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1.
Toxicon ; 188: 117-121, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33122156

RESUMO

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones (AQs). Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.


Assuntos
Emodina/toxicidade , Hepatócitos/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Senna (Planta) , Animais , Hepatócitos/fisiologia , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NADP , Intoxicação por Plantas , Quinona Redutases/antagonistas & inibidores , Ratos
2.
J Food Sci ; 85(6): 1752-1763, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32476138

RESUMO

Phenolics and carotenoids coexist in fruits and vegetables and could possess interaction effects after consumption. The present study aims to elucidate the possible mechanisms of the antioxidant interactions between anthocyanins and carotenoids using petunidin and lycopene as examples in hydrogen peroxide (H2 O2 )-induced heart myofibroblast cell (H9c2) line model. The results revealed that petunidin and lycopene showed antioxidant effects and petunidin in a larger proportion mixed with lycopene, for example, petunidin: lycopene = 9:1 significantly protected against the loss of the cell viability (8.98 ± 1.03%) and intracellular antioxidant enzyme activities of superoxide dismutase (SOD, 27.07 ± 3.51%), catalase (CAT, 29.51 ± 6.12%), and glutathione peroxidase (GSH-Px, 20.33 ± 2.65%). Moreover, the messenger RNA (mRNA) and protein expressions of NAD(P)H quinone reductase (NQO1) and heme oxygenase (HO-1) of the nuclear factor erythrocyte 2-related factor 2 (Nrf2) signaling pathway were significantly induced in petunidin, lycopene, and synergistic combinations, suggesting that the antioxidant action was through activating the Nrf2 antioxidant response pathway. This was further validated by Nrf2 siRNA, and the results that petunidin significantly induced more of NQO1 expression and lycopene more of HO-1 suggested that the synergism may be a result of concerted actions by the two compounds on these two different target genes of the Nrf2 pathway. The two compounds also significantly increased the phosphorylation of Akt in synergistic combinations. Findings of the present study demonstrated that petunidin and lycopene exerted synergistic antioxidant effects when petunidin in a larger proportion in the combinations and contribute to the prevention of cellular redox homeostasis, which might provide a theoretical basis for phenolics and carotenoids playing beneficial effects on the cardiovascular risk. PRACTICAL APPLICATION: In this study, we revealed that the combined treatments of petunidin and lycopen inhibited H2 O2 -induced oxidative damage in myocardial cells. Moreover, the treatments contributed to the Nrf2 pathway and the restoration of cellular redox homeostasis might provide a theoretical basis for phenolics and carotenoids playing beneficial effects on the cardiovascular risk.


Assuntos
Antocianinas/farmacologia , Antioxidantes/farmacologia , Peróxido de Hidrogênio/toxicidade , Licopeno/farmacologia , Miofibroblastos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carotenoides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa Peroxidase/metabolismo , Miofibroblastos/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo
3.
Life Sci ; 256: 117966, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32535079

RESUMO

AIM: The present study aims to investigate the protective effects of artemisinin (ATZ) on early renal damage in experimental diabetic rats and its probable mechanism. METHODS: Models of diabetic nephropathy (DN) rats was established utilizing streptozotocin (STZ)-injection intraperitoneally (55 mg/kg) method. All rats were subsequently divided into normal control group, model group and ATZ (25, 50, 75 mg/kg) group randomly. Biochemical parameters including body weight, kidney index, blood glucose, 24 h UAER, Scr, BUN, T-SOD, GSH-Px and MDA were comprehensively determined after 8-week consecutive administrations. HE and PAS stainings were performed to observe the histopathological alterations of kidney. Western blot was conducted to detect the expressions of TGF-ß1, Nrf2, HQ-1 and NQO1. KEY FINDINGS: ATZ at three concentrations in ATZ group significantly increased the body weight. Biochemical parameters altered significantly between model group and ATZ group. Moreover, ATZ inhibited TGF-ß1 protein expression and activated the Nrf2 signaling pathway. Pathological histology results revealed the alterations including mesangial cells proliferation, thickness of glomerular capillary basement membrane, extracellular matrix (ECM) and the 24 h UAER. Western blot analysis demonstrated the increase of antioxidant proteins HO-1 and NQO1 and Nrf2-related proteins. SIGNIFICANCE: ATZ could reduce early renal oxidative stress damage in DN rats by inhibiting TGF-ß1 protein expression in kidney tissues as well as activating the Nrf2 signaling pathway and enhancing the expression of antioxidant proteins, thereby exerting the protective effects on DN kidney. The current study is the first report of ATZ on attenuating effects on kidney of DN rats, which could lay solid theoretical foundations on clinical application of ATZ to treat DN.


