Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
1.
Acta Pharmacol Sin ; 35(1): 33-40, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24141567

RESUMO

AIM: To investigate the roles of cysteinyl leukotriene receptors CysLT1R and CysLT2R in leukotriene D4 (LTD4)-induced activation of microglial cells in vitro. METHODS: Mouse microglial cell line BV2 was transfected with pcDNA3.1(+)-hCysLT1R or pcDNA3.1(+)-hCysLT2R. The expression of relevant mRNAs and proteins in the cells was detected using RT-PCR and Western blotting, respectively. Phagocytosis was determined with flow cytometry analysis. The release of interleukin-1ß (IL-1ß) from the cells was measured using an ELISA assay. RESULTS: The expression of CysLT1R or CysLT2R was considerably increased in the transfected BV2 cells, and the receptors were mainly distributed in the plasma membrane and cytosol. Treatment of the cells expressing CysLT1R or CysLT2R with CysLT receptor agonist LTD4 (0.1-100 nmol/L) concentration-dependently enhanced the phagocytosis, and increased mRNA expression and release of IL-1ß. Moreover, the responses of hCysLT1R-BV2 cells to LTD4 were significantly larger than those of hCysLT2R-BV2 or WT-BV2 cells. Pretreatment of hCysLT1R-BV2 cells with the selective CysLT1R antagonist montelukast (1 µmol/L) significantly blocked LTD4-induced phagocytosis as well as the mRNA expression and release of IL-1ß, whereas the selective CysLT2R antagonist HAMI 3379 (1 µmol/L) had no such effects. CONCLUSION: CysLT1R mediates LTD4-induced activation of BV2 cells, suggesting that CysLT1R antagonists may exert anti-inflammatory activity in brain diseases.


Assuntos
Leucotrieno D4/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Receptores de Leucotrienos/agonistas , Receptores de Leucotrienos/fisiologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos
2.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 43(3): 265-72, 2014 05.
Artigo em Zh | MEDLINE | ID: mdl-24998648

RESUMO

OBJECTIVE: To investigate the protective effect of histone deacetylase inhibitor NL101 on L-homocysteine (HCA)-induced toxicity in rat neurons, and the toxic effect on normal rat neurons. METHODS: In the presence of NL101 at various concentrations, HCA (5 mmol/L)-induced changes in cell density, necrosis, and viability were determined in the mixed cultures of rat cortical cells and the primary cultures of rat neurons. The direct effect of NL101 on primary neurons was also observed in the absence of HCA. Histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was used as the control. After the treatments, cell viability, the density, and morphology of neurons and glial cells, and cell necrosis were determined. RESULTS: In the mixed cultures of cortical cells, NL101 had no effect on HCA (5 mmol/L)-induced cell number reduction at 0.001-10µmol/L; however, it significantly attenuated necrosis at 1-10 µmol/L, and increased neuronal number at 1 µmol/L. NL101 had no effect on the mixed cortical cells in the absence of HCA. In the primary neurons, NL101 reduced neuronal viability and mildly increased necrosis at 1-10 µmol/L in the absence of HCA, while it significantly attenuated HCA-induced neuronal viability reduction at 0.01-10 µmol/L and reduced neuronal necrosis at 1-10 µmol/L. The effects of NL101 were apparently similar to those of SAHA. CONCLUSION: NL101 has protective effect on HCA-induced neuronal injury but it is neurotoxic at high concentrations, which is similar to the typical histone deacetylase inhibitor SAHA.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Neurônios/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ratos
3.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 43(3): 287-92, 2014 05.
Artigo em Zh | MEDLINE | ID: mdl-24998651

