RESUMO
Chinese artichoke tuber (Stachys sieboldii Miq.) is used as an herbal medicine as well as edible food. This study examined the effect of the Chinese artichoke extracts on the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway that induces the expression of antioxidant enzymes to explore its novel characteristics. Hot water extracts exhibited relatively high ARE activity. ARE activity was observed in two fractions when the hot water extracts were separated in the presence of trifluoroacetic acid using HPLC. Conversely, the highly active fraction disappeared when the hot water extracts were separated in the absence of trifluoroacetic acid. These results indicate that acidic degradation produces active ingredients. The structural analysis of the two active fractions identified harpagide, which is an iridoid glucoside, and harpagogenin. In vitro experiments revealed that harpagide was converted into harpagogenin under acidic conditions and that harpagogenin, but not harpagide, had potent ARE activity. Therefore, this study identified harpagogenin, which is an acid hydrolysate of harpagide, as an ARE activator and suggests that Nrf2-ARE pathway activation by Chinese artichoke contributes to the antioxidative effect.
Assuntos
Stachys , Elementos de Resposta Antioxidante , Antioxidantes/farmacologia , Fator 2 Relacionado a NF-E2 , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Stachys/química , Ácido Trifluoracético , ÁguaRESUMO
The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Curcumina , Camundongos , Animais , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Curcumina/farmacologia , Curcumina/uso terapêutico , Antioxidantes/uso terapêutico , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/uso terapêutico , Camundongos Transgênicos , Superóxido Dismutase/genética , Modelos Animais de Doenças , Medula Espinal/metabolismoRESUMO
We previously generated an ischemic stroke in a zebrafish model using N2 gas perfusion; however, this model was an unsuitable drug screening system due to low throughput. In this study, we examined a zebrafish ischemic stroke model using an oxygen absorber to assess drug effects. Hypoxic exposure more than 2 h using the oxygen absorber significantly induced cell death in the brain and damage to the neuronal cells. To confirm the utility of the ischemic model induced by the oxygen absorber, we treated zebrafish with neuroprotective agents. MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly suppressed cell death in the brain, and edaravone, a free radical scavenger, significantly reduced the number of dead cells. These results suggest that the activation of NMDA receptors and the production of reactive oxygen species induce neuronal cell damage in accordance with previous mammalian reports. We demonstrate the suitability of an ischemic stroke model in zebrafish larvae using the oxygen absorber, enabling a high throughput drug screening.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Maleato de Dizocilpina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Edaravone/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Larva , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Peixe-Zebra , Animais , Encéfalo/patologia , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Edaravone/farmacologia , Sequestradores de Radicais Livres/farmacologia , Gases , Hipóxia/complicações , Hipóxia/patologia , Neurônios/patologia , NitrogênioRESUMO
Skin exposure to UV rays causes the production of reactive oxygen species (ROS), and it is a major risk factor for various skin disorders and diseases. In particular, exposure to UV-A is a major cause of photoaging. We have previously isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2)-antioxidant response element (ARE) and demonstrated the protective effects of DDC both in vitro and in vivo in PC12 cells and Parkinson's disease models, respectively. In this study, we used HaCaT cells to examine the effects of DDC on ROS production and cell damage induced by UV-A. Our results indicated that UV-A irradiation in HaCaT cells increased ROS production in an energy-dependent manner. In addition, cell viability decreased in an energy-dependent manner 24 h after UV-A irradiation. However, treatment with DDC 24 h prior to UV-A irradiation significantly suppressed UV-A radiation-induced ROS production. In addition, DDC showed cytoprotective effects when used 24 h before and after UV-A irradiation. Treatment with DDC for 24 h also increased the expression levels of heme oxygenase-1 (HO-1) in a concentration-dependent manner. Pretreatment with the HO-1 inhibitor followed by DDC treatment before UV-A irradiation for 24 h reduced ROS production and the cytoprotective effect. These results suggest that DDC increases the expression levels of HO-1 and protects HaCaT cells through the suppression of UV radiation-induced ROS production.
