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1.
Am J Physiol Renal Physiol ; 319(4): F686-F696, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32830535

RESUMO

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione-S-transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.


Assuntos
Apoptose , Células Epiteliais/enzimologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Túbulos Renais Proximais/enzimologia , Mitocôndrias/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Transporte Ativo do Núcleo Celular , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Metabolismo Energético , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/genética , Interferência de RNA , Transdução de Sinais , Marcadores de Spin
2.
Chem Biol Interact ; 329: 109210, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32726580

RESUMO

Cigarette smoke is a complex mixture capable of triggering inflammation and oxidative damage in animals at pulmonary and systemic levels. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) reduces tissue injury associated with inflammation in vivo by mechanisms that are not completely understood. Here we evaluated the effect of tempol on inflammation and oxidative damage induced by acute exposure to cigarette smoke in vivo. Male C57BL/6 mice (n = 32) were divided into 4 groups (n = 8 each): 1) control group exposed to ambient air (GC), 2) animals exposed to cigarette smoke for 5 days (CSG), mice treated 3) prior or 4) concomitantly with tempol (50 mg/kg/day) and exposed to cigarette smoke for 5 days. The results showed that the total number of leukocytes and neutrophils increased in the respiratory tract and lung parenchyma of mice exposed to cigarette smoke. Likewise, MPO levels and activity as well as lipid peroxidation and lung protein nitration and carbonylation also increased. Administration of tempol before or during exposure to cigarette smoke inhibited all the above parameters. Tempol also reduced the pulmonary expression of the inflammatory cytokines Il-6, Il-1ß and Il-17 to basal levels and of Tnf-α by approximately 50%. In contrast, tempol restored Il-10 and Tgf-ß levels and enhanced the expression of Nrf2-associated genes, such as Ho-1 and Gpx2. Accordingly, total GPx activity increased in lung homogenates of tempol-treated animals. Taken together, our results show that tempol protects mouse lungs from inflammation and oxidative damage resulting from exposure to cigarette smoke, likely through reduction of leukocyte infiltration and increased transcription of some of the Nrf2-controlled genes.


Assuntos
Óxidos N-Cíclicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fumar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/química , Interleucina-10/genética , Interleucina-10/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Nitritos/análise , Peroxidase/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Marcadores de Spin , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
3.
Am J Obstet Gynecol ; 223(5): 753.e1-753.e14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32416155

RESUMO

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.


Assuntos
Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Doenças Fetais/genética , Células-Tronco Neurais/metabolismo , Defeitos do Tubo Neural/genética , Gravidez em Diabéticas/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Resposta a Proteínas não Dobradas/genética , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Glicemia/metabolismo , Óxidos N-Cíclicos/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Doenças Fetais/etiologia , Doenças Fetais/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Células Neuroepiteliais/efeitos dos fármacos , Células Neuroepiteliais/metabolismo , Neurulação/genética , Estresse Oxidativo , Gravidez , Gravidez em Diabéticas/metabolismo , Marcadores de Spin , Resposta a Proteínas não Dobradas/efeitos dos fármacos
4.
Life Sci ; 254: 117819, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32442451

RESUMO

AIMS: Vascular dysfunction plays a key role in sepsis but the role of perivascular adipose tissue (PVAT) in this condition is relatively unknown. MAIN METHODS: Sepsis was induced by cecal ligation and puncture (CLP). The responses of the aorta and superior mesenteric artery to norepinephrine in the presence or absence of PVAT were evaluated. Fluorescent probes measured the production of nitric oxide (NO) and reactive oxygen species (ROS). NO synthases (NOS) and ß3-adrenoceptor expression were detected by immunofluorescence and S-nitrosylation by the biotin switch assay. KEY FINDINGS: Aorta and superior mesenteric arteries from septic animals with intact PVAT showed a worsened response to the vasoconstrictor compared to vessels without PVAT. PVAT from the aorta (APVAT) produced NO and ROS whereas PVAT from the superior mesenteric artery (MPVAT) produced only ROS. Septic APVAT exhibited a higher density of NOS-1 and NOS-3. S-nitrosylation was found in APVAT. Donor (PVAT obtained from normal or septic rats):Host (normal vessel without PVAT) experiments showed that L-NAME, ODQ and ß3-adrenergic receptor antagonist blocked the septic APVAT anti-contractile effect. None of these compounds affected MPVAT; tempol, but not apocynin, blocked its anti-contractile effect. SIGNIFICANCE: PVAT contributes to the anti-contractile effect in the aorta and mesenteric artery of septic rats through different pathways. ß3-Adrenergic receptor and NO appear to be key mediators of this effect in APVAT, but not in MPVAT where ROS seem to be a relevant mediator. Therefore, PVAT is a relevant player of sepsis vascular dysfunction.


