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1.
Braz J Med Biol Res ; 52(9): e8525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411316

RESUMO

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Assuntos
Condrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/efeitos dos fármacos , Degeneração do Disco Intervertebral/metabolismo , Adulto , Idoso , Agrecanas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Ginsenosídeos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Dor Lombar/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
2.
BMC Plant Biol ; 19(1): 368, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429706

RESUMO

BACKGROUND: We previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana, we examined the effects of HS on several groups of seedlings: wild type, cngc6, and cngc6 complementation and overexpression lines. RESULTS: After HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca2+) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca2+ ([Ca2+]cyt). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO. CONCLUSION: These data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Óxido Nítrico/metabolismo , Termotolerância , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Citosol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Mutação , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Plântula/genética , Plântula/metabolismo
3.
Life Sci ; 234: 116772, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31422097

RESUMO

AIMS: Ligation of the urethra to create partial bladder outlet obstruction has widely been used as an animal model of bladder obstruction, although obstructive bladder dysfunction may be due to both mechanical and functional obstruction. Previous studies in rodents have demonstrated that long-term nitric oxide (NO) deficiency can lead to detrusor overactivity, and lack of NO may thus cause impairment of bladder outlet relaxation. The aim of this study was to define the characteristics of bladder and urethral dysfunction induced by chronic NO deficiency through both in vivo and in vitro investigations. MAIN METHODS: Rats were divided into two groups, and one group received an NO synthase inhibitor (Nω-nitro-L-arginine methyl ester hydrochloride: L-NAME) in the drinking water for 4 weeks. Bladder and urethral function were evaluated by continuous cystometry and isovolumetric cystometry. In vitro functional studies of detrusor strips and measurement of the mRNA and protein expression of an ischemic marker and a gap junction protein were also performed in separate rats. KEY FINDINGS: L-NAME administration raised blood pressure and decreased plasma nitrite/nitrate level compared to the control group. L-NAME treatment increased the frequency of bladder contractions and the residual volume, and elevated urethral pressure and bladder contraction pressure. In addition, carbachol-induced contraction was reduced in isolated detrusor strips from the L-NAME group, and bladder expression of HIF-1 and connexin 43 showed upregulation. SIGNIFICANCE: These findings suggest that chronic administration of L-NAME to rats induces bladder hyperactivity with residual urine, and may provide a useful model of functional bladder obstruction.


Assuntos
NG-Nitroarginina Metil Éster , Óxido Nítrico Sintase/antagonistas & inibidores , Obstrução do Colo da Bexiga Urinária/induzido quimicamente , Obstrução do Colo da Bexiga Urinária/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Nitratos/sangue , Óxido Nítrico Sintase/metabolismo , Nitritos/sangue , Ratos , Ratos Sprague-Dawley , Uretra/metabolismo , Uretra/fisiopatologia , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologia , Obstrução do Colo da Bexiga Urinária/sangue
4.
J Phys Chem A ; 123(32): 7075-7086, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31310526

RESUMO

The nitric oxide synthase (NOS) enzyme consists of multiple domains connected by flexible random coil tethers. In a catalytic cycle, the NOS domains move within the limits determined by the length and flexibility of the interdomain tethers and form docking complexes with each other. This process represents a key component of the electron transport from the flavin adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate binding domain to the catalytic heme centers located in the oxygenase domain. Studying the conformational behavior of NOS is therefore imperative for a full understanding of the overall catalytic mechanism. In this work, we have investigated the equilibrium positional distributions of the NOS domains and the bound calmodulin (CaM) by using Monte Carlo calculations of the NOS conformations. As a main experimental reference, we have used the magnetic dipole interaction between a bifunctional spin label attached to T34C/S38C mutant CaM and the NOS heme centers, which was measured by pulsed electron paramagnetic resonance. In general, the calculations of the conformational distributions allow one to determine the range and statistics of positions occupied by the tethered protein domains, assess the crowding effect of the multiple domains on each other, evaluate the accessibility of various potential domain docking sites, and estimate the interaction energies required to achieve target populations of the docked states. In the particular application described here, we have established the specific mechanisms by which the bound CaM facilitates the flavin mononucleotide (FMN)/heme interdomain docking in NOS. We have also shown that the intersubunit FMN/heme domain docking and electron transfer in the homodimeric NOS protein are dictated by the existing structural makeup of the protein. Finally, from comparison of the calculated and experimental docking probabilities, the characteristic stabilization energies for the CaM/heme domain and the FMN domain/heme domain docking complexes have been estimated as -4.5kT and -10.5kT, respectively.


