RESUMO
Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Poli(ADP-Ribose) Polimerases , Biotinilação , Colorimetria , Desenho de Fármacos , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Miniaturização , NAD/química , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/químicaRESUMO
BACKGROUND: Brain astrocytes play a pivotal role in neuronal activities. METHODS: An investigation was undertaken to determine whether juniper oil inhibits heat shock-induced apoptosis of astrocytes. RESULTS: Juniper oil inhibited the heat shock-induced apoptosis in human astrocyte CCF-STTG1 cells. Pretreatment of the cells with juniper oil inhibited the heat shock-induced DNA fragmentation and condensation of nuclear chromatin. Juniper oil alone did not affect the apoptosis. Juniper oil inhibited the heat shock-induced caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation in the human astrocytes. CONCLUSIONS: Juniper oil may inhibit the apoptosis of astrocytes by preventing the caspase-3 activation.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/enzimologia , Caspases/metabolismo , Temperatura Alta/efeitos adversos , Juniperus/química , Óleos de Plantas/farmacologia , Choque/patologia , Astrócitos/efeitos dos fármacos , Western Blotting , Encéfalo/citologia , Encéfalo/enzimologia , Caspase 3 , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/metabolismo , DNA/biossíntese , DNA/química , Fragmentação do DNA/efeitos dos fármacos , Depressão Química , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
The objective of the currently study was to determine the effect of Kunbi-Boshin-Hangam-Tang (KBH-Tang) on the production of nitric oxide (NO). Stimulation of RAW 264.7 cells with KBH-Tang after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in increased NO synthesis. KBH-Tang partially increased NO synthesis by itself. When KBH-Tang was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) protein. NO production was inhibited by NG-monomethyl-L-arginine (NGMMA). Furthermore, activation of nuclear factor (NF)-kappaB was increased by KBH-Tang. These results suggest that KBH-Tang may stimulate the NO production through the activation of the NF-kappaB.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Humanos , Interferon gama/administração & dosagem , Interferon gama/farmacologia , Coreia (Geográfico) , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Proteínas Recombinantes , ômega-N-Metilarginina/farmacologiaRESUMO
The crude drug "Siberian Ginseng (SG)" has long been used in empirical Oriental medicine for the nonspecific enhancement of resistance in humans and animals. In this study, we investigated the effect of cell cultured SG by oral administration in mast cell-mediated allergic reactions. SG dose-dependently inhibited compound 48/80-induced systemic allergy with doses of 10(-2) to 1 g/kg 1 h before oral administration. Of special note, SG inhibited systemic allergy with the dose of 1 g/kg by 25%. SG (1 g/kg) also inhibited passive cutaneous allergic reaction by 51%. SG dose-dependently inhibited histamine release from rat peritoneal mast cells. When SG (0.01 mg/ml) was added, the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in antidinitrophenyl (DNP) IgE antibody-stimulated mast cells was inhibited 39.5% and 23.3%, respectively. In addition, SG inhibited anti-DNP IgE antibody-stimulated TNF-alpha protein expression in mast cells. Our studies provide evidence that SG may be beneficial in the treatment of various types of allergic diseases.
Assuntos
Anafilaxia/prevenção & controle , Hipersensibilidade/prevenção & controle , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Extratos Vegetais/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Anafilaxia/induzido quimicamente , Animais , Western Blotting , Células Cultivadas , Dinitrofenóis/farmacologia , Relação Dose-Resposta a Droga , Eleutherococcus , Ensaio de Imunoadsorção Enzimática , Liberação de Histamina/efeitos dos fármacos , Hipersensibilidade/metabolismo , Imunoensaio/métodos , Imunoglobulina E/imunologia , Interleucina-6/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Ratos , Ratos Wistar , p-Metoxi-N-metilfenetilamina/administração & dosagem , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Exposure to environmental stresses and toxins is linked to the pathogenesis of neuropsychiatric disorders. Astrocytes, the most abundant glial-cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We have investigated whether peppermint oil inhibits heat shock-induced apoptosis of astrocytes. We found that peppermint oil inhibits the heat shock-induced apoptosis in both human astrocyte CCF-STTG1 cells and rat astrocytes. Pretreatment of the cells with peppermint oil inhibited the heat shock-induced DNA fragmentation and condensation of nuclear chromatin. Peppermint oil also inhibited the caspase-3 activation and poly-ADP-ribose polymerase fragmentation in CCF-STTG1 cells. These results suggest that peppermint oil may modulate the apoptosis of astrocytes via the activation of the caspase-3.
Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/metabolismo , Temperatura Alta/efeitos adversos , Óleos de Plantas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Técnicas de Cultura de Células , Fragmentação do DNA/efeitos dos fármacos , Eletroforese , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mentha piperita , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , RatosRESUMO
A human hepatoma cell line, Hep G2 cells, is a reliable system for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Asparagus cochinchinensis(MERRIL) (Liliaceae) roots (ACAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. ACAE (1-100 microg/ml) dose-dependently inhibited the EtOH-induced tumor necrosis factor-alpha (TNF-alpha) secretion. ACAE (1-100 microg/ml) also inhibited the EtOH and TNF-alpha-induced cytotoxicity. Furthermore, we found that ACAE inhibited the TNF-alpha-induced apoptosis of Hep G2 cells. These results suggest that ACAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Etanol/toxicidade , Liliaceae , Neoplasias Hepáticas/metabolismo , Extratos Vegetais/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Etanol/antagonistas & inibidores , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Raízes de Plantas , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A human hepatoma cell line, Hep G2 cells are reliable for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Polygala tenuifolia WILLDENOW (Polygalaceae) roots (PTAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. PTAE (0.01-1 microg/ml) dose-dependently inhibited the EtOH-induced interleukin-1alpha (IL-1alpha) secretion. PTAE (0.01-1 microg/ml) also inhibited the EtOH- and IL-1alpha-induced cytotoxicity. Furthermore, we found that PTAE inhibited the IL-1alpha-induced apoptosis of Hep G2 cells. These results suggest that PTAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.