Assuntos
Aorta/metabolismo , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta 3/fisiologia , Sepse/fisiopatologia , Acetofenonas/farmacologia , Tecido Adiposo/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Óxidos N-Cíclicos/farmacologia , Feminino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Norepinefrina/farmacologia , Oxidiazóis/farmacologia , Fenótipo , Quinoxalinas/farmacologia , Ratos , Receptores Adrenérgicos beta 3/biossíntese , Marcadores de Spin , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia
5.
Am J Respir Cell Mol Biol ; 62(6): 732-746, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048876

RESUMO

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Mitocôndrias/fisiologia , Proteína Quinase C beta/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Depuradores de Radicais Livres/farmacologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Canais de Potássio/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Síndromes da Apneia do Sono/fisiopatologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia
6.
Biomater Sci ; 8(6): 1695-1701, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-31989134

RESUMO

Spinal cord injury is one of the most serious traumatic diseases. The current available clinical therapies are unable to provide effective recovery of nerve functions. Implantation of biomaterial scaffolds is a promising approach to bridge the damaged nerve tissue in the absence of the extracellular matrix. However, the treatments have been impaired by the increased generation of reactive oxygen species in the microenvironment of acute spinal cord injury. Efficient delivery of antioxidants and biocompatible materials and reagents has been a challenge. Herein, a novel hyaluronic acid (HA) hydrogel functionalized with the antioxidant compound 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) is fabricated for nerve tissue regeneration after serious spinal cord transection in rats. TEMPO is tethered onto HA chains to form HA-TEMPO through a Schiff base reaction between 4-amino-TEMPO and aldehyde modified HA chains. The TEMPO-hydrogel is constructed with a highly porous three-dimensional structure via the gelation between the residue aldehydes in HA-TEMPO and the amines in adipic dihydrazide modified HA. The functional TEMPO-hydrogel exhibits the antioxidant effect in an H2O2 simulated in vitro peroxidative microenvironment. Implantation of the functional hydrogel in vivo induces a significant motor function restoration, which could be attributed to the effective functions of the TEMPO-hydrogel in tissue reconnection as well as nerve fiber regeneration of the central nervous spinal cord tissue. Importantly, the treatment with the TEMPO-hydrogel effectively protects the bladder tissue from neurogenic damage. Therefore, the functional TEMPO-hydrogel provides a promising strategy for the treatment of central nervous system diseases through the antioxidant and lesion-bridging regulation of the pathological microenvironment.


Assuntos
Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Traumatismos da Medula Espinal/terapia , Bexiga Urinária/efeitos dos fármacos , Animais , Antioxidantes/química , Óxidos N-Cíclicos/química , Modelos Animais de Doenças , Ácido Hialurônico/química , Hidrogéis/química , Peróxido de Hidrogênio/efeitos adversos , Masculino , Ratos , Bases de Schiff/química , Tecidos Suporte/química , Transplante de Tecidos , Resultado do Tratamento
7.
J Pharmacol Toxicol Methods ; 102: 106677, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978535