Assuntos
Óxido Nítrico Sintase/química , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Método de Monte Carlo , Óxido Nítrico Sintase/metabolismo , Conformação Proteica
5.
Chem Biol Interact ; 309: 108710, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31199930

RESUMO

Formic acid is a common organic acid used in many industrial processes. There is a paucity of research on the direct toxicity of formic acid and how it might affect the cardiovascular system. This study aimed to understand the effect of formic acid on vascular tension in an animal model and the underlying mechanism. Results found that the vasodilation induced by formic acid was related to the endothelium. When the dosage of formic acid was 1 mM or 5 mM, the vasodilation of endothelium-intact rings was partially suppressed by l-NAME, NS-2028 and nifedipine, and vasoconstriction caused by CaCl2 was inhibited, and the mRNA levels of eNOS, the activity of NOS (tNOS, iNOS and cNOS) and the level of NO and cGMP were significantly increased. Results also found that eNOS protein expression was significantly enhanced by 5 mM of formic acid. These results suggest formic acid can relax the aortic vessels of rats in a dose-dependent pattern. Further, the mechanism of the formic acid-induced vasodilatation likely involved the NO/cGMP pathway. Finally, the current study has revealed that vasodilation induced by high concentrations of formaldehyde may be the effect of the metabolite formic acid. This study will help further inform the etiologies of formic acid-related angiocardiopathies.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Formiatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/metabolismo , Cloreto de Cálcio/farmacologia , GMP Cíclico/metabolismo , Endotélio Vascular/fisiologia , Expressão Gênica/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
6.
Life Sci ; 231: 116581, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31220524

RESUMO

AIMS: The aims of this study were to investigate the effect of colonic electrical stimulation (CES) on delayed colonic transit in Parkinson's disease (PD) model induced by rotenone and its possible mechanisms. MAIN METHODS: Sprague-Dawley male rats were implanted with a pair of electrodes on the serosa at the proximal colon and rotenone was subcutaneously injected for 6 weeks to induce the PD model. Behavior activity, stool volume and open-field test were recorded during the injection. Colonic propulsion rate was measured 6 weeks after rotenone injection. Colon samples of all rats were collected for the measurement of phosphorylated alpha-synuclein, choline acetyltransferase (CHAT), neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH). The protocols of control rats were the same as the PD rats except that no electrodes were implanted and no rotenone was injected. KEY FINDINGS: (1) Rotenone-induced PD rats demonstrated weight loss, significant decrease of the dopaminergic neurons in substantia nigra, and impairment of colon movement. (2) CES significantly accelerated the delayed colonic transmit (91.67 ±â€¯5.58% vs 51.33 ±â€¯4.18%), superior to Macrogol-4000. (3) CES significantly upregulated the expression of CHAT, nNOS and TH protein in colon of PD rats. (4) In colon of PD rats, the phosphorylated alpha-synuclein was significantly upregulated, but CES had no significant effect on phosphorylated alpha-synuclein. SIGNIFICANCE: Our data show that CES can normalize the delayed colonic transit and this normalization may attribute to affecting enteric excitatory and inhibitory neurons.