Assuntos
Artemisininas/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Rim/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Artemisininas/farmacologia , Glicemia/metabolismo , Nitrogênio da Ureia Sanguínea , Peso Corporal/efeitos dos fármacos , Creatinina/sangue , Nefropatias Diabéticas/sangue , Heme Oxigenase-1/metabolismo , Rim/efeitos dos fármacos , Masculino , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 15(5): e0223344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365104

RESUMO

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role.


Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Colo/efeitos dos fármacos , Citoproteção , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Mutagênicos/toxicidade , Estilbenos/farmacologia , Linhagem Celular , Colo/citologia , Reparo do DNA/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Furanos/química , Humanos , Mitocôndrias/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estilbenos/química
5.
J Neurosci ; 40(23): 4609-4619, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32350039

RESUMO

Idebenone is a synthetic quinone that on reduction in cells can bypass mitochondrial Complex I defects by donating electrons to Complex III. The drug is used clinically to treat the Complex I disease Leber's hereditary optic neuropathy (LHON), but has been less successful in clinical trials for other neurodegenerative diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) appears to be the main intracellular enzyme catalyzing idebenone reduction. However, NQO1 is not universally expressed by cells of the brain. Using primary rat cortical cells pooled from both sexes, we tested the hypotheses that the level of endogenous NQO1 activity limits the ability of neurons, but not astrocytes, to use idebenone as an electron donor to support mitochondrial respiration. We then tested the prediction that NQO1 induction by pharmacological activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) enables idebenone to bypass Complex I in cells with poor NQO1 expression. We found that idebenone stimulated respiration by astrocytes but reduced the respiratory capacity of neurons. Importantly, idebenone supported mitochondrial oxygen consumption in the presence of a Complex I inhibitor in astrocytes but not neurons, and this ability was reversed by inhibiting NQO1. Conversely, recombinant NQO1 delivery to neurons prevented respiratory impairment and conferred Complex I bypass activity. Nrf2 activators failed to increase NQO1 in neurons, but carnosic acid induced NQO1 in COS-7 cells that expressed little endogenous enzyme. Carnosic acid-idebenone combination treatment promoted NQO1-dependent Complex I bypass activity in these cells. Thus, combination drug strategies targeting NQO1 may promote the repurposing of idebenone for additional disorders.SIGNIFICANCE STATEMENT Idebenone is used clinically to treat loss of visual acuity in Leber's hereditary optic neuropathy. Clinical trials for several additional diseases have failed. This study demonstrates a fundamental difference in the way idebenone affects mitochondrial respiration in cortical neurons compared with cortical astrocytes. Cortical neurons are unable to use idebenone as a direct mitochondrial electron donor due to NQO1 deficiency. Our results suggest that idebenone behaves as an NQO1-dependent prodrug, raising the possibility that lack of neuronal NQO1 activity has contributed to the limited efficacy of idebenone in neurodegenerative disease treatment. Combination therapy with drugs able to safely induce NQO1 in neurons, as well as other brain cell types, may be able to unlock the neuroprotective therapeutic potential of idebenone or related quinones.


Assuntos
Antioxidantes/farmacologia , Astrócitos/enzimologia , Respiração Celular/fisiologia , Mitocôndrias/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ubiquinona/análogos & derivados , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células COS , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ubiquinona/farmacologia
6.
PLoS One ; 15(4): e0231403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271832

RESUMO

A flavone luteolin has various health-promoting activities. Several studies reported that high dose of luteolin activates the Nrf2/ARE pathway in the liver. However, the effect of the low dose of luteolin that can be taken from a dietary meal on the Nrf2 activation remain unclear. It is expected that the flavonoid metabolism possesses a circadian rhythm, since nutritional metabolism processes daily cycle. In this study we investigated whether an administration affects the Nrf2 activation. ICR mice were orally administered 0.01-10 mg/kg body weight of luteolin once a day for 7 days at two time-points: at the start of active phase (ZT12) or at that of inactive phase (ZT0). Luteolin increased the nuclear translocation of Nrf2, resulting in the increases in its target gene products HO-1 and NQO1 at ZT12 but not at ZT0. The expression level of Nrf2 was lower at ZT12 than at ZT0 in the liver. We also found that the level of luteolin aglycon in the plasma is higher at ZT12 than at ZT0. These results suggest that the low dose of luteolin can activate Nrf2 pathway and the aglycon form of luteolin may mainly contribute to activate the Nrf2 pathway at ZT12 in the liver.