RESUMO

The present paper describes the regulatory safety pharmacology studies that were carried out to support the clinical trial application for BIA 10-2474. Animal studies complied with worldwide regulatory guidelines (e.g. EEC Council Directive 75/318/EEC and subsequent amendments). Oral administration of BIA 10-2474 at doses of 30, 100 and 300 mg/kg to male Han Wistar rats did not cause any significant physiological or behavioral changes, affect the respiration rate or the tidal volume, the gastrointestinal transit, urinary output volume or pH, nor urinary sodium, potassium or chloride excretion. BIA 10-2474, at 30 µg/mL, slightly, but significantly, reduced the hERG outward tail currents by 10.62%, but had no effect at 1, 3 or 10 µg/mL. BIA 10-2474 (1.5, 4.5 and 15 µg/mL) had no substantial effects on resting membrane potential (RMP), maximal upstroke velocity (Vmax), action potential amplitude (APA), action potential duration at 30%, 60% and 90% or action potential triangulation over the 30-min superfusion period in the dog isolated Purkinje fiber. In conscious dogs monitored by telemetry, BIA 10-2474 (20, 50 and 100 mg/kg p.o.) did not significantly modify arterial blood pressure as compared with controls (inter-group comparison), although a tendency toward an increase in arterial blood pressure was observed at 100 mg/kg, with systolic blood pressure being the most affected parameter. In conclusion, BIA 10-2474 had no adverse effects on any of the major vital organ systems, and, unfortunately, the data shed no further light on the mechanism(s) responsible for the clinical trial accident with BIA 10-2474.


Assuntos
Óxidos N-Cíclicos/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Piridinas/administração & dosagem , Administração Oral , Animais , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Óxidos N-Cíclicos/toxicidade , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Masculino , Piridinas/farmacologia , Piridinas/toxicidade , Ratos , Ratos Wistar , Telemetria , Testes de Toxicidade
8.
Carbohydr Polym ; 230: 115587, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887889

RESUMO

Stability of cotton fibers oxidized by the 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO)-mediated system during natural aging was monitored; the oxidized and five-year-aged fibers were characterized in terms of molecular weight distribution, quantity and distribution of functionalities, tensile strength, color difference, and yellowness index. The distribution of functionalities introduced in cotton fibers by TEMPO-mediated oxidation and subsequent aging was investigated by means of gel permeation chromatography using multiple detection and group-selective labeling. The mild TEMPO-mediated oxidation retains the molar mass of oxidized cotton, but this status is not stable during natural aging. For highly oxidized cotton, the molar mass of aged fibers is low but stable. Aged TEMPO-oxidized cotton shows a prominent discoloration and an increased yellowness index. For tensile strength, the limiting value was obtained for aged TEMPO-oxidized cotton, regardless of whether the samples had initially been oxidized with a low or high degree of oxidation.


Assuntos
Celulose/química , Fibra de Algodão/análise , Nanofibras/química , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacologia , Peso Molecular , Oxirredução/efeitos dos fármacos , Resistência à Tração
9.
Org Biomol Chem ; 18(3): 557-568, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31894828

RESUMO

The isoquinolinequinone (IQQ) pharmacophore is a privileged framework in known cytotoxic natural product families, caulibugulones and mansouramycins. Exploiting both families as a chemical starting point, we report on the structured development of an IQQ N-oxide anticancer framework which exhibits growth inhibition in the nM range across melanoma, ovarian and leukaemia cancer cell lines. A new lead compound (16, R6 = benzyl, R7 = H) exhibits nM GI50 values against 31/57 human tumour cell lines screened as part of the NCI60 panel and shows activity against doxorubicin resistant tumour cell lines. An electrochemical study highlights a correlation between electropositivity of the IQQ N-oxide framework and cytotoxicity. Adduct binding to sulfur based biological nucleophiles glutathione and cysteine was observed in vitro. This new framework possesses significant anticancer potential.


Assuntos
Antineoplásicos/farmacologia , Óxidos N-Cíclicos/farmacologia , Isoquinolinas/farmacologia , Quinonas/farmacologia , Antineoplásicos/síntese química , Benzilaminas/síntese química , Benzilaminas/farmacologia , Linhagem Celular Tumoral , Óxidos N-Cíclicos/síntese química , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoquinolinas/síntese química , Quinonas/síntese química
10.
Am J Physiol Heart Circ Physiol ; 318(2): H470-H483, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922892

RESUMO

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.