Assuntos
Colo/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Doença de Parkinson/fisiopatologia , Animais , Colina O-Acetiltransferase/metabolismo , Colo/fisiopatologia , Constipação Intestinal/fisiopatologia , Modelos Animais de Doenças , Estimulação Elétrica , Motilidade Gastrointestinal/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos Sprague-Dawley , Rotenona/metabolismo , Rotenona/farmacologia , Substância Negra/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo
7.
Molecules ; 24(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052164

RESUMO

Myocardial infarction (MI) remains the leading cause of death worldwide. We aimed to investigate the effect of NO deficiency on selective biochemical parameters within discreet myocardial zones after experimentally induced MI. To induce MI, the left descending coronary artery was ligated in two groups of 16-week-old WKY rats. In one group, NO production was inhibited by L-NAME (20 mg/kg/day) administration four weeks prior to ligation. Sham operations were performed on both groups as a control. Seven days after MI, we evaluated levels of nitric oxide synthase (NOS) activity, eNOS, iNOS, NFÒ¡B/p65 and Nrf2 in ischemic, injured and non-ischemic zones of the heart. Levels of circulating TNF-α and IL-6 were evaluated in the plasma. MI led to increased NOS activity in all investigated zones of myocardium as well as circulating levels of TNF-α and IL-6. L-NAME treatment decreased NOS activity in the heart of sham operated animals. eNOS expression was increased in the injured zone and this could be a compensatory mechanism that improves the perfusion of the myocardium and cardiac dysfunction. Conversely, iNOS expression increased in the infarcted zone and may contribute to the inflammatory process and irreversible necrotic changes.


Assuntos
Adaptação Fisiológica , Pressão Sanguínea , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Óxido Nítrico/metabolismo , Animais , Biomarcadores , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal , Citocinas/metabolismo , Ativação Enzimática , Expressão Gênica , Hipertensão/etiologia , NG-Nitroarginina Metil Éster/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Tamanho do Órgão , Ratos
8.
J Toxicol Sci ; 44(5): 317-326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068537

RESUMO

The purpose of this study was to explore whether renal endothelial cell injury is associated with oxidative stress in trichloroethylene (TCE)-induced immune kidney damage by detecting adhesion molecules and oxidative stress indexes. In this study, a mouse model of skin sensitization with the antioxidant Tempol was used to explore the mechanism. Blood urea nitrogen (BUN), creatinine (Cre), and histological examination were used for kidney function evaluation. Kidney homogenates were used for detecting renal nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD) and malondialdehyde (MDA). Renal endothelial nitric oxide synthase (eNOS), E-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1) protein levels were measured by immunohistochemical and Western blot. We found that BUN and Cre levels increased in the TCE sensitization positive group and the TCE+Tempol sensitization positive group. In the TCE sensitization positive group, a partial area of vacuolar degeneration and lysed epithelial cells were observed in renal tubules. In TCE+Tempol sensitization positive group, small areas were also found to be vacuolar degenerated and renal tubules were dissolved. Renal NO, NOS, SOD and eNOS levels decreased and MDA levels increased, renal E-selectin, VCAM-1and ICAM-1 protein levels increased in the TCE sensitization positive group and the TCE+Tempol sensitization positive group. Tempol attenuated TCE induced up-regulation of MDA, E-selectin, VCAM-1and ICAM-1 and down-regulation of NO, NOS, SOD and eNOS. In conclusion, trichloroethylene-sensitized mice renal immune injury is associated with the renal endothelial cells' oxidative stress state.


Assuntos
Células Endoteliais/efeitos dos fármacos , Haptenos/toxicidade , Nefropatias/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Solventes/toxicidade , Tricloroetileno/toxicidade , Animais , Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Dermatite Alérgica de Contato/metabolismo , Dermatite Alérgica de Contato/patologia , Selectina E/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Malondialdeído/metabolismo , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Marcadores de Spin , Superóxido Dismutase/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo
9.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022832

RESUMO

Nitrergic enteric neurons are key players of the descending inhibitory reflex of intestinal peristalsis, therefore loss or damage of these neurons can contribute to developing gastrointestinal motility disturbances suffered by patients worldwide. There is accumulating evidence that the vulnerability of nitrergic enteric neurons to neuropathy is strictly region-specific and that the two main enteric plexuses display different nitrergic neuronal damage. Alterations both in the proportion of the nitrergic subpopulation and in the total number of enteric neurons suggest that modification of the neurochemical character or neuronal death occurs in the investigated gut segments. This review aims to summarize the gastrointestinal region and/or plexus-dependent pathological changes in the number of nitric oxide synthase (NOS)-containing neurons, the NO release and the cellular and subcellular expression of different NOS isoforms. Additionally, some of the underlying mechanisms associated with the nitrergic pathway in the background of different diseases, e.g., type 1 diabetes, chronic alcoholism, intestinal inflammation or ischaemia, will be discussed.