Assuntos
Fígado/efeitos dos fármacos , Luteolina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Relógios Biológicos/genética , Núcleo Celular/metabolismo , Heme Oxigenase-1/metabolismo , Fígado/metabolismo , Luteolina/sangue , Masculino , Camundongos , Camundongos Endogâmicos ICR , NAD(P)H Desidrogenase (Quinona)/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Curr Med Sci ; 40(1): 55-62, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166665

RESUMO

The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1 (NQO1) and γ-glutamylcysteine synthetase (γ-GCS) in brain tissues after intracerebral hemorrhage (ICH). The microglial cells obtained from newborn rats were cultured and then randomly divided into the normal control group (NC group), model control group (MC group), rosiglitazone (RSG) intervention group (RSG group), retinoic-acid intervention group (RSG+RA group), and sulforaphane group (RSG+SF group). The expression levels of NQO1, γ-GCS, and nuclear factor E2-related factor 2 (Nrf2) were measured by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. The results showed that the levels of NQO1, γ-GCS and Nrf2 were significantly increased in the MC group and the RSG group as compared with those in the NC group (P<0.01). They were found to be markedly decreased in the RSG+RA group and increased in the RSG+SF group when compared with those in the MC group or the RSG group (P<0.01). The RSG+SF group displayed the highest levels of NQO1, γ-GCS, and Nrf2 among the five groups. In conclusion, a medium dose of RSG increased the anti-oxidative ability of thrombin-activated microglia by increasing the expression of NQO1 and γ-GCS. The molecular mechanisms underlying the increase of NQO1 and γ-GCS in thrombin-activated microglia may be associated with the activation of Nrf2.


Assuntos
Hemorragia Cerebral/genética , Glutamato-Cisteína Ligase/genética , Microglia/citologia , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , PPAR gama/genética , Trombina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/metabolismo , Modelos Animais de Doenças , Feminino , Glutamato-Cisteína Ligase/metabolismo , Isotiocianatos/administração & dosagem , Isotiocianatos/farmacologia , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , PPAR gama/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Rosiglitazona/administração & dosagem , Rosiglitazona/farmacologia , Tretinoína/administração & dosagem , Tretinoína/farmacologia
8.
Int J Nanomedicine ; 15: 65-80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021165

RESUMO

Introduction: Poor cell uptake and incomplete intracellular drug release are the two major challenges for polymeric prodrug-based drug delivery systems (PPDDSs) in cancer treatment. Methods: Herein, a PPDDS with pH-induced surface charge-reversal and reactive oxygen species (ROS) amplification for ROS-triggered self-accelerating drug release was developed, which was formed by encapsulating a ROS generation agent (vitamin K3 (VK3)) in pH/ROS dual-sensitive polymetric prodrug (PEG-b-P(LL-g-TK-PTX)-(LL-g-DMA)) based micelle nanoparticles (denoted as PVD-NPs). Results: The surface charge of the PVD-NPs can change from negative to positive for enhanced cell uptake in response to tumor extracellular acidity pH. After internalization by cancer cells, PVD-NPs demonstrate dual drug release in response to intracellular ROS-rich conditions. In addition, the released VK3 can produce ROS under the catalysis by NAD(P)H:quinone oxidoreductase-1, which facilitates tumor-specific ROS amplification and drug release selectively in cancer cells to enhance chemotherapy. Conclusion: Both in vitro and in vivo experiments demonstrated that the PVD-NPs showed significant antitumor activity in human prostate cancer.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Pró-Fármacos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Micelas , NAD(P)H Desidrogenase (Quinona)/metabolismo , Células NIH 3T3 , Nanopartículas/administração & dosagem , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Polímeros/síntese química , Polímeros/química , Pró-Fármacos/farmacocinética , Vitamina K 3/administração & dosagem , Vitamina K 3/farmacocinética
9.
J Biol Chem ; 295(10): 3055-3063, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32001619

RESUMO

In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.