RESUMO

OBJECTIVE: To investigate the effects of cysteinyl leukotriene (CysLT) receptor agonist leukotriene D4 (LTD4) on proliferation and migration in lung epithelial A549 cells. METHODS: The expression of CysLT1 receptor and CysLT2 receptor was determined by immunofluoresence staining in A549 cells. A549 cells were treated with LTD4 (0.01-100 nmol/L) for 24-72 h. Cell viability was detected by MTT reduction assay. Cell migration was determined by modified scratch and healing model. RESULTS: In A549 cells, CysLT1 receptor and CysLT2 receptor were mainly expressed in the cytoplasm, membrane and few in the nuclei. The treatment of LTD4 (0.01-100 nmol/L) for 24-72 h caused no effect on cell viability (Ps>0.05); when A549 cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h the cell viability was (103.00±4.46)%,(107.00±9.45)% and (105.00±9.02)% of control, respectively (Ps>0.05). The migration rate of A549 cells after scratching during the first 24 h was markedly greater than that during the second and third 24 h in the same concentration groups; however, no significant difference in migration rate was noticed when the cells were treated with different concentrations of LTD4 (0.01-100 nmol/L)(Ps>0.05). The migration of A549 cells was 1.15-fold, 1.21-fold and 1.06-fold of that of control when the cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h, respectively (Ps>0.05). CONCLUSION: The proliferation and migration of A549 cells are not changed when treated with 0.01-100 nmol LTD4 for up to 72h.


Assuntos
Células Epiteliais/citologia , Leucotrieno D4/farmacologia , Alvéolos Pulmonares/citologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos
4.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 43(3): 257-64, 2014 05.
Artigo em Zh | MEDLINE | ID: mdl-24998647

RESUMO

OBJECTIVE: To investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro. METHODS: Cultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R)or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R,and the effects were observed. RESULTS: ROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1µmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05),but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1µmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above. CONCLUSION: In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.


Assuntos
Acetatos/farmacologia , Antioxidantes/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Neurônios/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Quinolinas/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Ciclopropanos , Antagonistas de Leucotrienos/farmacologia , Neurônios/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sulfetos
5.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 43(3): 281-6, 2014 05.
Artigo em Zh | MEDLINE | ID: mdl-24998650

RESUMO

OBJECTIVE: To evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice. METHODS: In wild type and AQP4 gene knockout (AQP4-/-) mice, lung fibrosis was induced by injection of bleomycin (3 mg/kg) into the trachea and saline injection was used as a control. At d3, 7, 14, 28 after bleomycin-treatment, mice were randomly sacrificed in batch and the lung coefficient was determined. Serum levels of TGF-ß1 and TNF-α were measured by ELISA and hydroxyproline contents in lung tissue were determined by Alkaline hydrolysis method. H-E staining and Masson's staining were performed to examine the pathological changes of lung tissues after bleomycin-treatment. RESULTS: On d14 after bleomycin-treatment, the lung coefficients in wild type mice and AQP4-/- mice were 1.9-fold (12.69 ± 6.05 vs 6.80 ± 0.82, q=4.204, P<0.05) and 2.3-fold (14.05 ± 5.82 vs 6.05± 0.58, q=5.172, P<0.01) of that in control, respectively, but no significant difference was found between wild type and AQP4-/- mice in the lung coefficient value (P>0.05). The hydroxyproline contents in the lung increased after bleomycin-treatment; on d28, the lung hydroxyproline contents in wild type and in AQP4-/- mice were 1.55-fold (0.85 ± 0.22 g/mg vs 0.55 ± 0.14 µg/mg, q=4.313, P<0.05) and 1.4-fold (0.84 ± 0.13 µg/mg vs 0.60 ± 0.14µg/mg, q=4.595,P<0.05) of that in control, respectively, but no significant difference was noticed between wild type and AQP4-/- mice in lung hydroxyproline contents. There was a tendency that serum TGF-ß1 and TNF-α levels increased in bleomycin-treated mice, but no significant difference was found between wild type and AQP4-/- mice. AQP4-knockout showed no effects on pathological changes of lung tissues with H-E staining and Masson's staining in mice with bleomycin-induced lung fibrosis. CONCLUSION: AQP4 might not be involved in bleomycin-induced lung fibrosis in mice.


Assuntos
Aquaporina 4/genética , Bleomicina/toxicidade , Fibrose Pulmonar/induzido quimicamente , Animais , Masculino , Camundongos , Camundongos Knockout , Fibrose Pulmonar/genética
6.
J Pharmacol Exp Ther ; 346(2): 328-41, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23750020

RESUMO

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonist HAMI 3379 [3-({[(1S,3S)-3- carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy)benzoic acid] in comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1ß and tumor necrosis factor-α) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R- and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.