Assuntos
Neurônios Nitrérgicos/citologia , Neurônios Nitrérgicos/patologia , Alcoolismo/metabolismo , Alcoolismo/patologia , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Intestinos/inervação , Intestinos/patologia , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 218: 374-387, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31030004

RESUMO

The inhibition of the enzyme Nitric Oxide Synthase by a bioactive compounds results in it possessing anti-inflammatory property. The ability of Andrographolide and its derivative Isoandrographolide to inhibit Nitric Oxide Synthase was studied using computational and experimental techniques. A combination of UV Spectroscopic and DFT computational techniques were used to calculate the molecular descriptors of the title compounds which were used to establish relationship with its biological activity. The drug-likeness of the compounds was estimated using Lipinski's rule. Molecular dynamics and docking studies were carried out to test for the structural and energetic favourability of the title compounds(ligand) being bound to Nitric Oxide Synthase(Protein) to induce inhibition. The force constant data obtained from IR spectroscopy was used in aid to parametrize force fields used in molecular dynamics simulation. The DFT method was used to perform NBO analysis that revealed the charge transfer interactions responsible for its biological properties. The Molecular Electrostatic Potential (MEP) plot revealed the regions of electrophilic and nucleophilic reactivity of the title compounds. MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was carried out which revealed the cytotoxicity at different concentrations of the title compounds by which means the biologically safe concentration was determined and therefore at this biologically safe concentration the ability of the compounds to inhibit Nitric Oxide formation was determined. Quantitative Structure-Activity Studies (QSAR) were used to furnish relationship between molecular descriptors and the Nitric Oxide Synthase inhibition activity resulting in anti-inflammatory property, based on the chosen molecular descriptors suggestions were made for the search of more potent Nitric Oxide Synthase inhibitors in the Andrographolide derivative family of compounds.


Assuntos
Diterpenos/química , Diterpenos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Relação Quantitativa Estrutura-Atividade , Células RAW 264.7 , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
11.
Biosci Biotechnol Biochem ; 83(7): 1205-1215, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30999826

RESUMO

Panax ginseng C. A. Meyer has been widely used in skin care. Our previous study showed that the phenolic acids in ginseng root extract (GRE) impart inhibitory effects on melanogenesis. In this study, we found that as the most abundant component of phenolic acids in GRE, vanillic acid decreased tyrosinase activity and melanin levels with or without α-MSH stimulation and suppressed the expression of microphthalmia-associated transcription factor (MITF) and melanogenic enzymes in B16F10 cells. Furthermore, vanillic acid downregulated NOS activity, nitric oxide (NO) content, cGMP level, guanylate cyclase (GC) and protein kinase G (PKG) activity, and the phosphorylation of cAMP-response element-binding protein (CREB), whereas arbutin had no effect on the NO/PKG pathway. These findings indicate that vanillic acid in GRE suppressed melanogenesis by inhibiting the NO/PKG signaling pathways. This study provides a potential mechanism underlying the inhibitory effect of ginseng on melanogenesis.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Melaninas/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Panax/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Transdução de Sinais/efeitos dos fármacos , Ácido Vanílico/farmacologia , Animais , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Oxirredutases Intramoleculares/metabolismo , Melaninas/biossíntese , Melaninas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Oxirredutases/metabolismo , Regulação para Cima/efeitos dos fármacos , alfa-MSH/farmacologia
12.
BMC Plant Biol ; 19(1): 108, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894123