Assuntos
Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
10.
Oxid Med Cell Longev ; 2020: 5967434, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32082480

RESUMO

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3ß- (GSK-3ß-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3ß and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3ß at Ser9 without affecting the expression level of total GSK-3ß protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3ß siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3ß inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3ß, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3ß and further activating the Nrf2-ARE signaling pathway.


Assuntos
Elementos de Resposta Antioxidante/genética , Cromonas/farmacologia , Células Endoteliais/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Fator de Transcrição NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromonas/química , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/enzimologia , Endotélio/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Cinética , Cloreto de Lítio/farmacologia , Simulação de Acoplamento Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator de Transcrição NF-E2/genética , Estresse Oxidativo/genética , Fosforilação , RNA Interferente Pequeno , Transdução de Sinais/genética
11.
Oxid Med Cell Longev ; 2020: 2576823, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104530

RESUMO

UVA irradiation induced ROS-mediated photo damage to the human skin leading to coarseness, wrinkling, pigmentation, and cutaneous malignancies. We investigated the dermatoprotective efficacies of submicromolar concentrations of ergothioneine (EGT, 0.125-0.5 µM), which occurs naturally as a sulfur-containing amino acid, in the mechanisms in human skin fibroblast (HSF) cells. UVA-induced AP-1 (c-Fos and c-Jun) translocation was found to be inhibited by EGT treatments with the parallel inhibition of the collagenolytic matrix metalloproteinase- (MMP-) 1 activation and type I procollagen degradation. Moreover, EGT mitigated UVA-induced ROS generation. An increase in the amount of antioxidant genes (HO-1, NQO-1, and γ-GCLC) from EGT and were associated with upregulated Nrf2 expressions in a dose-dependent or time-dependent manner. We confirmed this from Nrf2 translocation and increased nuclear ARE promoter activity that underlie EGT dermatoprotective activities. Also, glutathione (GSH) levels (from γ-GCLC) were significantly increased. Moreover, we showed that mediated by ERK, JNK, and PKC, signaling cascades mediate Nrf2 translocation. We confirmed this phenomenon by the suppressed nuclear Nrf2 activation in cells that were treated with respective inhibitors (PD98059, SP600125, and GF109203X). However, antioxidant protein expressions were impaired in Nrf2 knockdown cells to confirm that ARE/Nrf2 pathways and the inhibition of AP-1 had significant roles in EGT-mediated protective effects. We can conclude that ergothioneine ameliorated UVA-induced skin aging and is a useful food supplement for skin care products.


Assuntos
Ergotioneína/farmacologia , Fibroblastos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Antioxidantes/farmacologia , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Pró-Colágeno/metabolismo , Proteína Quinase C/metabolismo , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiação , Transdução de Sinais/genética , Fator de Transcrição AP-1/antagonistas & inibidores , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos
12.
Life Sci ; 248: 117467, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105706

RESUMO

BACKGROUND: NQO1 protein acts as a cellular protective system, on account of its role as a quinone reductase and redox regulator. Nonetheless, new NQO1 roles are emerging-including its regulation of the cellular proliferation of many tumor cells-and this enzyme has been found to relate to the incidence of various diseases, including chronic myeloid leukemia. However, the mechanisms through which NQO1 influences leukemia progression remain unclear. MARTIAL AND METHODS: The current study looks to name NQO1 as a novel molecular target that modulates DNA synthesis and chronic myeloid leukemia growth. RESULTS AND CONCLUSION: Our results indicate that the frequency of the T allele of NQO1 polymorphism in chronic myeloid leukemia patients is higher than that among healthy East Asian individuals (0.492 vs. 0.419) and much higher than the average level of the general population (0.492 vs. 0.289) (1000 Genomes). Functionally, NQO1 knockdown increases the protein expression of the TOP2A and MCM complex, and consequently promotes DNA synthesis and K562 cell growth. NQO1 knockdown also promotes tumorigenesis in a xenograft model. NQO1 overexpression, on the other hand, was found to have the opposite effects. SIGNIFICANCE: Our results show that NQO1 downregulation promotes K562 cellular proliferation via the elevation of DNA synthesis.