Assuntos
Chalconas/farmacologia , Raios Ultravioleta/efeitos adversos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Humanos , Queratinócitos , Fator 2 Relacionado a NF-E2 , Perilla , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Pele/metabolismoRESUMO
Aggregation of amyloid-ß (Aß) and tau plays a crucial role in the onset and progression of Alzheimer's disease (AD). Therefore, the inhibition of Aß and tau aggregation may represent a potential therapeutic target for AD. Herein, we designed and synthesized both Aß and tau dual aggregation inhibitors based on the structure of curcumin and developed the novel curcumin derivative PE859. In this study, we investigated the inhibitory activity of PE859 on Aß aggregationin vitro and the therapeutic effects of PE859 on cognitive dysfunction via dual inhibition of Aß and tau aggregation in vivo. PE859 inhibited Aß aggregation in vitro and protected cultured cells from Aß-induced cytotoxicity. Furthermore, PE859 ameliorated cognitive dysfunction and reduced the amount of aggregated Aß and tau in brains of senescence-accelerated mouse prone 8 (SAMP8). These results warrant consideration of PE859 as a candidate drug for AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/metabolismo , Indóis/uso terapêutico , Agregados Proteicos/efeitos dos fármacos , Pirazóis/uso terapêutico , Proteínas tau/metabolismo , Envelhecimento/genética , Peptídeos beta-Amiloides/ultraestrutura , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Linhagem Celular Tumoral , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Atividade Motora/efeitos dos fármacos , Neuroblastoma/patologia , Técnicas de Microbalança de Cristal de Quartzo , Fatores de Tempo , Proteínas tau/ultraestruturaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN), and oxidative stress is thought to contribute to the pathogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which is a cellular defense system against oxidative stress, is a promising target for therapeutics aimed at reducing neuronal death in PD. Previously, we have isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as an activator of the Nrf2-ARE pathway. The present study showed the protective effect of DDC on PD models in vivo and in vitro. In a 6-hydroxydopamine (6-OHDA)-induced hemiparkinson's disease mouse model, intracerebral administration of DDC suppressed the dopaminergic neuronal loss and behavioral dysfunction. DDC upregulated the expression of heme oxygenase-1 (HO-1), one of the ARE-driven antioxidant enzymes, in astrocytes and microglia of the SN. In primary mesencephalic cultures, treatment with DDC also increased the HO-1 expression in astrocytes and microglia. DDC showed a protective effect against 6-OHDA-induced dopaminergic neuronal death, and the effect was suppressed by an HO-1 inhibitor. These results suggest that DDC prevents dopaminergic neurons from oxidative stress by upregulation of glial expression of HO-1.
Assuntos
Antioxidantes/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Doença de Parkinson/patologia , Perilla/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Masculino , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Oxidopamina/toxicidade , Elementos de Resposta/efeitos dos fármacosRESUMO
The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.
Assuntos
Elementos de Resposta Antioxidante , Chalconas/isolamento & purificação , Fator 2 Relacionado a NF-E2/genética , Perilla frutescens/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Ativação Transcricional/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Chalconas/farmacologia , Citoproteção , Indução Enzimática/efeitos dos fármacos , Frutas/química , Genes Reporter , Glutationa/metabolismo , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Células PC12 , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Verduras/química , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Thrombin activates immunocompetent microglia and increases release of inflammatory cytokines under intracerebral hemorrhage (ICH) insults. Also, thrombin injection into the striatum evokes acute necrosis and delayed apoptosis of neurons. A nucleoprotein high-mobility group box 1 (HMGB1) that is released from necrotic cells has been suggested to behave like a cytokine and cause over-facilitation of immune functions. Here we examined the effect of glycyrrhizin, known as an inhibitor of HMGB1, on thrombin-induced injury in rat cortico-striatal slice cultures and in vivo rat ICH model. In slice cultures, thrombin-induced a drastic increase in propidium iodide fluorescence indicating necrotic cell death in the cortical region, and robust shrinkage of the striatal tissue. Glycyrrhizin (10-100 µM) attenuated thrombin-induced cortical injury in a concentration-dependent manner. The protective effect of glycyrrhizin was not mediated by glucocorticoid receptors or modulation of nitric oxide production, but was reversed by exogenous HMGB1 application. The injury induced by a high concentration of HMGB1 was suppressed by glycyrrhizin. In vivo, unilateral injection of type IV collagenase into rat striatum induced ICH associated with brain edema formation, contralateral paralysis and neuron death. Once daily intraperitoneal administration of glycyrrhizin attenuated ICH-induced edema in both the cortex and the basal ganglia, and improved behavioral performance of rats in forelimb placing. Moreover, glycyrrhizin partially but significantly ameliorated ICH-induced neuron loss inside hematoma. These findings suggest that an HMGB1 inhibitor glycyrrhizin is a potential candidate for a remedy for ICH.
Assuntos
Anti-Inflamatórios/farmacologia , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/patologia , Ácido Glicirrízico/farmacologia , Proteína HMGB1/antagonistas & inibidores , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Edema Encefálico/induzido quimicamente , Edema Encefálico/tratamento farmacológico , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Bovinos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Hemorragia Cerebral/induzido quimicamente , Colagenases/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Avaliação Pré-Clínica de Medicamentos , Ácido Glicirrízico/metabolismo , Ácido Glicirrízico/uso terapêutico , Proteína HMGB1/fisiologia , Hemostáticos/farmacologia , Masculino , Terapia de Alvo Molecular , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Trombina/farmacologia , Técnicas de Cultura de TecidosRESUMO
Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress-induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H(2)O(2)- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H(2)O(2) and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.
Assuntos
Glutationa/biossíntese , Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Tiocianatos/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corpo Estriado/citologia , Avaliação Pré-Clínica de Medicamentos , Glutamato-Cisteína Ligase/biossíntese , Heme Oxigenase-1/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Wistar , Elementos de Resposta , Transdução de Sinais , SulfóxidosRESUMO
A hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K(+) (+10-30 mM) for 24-72h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72h treatment with 1-10 microM veratridine, a Na(+) channel activator. The effect of elevated K(+) was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K(+) or veratridine. After treatment with elevated K(+) and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K(+) and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.