RESUMO

BACKGROUND: Nutrition with ammonium (NH4+) can enhance the drought tolerance of rice seedlings in comparison to nutrition with nitrate (NO3-). However, there are still no detailed studies investigating the response of nitric oxide (NO) to the different nitrogen nutrition and water regimes. To study the intrinsic mechanism underpinning this relationship, the time-dependent production of NO and its protective role in the antioxidant defense system of NH4+- or NO3--supplied rice seedlings were studied under water stress. RESULTS: An early NO burst was induced by 3 h of water stress in the roots of seedlings subjected to NH4+ treatment, but this phenomenon was not observed under NO3- treatment. Root oxidative damage induced by water stress was significantly higher for treatment with NO3- than with NH4+ due to reactive oxygen species (ROS) accumulation in the former. Inducing NO production by applying the NO donor 3 h after NO3- treatment alleviated the oxidative damage, while inhibiting the early NO burst by applying the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) increased root oxidative damage in NH4+ treatment. Application of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester(L-NAME) completely suppressed NO synthesis in roots 3 h after NH4+ treatment and aggravated water stress-induced oxidative damage. Therefore, the aggravation of oxidative damage by L-NAME might have resulted from changes in the NOS-mediated early NO burst. Water stress also increased the activity of root antioxidant enzymes (catalase, superoxide dismutase, and ascorbate peroxidase). These were further induced by the NO donor but repressed by the NO scavenger and NOS inhibitor in NH4+-treated roots. CONCLUSION: These findings demonstrate that the NOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by water stress by enhancing the antioxidant defenses in roots supplemented with NH4+.


Assuntos
Compostos de Amônio/farmacologia , Desidratação , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Oryza/fisiologia , Antioxidantes/metabolismo , Arginina/metabolismo , Citrulina/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Nitratos/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Oryza/efeitos dos fármacos , Oxirredução , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo
13.
Food Chem ; 288: 187-192, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902280

RESUMO

In this study, the activity, expression and localization of nitric oxide synthase (NOS) was investigated in beef semimembranosus muscle (SM) during postmortem aging. Five beef SM muscles were vacuum-packaged and aged for 1, 3, 7 and 14 d at 4 °C. Results showed that SM muscle retained NOS activity during the 14 d storage period. Compared to 1 d of storage, NOS activity and neuronal NOS (nNOS) content decreased significantly at 3 and 7 d (P < 0.05). Calpain played a major role in degrading nNOS during beef postmortem aging. In addition, immunofluorescence studies suggested that nNOS in beef SM muscle was mainly distributed in the sarcolemma. The S-nitrosothiol (SNO) content in SM muscle increased significantly at 1, 3 and 7 d post-slaughter. This manuscript is the first study to demonstrate the activity and expression of NOS in beef during postmortem aging.


Assuntos
Envelhecimento , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase/metabolismo , Mudanças Depois da Morte , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Caspases/metabolismo , Bovinos , Proteólise
14.
J Enzyme Inhib Med Chem ; 34(1): 753-760, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30829084

RESUMO

The aerial parts of Tetrastigma hemsleyanum (APTH) have been used as a functional tea in China. The purpose of the current study was to identify the bioactive constituents with inhibitory activity against soluble epoxide hydrolase (sEH) and inducible nitric oxide synthase (iNOS), which are jointly considered potential therapeutic targets for vascular system diseases. In the present study, 39 compounds (1-39) were isolated from the APTH. Among them, compounds 8, 10, 12, 16, 17, 19, and 32 displayed potential activities, with IC50 values ranging from 4.5 to 9.5 µM, respectively, and all in non-competitive inhibition mode. Compounds 5, 10, 12, 19, and 32 displayed potent iNOS inhibitory effects, with IC50 values ranging from 15.6 to 47.3 µM. The results obtained in this work contribute to a better understanding of the pharmacological activities of T. hemsleyanum and its potential application as a functional food.


Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Óxido Nítrico Sintase/antagonistas & inibidores , Fenóis/farmacologia , Componentes Aéreos da Planta/química , Extratos Vegetais/farmacologia , Vitaceae/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Epóxido Hidrolases/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Óxido Nítrico Sintase/metabolismo , Fenóis/química , Fenóis/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Células RAW 264.7 , Solubilidade , Relação Estrutura-Atividade
15.
J Vet Pharmacol Ther ; 42(3): 361-367, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30888081

RESUMO

Endothelial dysfunction contributes to the development of ungulate's laminitis. Although extensively studied in equines, the endothelial function is not fully examined in bovine digital veins (BDVs). BDVs were studied under isometric conditions to describe the acetylcholine (ACh) endothelium-dependent relaxation. Concentration-response curves were constructed to phenylephrine, ACh, and sodium nitroprusside (SNP). Relaxation responses were evaluated using either phenylephrine or depolarizing high-potassium Krebs solution (DKS) as precontraction agents. Endothelium denudation and incubation with L-NAME (300 µM), indomethacin (10 µM) or both were used to explore endothelial-mediated mechanisms. Endothelium denudation did not modify phenylephrine and SNP responses, however, significantly (p < 0.05) converted a relaxation (63.2 ± 5%) response to ACh into a contraction (30.3±9%). The ACh-evoked relaxation was significantly (p < 0.05) reduced in the presence of indomethacin (37.5 ± 6%) and L-NAME (6.40 ± 2%). The presence of both inhibitors abolished the ACh-evoked relaxation. Although DKS caused a higher precontraction than phenylephrine, ACh-evoked relaxation (22.4 ± 3.4%) was still observed and was reduced by the combination of inhibitors (7.0 ± 1.0%). The ACh endothelium-dependent relaxation in BDVs is essentially mediated by nitric oxide and endothelium-derived prostanoids. The BDV endothelium function is a dynamic component in the control of the bovine digital blood flow, particularly under endothelial dysfunction conditions when venoconstriction might lead to postcapillary resistance increase.


Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico/fisiologia , Prostaglandinas/fisiologia , Vasodilatação , Veias/metabolismo , Acetilcolina/farmacologia , Animais , Bovinos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Casco e Garras/irrigação sanguínea , Indometacina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Fenilefrina/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Veias/efeitos dos fármacos , Veias/fisiologia
16.
Food Funct ; 10(3): 1726-1735, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30848260

RESUMO

Acylated anthocyanins are more stable than monomeric anthocyanins, but little is known about their physiological effects. We evaluated the hemodynamic effects of single intragastric doses of purple carrot (Daucus carota L.) anthocyanin (PCA) and two monomeric anthocyanins, cyanidin 3-O-glycoside (C3G) and delphinidin 3-O-ruthenoside (D3R). PCA, C3G, or D3R was administered orally to rat and blood flow in the cremaster artery was measured for 60 min using a laser Doppler blood flow meter. After measurements, the aorta of the animal was removed and the extent of phosphorylation of aortic epithelial nitric oxide synthase (eNOS) and Akt were determined by western blotting. PCA (10 mg kg-1) or C3G (1 mg kg-1) significantly increased rat cremaster arteriole blood flow and phosphorylation of eNOS and Akt; D3G (1 mg kg-1) only slightly altered cremaster arteriole blood flow and did not affect the phosphorylation of eNOS and Akt in the aorta. These results suggest that hemodynamic alterations depend more on the chemical structure of anthocyanins, particularly the aglycon, than on the glycoside. In addition, increase of blood flow by a single oral dose of PCA was practically reduced with treatment of carvedilol (CR), a non-specific adrenaline blocker. Blood concentrations of cyanidin or its glycoside 15, 30, or 60 min after the administration of 10 mg kg-1 PCA were below the limit of detection. These hemodynamic changes may have been associated with an indirect adrenergic action induced following a single dose of PCA.