Assuntos
Hipóxia/metabolismo , Proteína Quinase C beta/metabolismo , Animais , Animais Recém-Nascidos , Doença Crônica , Óxidos N-Cíclicos/farmacologia , Inibidores Enzimáticos , Feminino , Depuradores de Radicais Livres , Indóis/farmacologia , Maleimidas/farmacologia , Compostos Organofosforados/farmacologia , Estresse Oxidativo , Gravidez , Proteína Quinase C beta/antagonistas & inibidores , Artéria Pulmonar/efeitos dos fármacos , Circulação Pulmonar , Ratos , Espécies Reativas de Oxigênio , Marcadores de Spin , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Vasoconstrição , Vasoconstritores/farmacologia
11.
Mater Sci Eng C Mater Biol Appl ; 108: 110189, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924023

RESUMO

Particulate pollution in the air has strong links with increased morbidity of cardiopulmonary diseases. Iron is one of the major carcinogens in air pollution and can produce hydroxyl radical which induce oxidative stress, lead to cell damage and even to cancer. In this work, a novel nitronyl nitroxide radical NITPh(OMe)2 (2-(2,4-dimethoxyphenyl) -4,4,5,5- tetramethylimidazoline- 1- oxyl-3- oxide) was prepared and characterized by electron spin-resonance spectroscopy (ESR), X-ray crystal diffraction, Fourier transform infrared (IR), X-ray powder diffraction (XRD), elemental analysis, ultraviolet and visible spectra (UV-Vis), and the electronic transition processes was also calculated by time-dependent density functional theory (TDDFT) to analysis UV-Vis spectrum. In vitro cell model of oxidative damage was established by ferric ammonium citrate (FAC) overload, and NITPh(OMe)2 was studied as a free radical scavenger to protect peroxidation of A549 cells. Results showed that NITPh(OMe)2 could significantly alleviate the damage of A549 cells by iron overload in cell morphology, cell viability, cell proliferation and cell apoptosis. The apoptotic signaling pathway of A549 cells induced by FAC and the protection mechanism of NITPh(OMe)2 were all discussed through the expression of three relating proteins, Bcl-2, Bax and DDIT3. This work confirms that nitroxide radicals are effective antioxidants, and have potential application in clinical practice as therapeutic agents.


Assuntos
Antioxidantes , Apoptose/efeitos dos fármacos , Óxidos N-Cíclicos , Sobrecarga de Ferro/metabolismo , Óxidos de Nitrogênio , Células A549 , Antioxidantes/química , Antioxidantes/farmacologia , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacologia , Humanos , Sobrecarga de Ferro/patologia , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/farmacologia , Oxirredução/efeitos dos fármacos
12.
Regul Toxicol Pharmacol ; 111: 104556, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31866344

RESUMO

In 2016 one person died and others had neurological sequelae during a clinical trial with BIA 10-2474 (3-(1-(cyclohexyl(methyl)carbamoyl)-lH-imidazol-4-yl)pyridine 1-oxide), a novel fatty acid amide hydrolase (FAAH) inhibitor being developed for the treatment of medical conditions such as pain. Prior to the clinical trial a full battery of regulatory toxicology tests were carried out and this paper describes the genotoxicity/mutagenicity tests undertaken with BIA 10-2474 using the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli WP2uvrA forward mutation test, an in vitro chromosome damage assay in human lymphocytes, and an in vivo micronucleus test in mice. All tests were conducted with and without a rat liver S9 metabolic activation system. None of the test results were judged to be positive with regards to the mutagenicity/genotoxicity of BIA 10-2474 making it unlikely that any such effect was involved in the toxicity observed in the clinic.


Assuntos
Antibacterianos/farmacologia , Óxidos N-Cíclicos/farmacologia , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Células Cultivadas , Aberrações Cromossômicas , Feminino , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Testes de Mutagenicidade , Ratos , Ratos Endogâmicos F344 , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
13.
Redox Biol ; 28: 101333, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31593888

