposed by ceria NPs to produce NO. Ceria NPs maintain a high NO release recovery rate and retain their crystalline structure for at least 4 weeks. Importantly, the mechanism of this newly discovered NO generation capability of ceria NPs from GSNO is deciphered to be attributed to the oxidation of Ce3+ to Ce4+ on their surface, which is supported by X-ray photoelectron spectroscopy and density functional theory analysis. The prospective therapeutic effect of NO-generating ceria NPs is evaluated by the suppression of cancer cells, displaying a significant reduction of 93% in cell viability. Overall, this report is, to the authors' knowledge, the first study to identify the capability of ceria NPs to induce NO generation from GSNO, which overturns the conventional concept of them acting solely as a NO-scavenging agent. This study will deepen our knowledge about the therapeutic effects of ceria NPs and open a new route toward the NO-generating systems for biomedical applications.


Assuntos
Cério , Nanopartículas , Catálise , Cério/química , Nanopartículas/química , Óxido Nítrico , S-Nitrosoglutationa
17.
Ann Thorac Surg ; 114(4): 1468-1474, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-34416229

RESUMO

BACKGROUND: During hypoxia or acidosis, S-nitrosoglutathione (GSNO) has been shown to protect the cardiomyocyte from ischemia-reperfusion injury. In a randomized double-blinded control study of a porcine model of paediatric cardiopulmonary bypass (CPB), we aimed to evaluate the effects of 2 different doses (low and high) of GSNO. METHODS: Pigs weighing 15-20 kg were exposed to CPB with 1 hour of aortic cross-clamp. Prior to and during CPB, animals were randomized to receive low-dose (up to 20 nmol/kg/min) GSNO (n = 8), high-dose (up to 60 nmol/kg/min) GSNO (n = 6), or normal saline (n = 7). Standard cardiac intensive care management was continued for 4 hours post-bypass. RESULTS: There was a reduction in myocyte apoptosis after administration of GSNO (P = .04) with no difference between low- and high-dose GSNO. The low-dose GSNO group had lower pulmonary vascular resistance post-CPB (P = .007). Mitochondrial complex I activity normalized to citrate synthase activity was higher after GSNO compared with control (P = .02), with no difference between low- and high-dose GSNO. CONCLUSIONS: In a porcine model of CPB, intravenous administration of GSNO limits myocardial apoptosis through preservation of mitochondrial complex I activity, and improves pulmonary vascular resistance. There appears to be a dose-dependent effect to this protection.


Assuntos
S-Nitrosoglutationa , Solução Salina , Animais , Apoptose , Ponte Cardiopulmonar/efeitos adversos , Citrato (si)-Sintase , Humanos , S-Nitrosoglutationa/farmacologia , S-Nitrosoglutationa/uso terapêutico , Suínos
18.
Biomolecules ; 11(11)2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34827584

RESUMO

The COVID-19 pandemic has escalated the occurrence of hypoxia including thrombotic stroke worldwide, for which nitric oxide (NO) therapy seems very promising and translatable. Therefore, various modes/routes of NO-delivery are now being tested in different clinical trials for safer, faster, and more effective interventions against ischemic insults. Intravenous (IV) infusion of S-Nitrosoglutathione (GSNO), the major endogenous molecular pool of NO, has been reported to protect against mechanical cerebral ischemia-reperfusion (IR); however, it has been never tested in any kind of "clinically" relevant thromboembolic stroke models with or without comorbidities and in combination with the thrombolytic reperfusion therapy. Moreover, "IV-effects" of higher dose of GSNO following IR-injury have been contradicted to augment stroke injury. Herein, we tested the hypothesis that nebulization of low-dose GSNO will not alter blood pressure (BP) and will mitigate stroke injury in diabetic mice via enhanced cerebral blood flow (CBF) and brain tissue oxygenation (PbtO2). GSNO-nebulization (200 µg/kgbwt) did not alter BP, but augmented the restoration of CBF, improved behavioral outcomes and reduced stroke injury. Moreover, GSNO-nebulization increased early reoxygenation of brain tissue/PbtO2 as measured at 6.5 h post-stroke following thrombolytic reperfusion, and enervated unwanted effects of late thrombolysis in diabetic stroke. We conclude that the GSNO-nebulization is safe and effective for enhancing collateral microvascular perfusion in the early hours following stroke. Hence, nebulized-GSNO therapy has the potential to be developed and translated into an affordable field therapy against ischemic events including strokes, particularly in developing countries with limited healthcare infrastructure.