Assuntos
DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucócitos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Adulto , Alelos , Animais , Grupo com Ancestrais do Continente Asiático , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/biossíntese , Feminino , Xenoenxertos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/etnologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Polimorfismo Genético , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
13.
Oxid Med Cell Longev ; 2020: 6325378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064027

RESUMO

In addition to the lung, the liver is considered another major target for paraquat (PQ) poisoning. Hydrogen sulfide (H2S) has been demonstrated to be effective in the inhibition of oxidative stress and inflammation. The aim of this study was to investigate the protective effect of exogenous H2S against PQ-induced acute liver injury. The acute liver injury model was established by a single intraperitoneal injection of PQ, evidenced by histological alteration and elevated serum aminotransferase levels. Different doses of NaHS were administered intraperitoneally one hour before exposure to PQ. Analysis of the data shows that exogenous H2S attenuated the PQ-induced liver injury and oxidative stress in a dose-dependent manner. H2S significantly suppressed reactive oxygen species (ROS) generation and the elevation of malondialdehyde content while it increased the ratio of GSH/GSSG and levels of antioxidant enzymes including SOD, GSH-Px, HO-1, and NQO-1. When hepatocytes were subjected to PQ-induced oxidative stress, H2S markedly enhanced nuclear translocation of Nrf2 via S-sulfhydration of Keap1 and resulted in the increase in IDH2 activity by regulating S-sulfhydration of SIRT3. In addition, H2S significantly suppressed NLRP3 inflammasome activation and subsequent IL-1ß excretion in PQ-induced acute liver injury. Moreover, H2S cannot reverse the decrease in SIRT3 and activation of the NLRP3 inflammasome caused by PQ in Nrf2-knockdown hepatocytes. In summary, H2S attenuated the PQ-induced acute liver injury by enhancing antioxidative capability, regulating mitochondrial function, and suppressing ROS-induced NLRP3 inflammasome activation. The antioxidative effect of H2S in PQ-induced liver injury can at least partly be attributed to the promotion of Nrf2-driven antioxidant enzymes via Keap1 S-sulfhydration and regulation of SIRT3/IDH2 signaling via Nrf2-dependent SIRT3 gene transcription as well as SIRT3 S-sulfhydration. Thus, H2S supplementation can form the basis for a promising novel therapeutic strategy for PQ-induced acute liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Inflamação/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Isocitrato Desidrogenase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Mitocôndrias/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/metabolismo , Sulfetos/administração & dosagem , Superóxido Dismutase-1/metabolismo , Transaminases/metabolismo
14.
Sci Rep ; 10(1): 2477, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051471

RESUMO

Oxidative stress has been associated with the etipathogenesis of Diabetic retinopathy (DR). Studies have shown that DJ-1 plays an important role in regulating the reactive oxygen species (ROS) production and resistance to oxidative stress-induced apoptosis. This study aimed to investigate whether DJ-1 upregulates oxidative stress and prevents damage to retinal capillary pericytes by increasing antioxidant capacity through the Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 is a redox-sensitive transcription factor that encode antioxidant enzymes and phase II metabolic enzymes, activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many tissues. Our results showed after DJ-1 overexpression, apoptosis of rat retinal pericytes (RRPs) decreased, the ratio of B-cell lymphoma-2 (Bcl-2) to BCL2-Associated X Protein (BAX) increased, the production of ROS decreased, and the protein expression and activity of manganese superoxide dismutase (MnSOD, also called SOD2) and catalase (CAT) increased. DJ-1 overexpression activated Nrf2 expression, however, after Nrf2 silencing, apoptosis of RRPs increased, the ratio of Bcl-2 to BAX decreased, the production of ROS increased, the protein expression of MnSOD and CAT decreased, and the expression of heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) decreased. These data suggest that enhancement of the Nrf2 pathway is a potential protective strategy for the treatment of DR. Therefore, DJ-1 may prevent high glucose-induced oxidative stress and RRPs apoptosis through the Nrf2 signaling pathway, thereby preventing the early onset and progression of DR.