Assuntos
Músculos Abdominais/irrigação sanguínea , Antocianinas/química , Antocianinas/farmacologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Daucus carota/química , Animais , Fator 2 de Liberação do Nucleotídeo Guanina/farmacologia , Masculino , Estrutura Molecular , Óxido Nítrico Sintase/classificação , Óxido Nítrico Sintase/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos
17.
Fish Physiol Biochem ; 45(3): 1153-1165, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30847628

RESUMO

The stress ameliorating effect of dietary supplementation of vitamin C, vitamin E, and tryptophan on rohu Labeo rohita fry was evaluated. Rohu fry (1.1 ± 0.03 g) were cultured under five different feeding regimes: enriched with 0.08% vitamin C (D1), 0.02% vitamin E (D2), 1.42% tryptophan (D3), a combination of these three ingredients at similar doses (D4), and control diet (D5). Rohu fry of D5 were divided into two groups-exposed to experimental light (D5FL) and ambient light (114 ± 4 lx, D5AL). All fry (except D5AL) were exposed at light intensity of 3442 ± 648 lx. Feeding of rohu with enriched diets significantly (P < 0.05) enhanced the survival rate and average weight. A 15-25% higher survival and 1.3-1.8-fold higher average weight were recorded in rohu fed with enriched diet compared to D5FL treatment. Supplementation of vitamin C in diet (D1) of rohu resulted in 4.1-fold and 6.9-fold higher nitric oxide synthase and reduced glutathione (GSH) levels, respectively compared to the D5FL treatment. The tryptophan-enriched diet (D3) showed 5.8-fold higher melatonin and 4.4-fold lower cortisol levels in rohu compared to the D5FL treatment. Significantly (P < 0.05) higher nitric oxide synthase, GSH and melatonin, and lower cortisol, glucose, thiobarbituric acid reactive substances, carbonyl protein, glutathione S-transferase, and glutathione peroxidase levels were found in D4 diet fed rohu compared to the other treatments. Reduced level of stress in D4 treatment resulted in best performance of rohu in terms of less swimming activity and higher survival and growth compared to the other treatments.


Assuntos
Ácido Ascórbico/farmacologia , Cyprinidae/crescimento & desenvolvimento , Suplementos Nutricionais , Triptofano/farmacologia , Vitamina E/farmacologia , Animais , Glicemia , Metabolismo Energético , Glutationa Peroxidase , Glutationa Transferase , Hidrocortisona , Larva/efeitos dos fármacos , Larva/efeitos da radiação , Longevidade , Melatonina , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico , Ganho de Peso
18.
Proc Natl Acad Sci U S A ; 116(14): 7129-7136, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894481

RESUMO

Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein-protein interactions but also metabolite-dependent interactions. Based on this protein-metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4 Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood-brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein-metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Descoberta de Drogas , NADPH Oxidase 4/metabolismo , Óxido Nítrico Sintase/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , NADPH Oxidase 4/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Pirazóis/farmacologia , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/prevenção & controle
19.
Int J Mol Sci ; 20(6)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909368

RESUMO

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.


Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina/metabolismo , Apoptose/genética , Biomarcadores , Espaço Extracelular/metabolismo , Expressão Gênica , Humanos , Hipóxia/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Transdução de Sinais , Estresse Fisiológico/genética
20.
Bull Exp Biol Med ; 166(5): 598-601, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903506

RESUMO

The cardioprotective and inotropic effects of standardized active natural substance based on high-molecular-weight compounds of humic origin were studied on the model of global ischemia (40 min) and reperfusion of isolated perfused rat heart. Preventive administration of the test substance (0.1 mg/ml) before ischemia/reperfusion modeling reduced reperfusion contracture and necrotic death of cardiomyocytes and promoted recovery of myocardial contractility. Blockade of NO synthase with L-NAME (100 µM) abolished the above effects of the test substance. It was hypothesized that NO synthase plays an important role in the development of the cardioprotective and inotropic effects of the test natural substance.


Assuntos
Isquemia/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Coração , Substâncias Húmicas , Isquemia/genética , Masculino , Traumatismo por Reperfusão Miocárdica/genética , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintase/genética , Ratos , Ratos Wistar
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