Assuntos
Complicações do Diabetes/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Hemorragia/prevenção & controle , S-Nitrosoglutationa/administração & dosagem , Acidente Vascular Cerebral/complicações , Terapia Trombolítica/efeitos adversos , Animais , Comportamento Animal , Pressão Sanguínea , Barreira Hematoencefálica , COVID-19/epidemiologia , Hemorragia/complicações , Hipóxia , Infusões Intravenosas , Fluxometria por Laser-Doppler , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Nebulizadores e Vaporizadores , Fármacos Neuroprotetores/farmacologia , Perfusão , Traumatismo por Reperfusão/tratamento farmacológico , Risco , Estresse Mecânico
19.
Biomed Khim ; 67(5): 394-401, 2021 Sep.
Artigo em Russo | MEDLINE | ID: mdl-34730552

RESUMO

Pregnan X receptor (PXR) is a nuclear receptor that plays an important role in the regulation of the expression of biotransformation and metabolic enzymes. The functioning and possible mechanisms of PXR regulation under conditions of nitrosative stress have not been studied, which served as the purpose of this study. The work was performed on Caco-2 cells. Nitrosative stress (NS) was modeled using S-nitrosoglutathione (GSNO) at concentrations of 1 µM, 10 µM, 50 µM, 100 µM, and 500 µM and incubation during of 3 h, 24 h, and 72 h. The amount of PXR was assessed byWestern blotting. Incubation of Caco-2 cells with all concentrations GSNO for 3 h led to a decrease in the amount of PXR. Incubation with GSNO (1-50 µM) for 24 h was accompanied by an increase in the amount of PXR, while at a concentration of 100 µM this indicator did not significantly differ from the control, at a concentration of 500 µM it was lower. Prolonged incubation (72 h) enhanced NS and led to a normalization (1 µM GSNO) or a decrease of the PXR level (10-500 µM GSNO). The induction of PXR by GSNO was mediated by the effect of the nitrosative stress product bityrosine on the transcription factor. It was shown that bityrosine at concentrations of 0,4 mM and 1 mM increased the amount of PXR.


Assuntos
Estresse Nitrosativo , S-Nitrosoglutationa , Células CACO-2 , Regulação da Expressão Gênica , Humanos , S-Nitrosoglutationa/metabolismo , Fatores de Transcrição
20.
Sci Rep ; 11(1): 20979, 2021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34697378

RESUMO

Among many other molecules, nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events. For a more comprehensive understanding of how this happens, we capacitated human spermatozoa from healthy men in the presence/absence of S-Nitrosoglutathione, a nitric oxide donor, two nitric oxide synthase inhibitors, NG-Nitro-L-arginine Methyl Ester Hydrochloride and Aminoguanidine Hemisulfate salt and, finally, with/without L-Arginine, the substrate for nitric oxide synthesis, and/or human follicular fluid. When analyzing the phosphorylation of protein kinase A substrates and tyrosine residues, we particularly observed how the inhibition of nitric oxide synthesis affects certain protein bands (~ 110, ~ 87, ~ 75 and ~ 62 kD) by lowering their phosphorylation degree, even when spermatozoa were incubated with L-Arginine and/or follicular fluid. Mass spectrometry analysis identified 29 proteins in these species, related to: spermatogenesis, binding to the zona pellucida, energy and metabolism, stress response, motility and structural organization, signaling and protein turnover. Significant changes in the phosphorylation degree of specific proteins could impair their biological activity and result in severe fertility-related phenotypes. These findings provide a deeper understanding of nitric oxide's role in the capacitation process, and consequently, future studies in infertile patients should determine how nitric oxide mediates phosphorylation events in the species here described.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Óxido Nítrico/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Espermatozoides/fisiologia , Arginina/farmacologia , Feminino , Técnicas de Inativação de Genes , Guanidinas/farmacologia , Voluntários Saudáveis , Humanos , Masculino , Espectrometria de Massas , NG-Nitroarginina Metil Éster/farmacologia , Fosforilação/efeitos dos fármacos , Proteômica/métodos , S-Nitrosoglutationa/farmacologia , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
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