RESUMO

Inflammatory bowel disease (IBD) is a chronic condition characterised by leukocyte recruitment to the gut mucosa. Leukocyte myeloperoxidase (MPO) produces the two-electron oxidant hypochlorous acid (HOCl), damaging tissue and playing a role in cellular recruitment, thereby exacerbating gut injury. We tested whether the MPO-inhibitor, 4-Methoxy-TEMPO (MetT), ameliorates experimental IBD. Colitis was induced in C57BL/6 mice by 3% w/v dextran-sodium-sulfate (DSS) in drinking water ad libitum over 9-days with MetT (15 mg/kg; via i. p. injection) or vehicle control (10% v/v DMSO+90% v/v phosphate buffered saline) administered twice daily during DSS challenge. MetT attenuated body-weight loss (50%, p < 0.05, n = 6), improved clinical score (53%, p < 0.05, n = 6) and inhibited serum lipid peroxidation. Histopathological damage decreased markedly in MetT-treated mice, as judged by maintenance of crypt integrity, goblet cell density and decreased cellular infiltrate. Colonic Ly6C+, MPO-labelled cells and 3-chlorotyrosine (3-Cl-Tyr) decreased in MetT-treated mice, although biomarkers for nitrosative stress (3-nitro-tyrosine-tyrosine; 3-NO2-Tyr) and low-molecular weight thiol damage (assessed as glutathione sulfonamide; GSA) were unchanged. Interestingly, MetT did not significantly impact colonic IL-10 and IL-6 levels, suggesting a non-immunomodulatory pathway. Overall, MetT ameliorated the severity of experimental IBD, likely via a mechanism involving the modulation of MPO-mediated damage.


Assuntos
Colite/etiologia , Colite/patologia , Óxidos N-Cíclicos/farmacologia , Sulfato de Dextrana/efeitos adversos , Suscetibilidade a Doenças , Substâncias Protetoras/farmacologia , Animais , Biópsia , Colite/diagnóstico por imagem , Colite/tratamento farmacológico , Modelos Animais de Doenças , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Camundongos , Imagem Óptica , Oxirredução , Estresse Oxidativo , Fenótipo
15.
J Neuroinflammation ; 16(1): 218, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727149

RESUMO

BACKGROUND: The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. METHODS: Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease. The animals were sacrificed at week 14 (initial stage of symptoms-ISS) and at the end stage (ES) of the disease. The lumbar spinal cord of the animals was dissected and processed for use in the following techniques: Nissl staining to evaluate neuronal survival; immunohistochemistry to evaluate astrogliosis and microgliosis (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS); and transmission electron microscopy to evaluate the alpha-motoneurons (ES). Behavioral analyses considering the survival of animals, bodyweight loss, and Rotarod motor performance test started on week 10 and were performed every 3 days until the end-stage of the disease. RESULTS: The results revealed that treatment with tempol promoted greater neuronal survival (23%) at ISS compared to untreated animals, which was maintained until ES. The intense reactivity of astrocytes and microglia observed in vehicle animals was reduced in the lumbar spinal cords of the animals treated with tempol. In addition, the groups treated with tempol showed reduced expression of proinflammatory cytokines (IL1ß and TNFα) and a three-fold decrease in the expression of TGFß1 at ISS compared with the group treated with vehicle. CONCLUSIONS: Altogether, our results indicate that treatment with tempol has beneficial effects, delaying the onset of the disease by enhancing neuronal survival and decreasing glial cell reactivity during ALS progression in SOD1G93A mice.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Óxidos N-Cíclicos/uso terapêutico , Inflamação/tratamento farmacológico , Destreza Motora/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Medula Espinal/efeitos dos fármacos , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Destreza Motora/fisiologia , Fármacos Neuroprotetores/farmacologia , Teste de Desempenho do Rota-Rod , Marcadores de Spin , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
16.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(8): 883-890, 2019 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-31511206

RESUMO

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size (P < 0.01) and a shorter lifespan (P < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis (P < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment (P < 0.05). CONCLUSIONS: Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.