Assuntos
Retinopatia Diabética/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Pericitos/metabolismo , Proteína Desglicase DJ-1/metabolismo , Animais , Apoptose , Catalase/genética , Catalase/metabolismo , Células Cultivadas , Glucose/metabolismo , Glucose/toxicidade , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADP/genética , NADP/metabolismo , Fator 2 Relacionado a NF-E2/genética , Pericitos/efeitos dos fármacos , Proteína Desglicase DJ-1/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
15.
Am J Physiol Heart Circ Physiol ; 318(4): H853-H866, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32108526

RESUMO

Right ventricular (RV) dysfunction is the main determinant of mortality in patients with pulmonary arterial hypertension (PAH) and while inflammation is pathogenic in PAH, there is limited information on the role of RV inflammation in PAH. Sulforaphane (SFN), a potent Nrf2 activator, has significant anti-inflammatory effects and facilitates cardiac protection in preclinical diabetic models. Therefore, we hypothesized that SFN might play a comparable role in reducing RV and pulmonary inflammation and injury in a murine PAH model. We induced PAH using SU5416 and 10% hypoxia (SuHx) for 4 wk in male mice randomized to SFN at a daily dose of 0.5 mg/kg 5 days per week for 4 wk or to vehicle control. Transthoracic echocardiography was performed to characterize chamber-specific ventricular function during PAH induction. At 4 wk, we measured RV pressure and relevant measures of histology and protein and gene expression. SuHx induced progressive RV, but not LV, diastolic and systolic dysfunction, and RV and pulmonary remodeling, fibrosis, and inflammation. SFN prevented SuHx-induced RV dysfunction and remodeling, reduced RV inflammation and fibrosis, upregulated Nrf2 expression and its downstream gene NQO1, and reduced the inflammatory mediator leucine-rich repeat and pyrin domain-containing 3 (NLRP3). SFN also reduced SuHx-induced pulmonary vascular remodeling, inflammation, and fibrosis. SFN alone had no effect on the heart or lungs. Thus, SuHx-induced RV and pulmonary dysfunction, inflammation, and fibrosis can be attenuated or prevented by SFN, supporting the rationale for further studies to investigate SFN and the role of Nrf2 and NLRP3 pathways in preclinical and clinical PAH studies.NEW & NOTEWORTHY Pulmonary arterial hypertension (PAH) in this murine model (SU5416 + hypoxia) is associated with early changes in right ventricular (RV) diastolic and systolic function. RV and lung injury in the SU5416 + hypoxia model are associated with markers for fibrosis, inflammation, and oxidative stress. Sulforaphane (SFN) alone for 4 wk has no effect on the murine heart or lungs. Sulforaphane (SFN) attenuates or prevents the RV and lung injury in the SUF5416 + hypoxia model of PAH, suggesting that Nrf2 may be a candidate target for strategies to prevent or reverse PAH.


Assuntos
Anti-Inflamatórios/uso terapêutico , Hipertensão Pulmonar/tratamento farmacológico , Isotiocianatos/uso terapêutico , Artéria Pulmonar/efeitos dos fármacos , Remodelação Vascular , Disfunção Ventricular Direita/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Hipertensão Pulmonar/complicações , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Artéria Pulmonar/patologia , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/prevenção & controle
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118038, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31945713

RESUMO

Depending on temperature, the 2-amino-2H-[1,2,3]triazolo[4,5-g]quinoline-4,9-dione forms two polymorphic structures, which differ in the spatial arrangement of the amine group. Both polymorphs were investigated using different experimental methods as well as various quantum chemical calculations in order to characterise their molecular structures. We used X-ray diffraction, FT-IR and NMR (solid-state and liquid) methods supplemented by the density functional theory (DFT) calculations, molecular electrostatic potential (MEP) and molecular orbital (HOMO, LUMO) analyses. It was found that the arrangement of the amine group affected the crystal structure, formation of H-bonds, the amine and carbonyl vibration bands in the FT-IR spectra, chemical shift of amine group in 15N CP/MAS NMR and chemical shift of amine protons in 1H NMR spectra. Both polymorphs were tested on anticancer activity against a panel of human cancer cell lines. Comparing the activity of both compounds showed that activity against MCF-7, MDA-MB-231 and Caco-2 lines depend on the arrangement of the amine group. Moreover, both polymorphs exhibited the highest activity against cell line with high NQO1 protein level, such as: A549, MCF-7 and Caco-2. The molecular docking was used to examine the probable interaction between the ligand of the tested polymorphs and the NQO1 enzyme. The analysis showed that ligands formed a hydrophobic interaction with tryptophan (Trp105), phenylalanine (Phe126 and Phe178) and tyrosine (Tyr 126).