Assuntos
Óxidos N-Cíclicos/farmacologia , Animais , Antineoplásicos , Antioxidantes , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Marcadores de Spin
17.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514290

RESUMO

In salt-sensitive hypertension, reactive oxygen species (ROS) play a major role in the progression of renal disease partly through the activation of the mineralocorticoid receptor (MR). We have previously demonstrated that urinary vanin-1 is an early biomarker of oxidative renal tubular injury. However, it remains unknown whether urinary vanin-1 might reflect the treatment effect. The objective of this study was to clarify the treatment effect for renal tubular damage in Dahl salt-sensitive (DS) rats. DS rats (six weeks old) were given one of the following for four weeks: high-salt diet (8% NaCl), high-salt diet plus a superoxide dismutase mimetic, tempol (3 mmol/L in drinking water), high-salt diet plus eplerenone (100 mg/kg/day), and normal-salt diet (0.3% NaCl). After four-week treatment, blood pressure was measured and kidney tissues were evaluated. ROS were assessed by measurements of malondialdehyde and by immunostaining for 4-hydroxy-2-nonenal. A high-salt intake for four weeks caused ROS and histological renal tubular damages in DS rats, both of which were suppressed by tempol and eplerenone. Proteinuria and urinary N-acetyl-ß-D-glucosaminidase exhibited a significant decrease in DS rats receiving a high-salt diet plus eplerenone, but not tempol. In contrast, urinary vanin-1 significantly decreased in DS rats receiving a high-salt diet plus eplerenone as well as tempol. Consistent with these findings, immunohistochemical analysis revealed that vanin-1 was localized in the renal proximal tubules but not the glomeruli in DS rats receiving a high-salt diet, with the strength attenuated by tempol or eplerenone treatment. In conclusion, these results suggest that urinary vanin-1 is a potentially sensitive biomarker for ameliorating renal tubular damage in salt-sensitive hypertension.


Assuntos
Amidoidrolases/metabolismo , Túbulos Renais/patologia , Estresse Oxidativo , Amidoidrolases/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Eplerenona/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/fisiopatologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos Dahl , Marcadores de Spin , Sístole/efeitos dos fármacos
18.
Am J Physiol Renal Physiol ; 317(4): F957-F966, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31432707

RESUMO

Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg-1·day-1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 µg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg-1·day-1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.


Assuntos
Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Circulação Renal/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Masculino , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Receptor 4 Toll-Like/antagonistas & inibidores , Vasoconstrição/efeitos dos fármacos
19.
Invest Ophthalmol Vis Sci ; 60(10): 3613-3624, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31433458

RESUMO

Purpose: POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-ß2-mediated remodeling of the ECM. Methods: Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-ß2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-ß2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Results: Primary or transformed human TM cells cultured in the presence of TGF-ß2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-ß2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 µM) or MitoQ (10 nM) attenuated TGF-ß2-mediated changes in Smad-dependent transcriptional activity. Conclusions: The multifunctional profibrotic cytokine TGF-ß2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-ß2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-ß2-mediated remodeling of the ECM within the TM.


Assuntos
Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/fisiologia , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo IV/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Óxidos N-Cíclicos/farmacologia , Humanos , Immunoblotting , Imuno-Histoquímica , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Malha Trabecular/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
20.
ACS Chem Biol ; 14(9): 2055-2064, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31465201

RESUMO

GPR84 is an orphan G-protein-coupled receptor that is expressed on immune cells and implicated in several inflammatory diseases. The validation of GPR84 as a therapeutic target is hindered by the narrow range of available chemical tools and consequent poor understanding of GPR84 pathophysiology. Here we describe the discovery and characterization of DL-175, a potent, selective, and structurally novel GPR84 agonist and the first to display significantly biased signaling across GPR84-overexpressing cells, primary murine macrophages, and human U937 cells. By comparing DL-175 with reported GPR84 ligands, we show for the first time that biased GPR84 agonists have markedly different abilities to induce chemotaxis in human myeloid cells, while causing similar levels of phagocytosis enhancement. This work demonstrates that biased agonism at GPR84 enables the selective activation of functional responses in immune cells and delivers a high-quality chemical probe for further investigation.


Assuntos
Fatores Quimiotáticos/farmacologia , Óxidos N-Cíclicos/farmacologia , Macrófagos/efeitos dos fármacos , Piridinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Animais , Células CHO , Linhagem Celular Tumoral , Fatores Quimiotáticos/química , Cricetulus , Óxidos N-Cíclicos/química , Humanos , Camundongos , Estrutura Molecular , Fagocitose/efeitos dos fármacos , Piridinas/química , Relação Quantitativa Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacos
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