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neoplasias/tratamento farmacológico , Quinolinas/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/química , Neoplasias/patologia , Células Tumorais Cultivadas
17.
Int J Mol Sci ; 21(2)2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940911

RESUMO

Oxidative stress is a key player of the inflammatory cascade responsible for the initiation of ulcerative colitis (UC). Although the short chain quinone idebenone is considered a potent antioxidant and a mitochondrial electron donor, emerging evidence suggests that idebenone also displays anti-inflammatory activity. This study evaluated the impact of idebenone in the widely used dextran sodium sulphate (DSS)-induced mouse model of acute colitis. Acute colitis was induced in C57BL/6J mice via continuous exposure to 2.5% DSS over 7 days. Idebenone was co-administered orally at a dose of 200 mg/kg body weight. Idebenone significantly prevented body weight loss and improved the disease activity index (DAI), colon length, and histopathological score. Consistent with its reported antioxidant function, idebenone significantly reduced the colonic levels of malondialdehyde (MDA) and nitric oxide (NO), and increased the expression of the redox factor NAD(P)H (nicotinamide adenine dinucleotide phosphate) dehydrogenase quinone-1 (NQO-1) in DSS-exposed mice. Immunohistochemistry revealed a significantly increased expression of tight junction proteins, which protect and maintain paracellular intestinal permeability. In support of an anti-inflammatory activity, idebenone significantly attenuated the elevated levels of pro-inflammatory cytokines in colon tissue. These results suggest that idebenone could represent a promising therapeutic strategy to interfere with disease pathology in UC by simultaneously inducing antioxidative and anti-inflammatory pathways.


Assuntos
Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Ubiquinona/análogos & derivados , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NAD(P)H Desidrogenase (Quinona)/metabolismo , Óxido Nítrico/metabolismo , Ubiquinona/administração & dosagem , Ubiquinona/farmacologia , Perda de Peso/efeitos dos fármacos
18.
Arch Biochem Biophys ; 681: 108266, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31953132

RESUMO

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na+-translocating (NQR) and H+-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na+ and Li+ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na+. These findings testified a primary Na+-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H+ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H+-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na+ or H+ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na+ transport in nitrogen fixation by A. vinelandii.


Assuntos
Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Sódio/metabolismo , Fixação de Nitrogênio
19.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G419-G427, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961719

RESUMO

The activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) pathway contributes to cancer progression in addition to oxidative stress responses. Loss-of-function Keap1 mutations were reported to activate Nrf2, leading to cancer progression. We examined the effects of Keap1 deletion in a cholangiocarcinoma mouse model using a mutant K-ras/p53 mouse. Introduction of the Keap1 deletion into liver-specific mutant K-ras/p53 expression resulted in the formation of invasive cholangiocarcinoma. Comprehensive analyses of the gene expression profiles identified broad upregulation of Nrf2-target genes such as Nqo1 and Gstm1 in the Keap1-deleted mutant K-ras/p53 expressing livers, accompanied by upregulation of cholangiocyte-related genes. Among these genes, the transcriptional factor Sox9 was highly expressed in the dysplastic bile duct. The Keap-Nrf2-Sox9 axis might serve as a novel therapeutic target for cholangiocarcinoma.NEW & NOTEWORTHY The Keap1-Nrf2 system has a wide variety of effects in addition to the oxidative stress response in cancer cells. Addition of the liver-specific Keap1 deletion to mice harboring mutant K-ras and p53 accelerated cholangiocarcinoma formation, together with the hallmarks of Nrf2 activation. This process involved the expansion of Sox9-positive cells, indicating increased differentiation toward the cholangiocyte phenotype.


Assuntos
Neoplasias dos Ductos Biliares/genética , Transformação Celular Neoplásica/genética , Colangiocarcinoma/genética , Deleção de Genes , Genes ras , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Diferenciação Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/deficiência , Masculino , Camundongos Transgênicos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Invasividade Neoplásica , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcriptoma , Proteína Supressora de Tumor p53/metabolismo
20.
Biochim Biophys Acta Gen Subj ; 1864(3): 129514, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911239

RESUMO

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.


Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , Xanthomonas/enzimologia , Benzoquinonas/metabolismo , Citrus/metabolismo , Citrus/microbiologia , Peróxido de Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NADP/metabolismo , Estresse Oxidativo , Folhas de Planta/metabolismo , Estresse Salino/genética , Estresse Salino/fisiologia , Virulência , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Xanthomonas/fisiologia
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