Assuntos
Ácidos Cicloexanocarboxílicos/farmacologia , Antagonistas de Leucotrienos/farmacologia , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Receptores de Leucotrienos/metabolismo , Acetatos/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Hipóxia Celular , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Ciclopropanos , Citocinas/metabolismo , Feminino , Glucose/metabolismo , Masculino , Microglia/metabolismo , Microglia/patologia , Necrose , Neurônios/metabolismo , Neurônios/patologia , Oxigênio/metabolismo , Fagocitose , Cultura Primária de Células , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Leucotrienos/agonistas , Sulfetos
7.
J Neuroinflammation ; 9: 145, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22734808

RESUMO

BACKGROUND: Transforming growth factor-ß 1 (TGF-ß 1) is an important regulator of cell migration and plays a role in the scarring response in injured brain. It is also reported that 5-lipoxygenase (5-LOX) and its products, cysteinyl leukotrienes (CysLTs, namely LTC4, LTD4 and LTE4), as well as cysteinyl leukotriene receptor 1 (CysLT1R) are closely associated with astrocyte proliferation and glial scar formation after brain injury. However, how these molecules act on astrocyte migration, an initial step of the scarring response, is unknown. To clarify this, we determined the roles of 5-LOX and CysLT1R in TGF-ß 1-induced astrocyte migration. METHODS: In primary cultures of rat astrocytes, the effects of TGF-ß 1 and CysLT receptor agonists on migration and proliferation were assayed, and the expression of 5-LOX, CysLT receptors and TGF-ß1 was detected. 5-LOX activation was analyzed by measuring its products (CysLTs) and applying its inhibitor. The role of CysLT1R was investigated by applying CysLT receptor antagonists and CysLT1R knockdown by small interfering RNA (siRNA). TGF-ß 1 release was assayed as well. RESULTS: TGF-ß 1-induced astrocyte migration was potentiated by LTD4, but attenuated by the 5-LOX inhibitor zileuton and the CysLT1R antagonist montelukast. The non-selective agonist LTD4 at 0.1 to 10 nM also induced a mild migration; however, the selective agonist N-methyl-LTC4 and the selective antagonist Bay cysLT2 for CysLT2R had no effects. Moreover, CysLT1R siRNA inhibited TGF-ß 1- and LTD4-induced astrocyte migration by down-regulating the expression of this receptor. However, TGF-ß 1 and LTD4 at various concentrations did not affect astrocyte proliferation 24 h after exposure. On the other hand, TGF-ß 1 increased 5-LOX expression and the production of CysLTs, and up-regulated CysLT1R (not CysLT2R), while LTD4 and N-methyl-LTC4 did not affect TGF-ß 1 expression and release. CONCLUSIONS: TGF-ß 1-induced astrocyte migration is, at least in part, mediated by enhanced endogenous CysLTs through activating CysLT1R. These findings indicate that the interaction between the cytokine TGF-ß 1 and the pro-inflammatory mediators CysLTs in the regulation of astrocyte function is relevant to glial scar formation.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Astrócitos/metabolismo , Movimento Celular/imunologia , Movimento Celular/fisiologia , Receptores de Leucotrienos/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Animais , Animais Recém-Nascidos , Araquidonato 5-Lipoxigenase/fisiologia , Astrócitos/citologia , Ativação Enzimática/fisiologia , Leucotrieno D4/farmacologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores de Leucotrienos/fisiologia , Fator de Crescimento Transformador beta1/farmacologia
8.
Acta Pharmacol Sin ; 33(12): 1511-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23085741

RESUMO

AIM: Cysteinyl leukotriene receptor 1 (CysLT(1) receptor) is located in epithelial cells, and translocates from the plasma membrane to the nucleus in a ligand-dependent manner. Here, we investigated whether CysLT(1) receptors translocated to the nucleus in endothelial cells after ischemic insult in vitro and whether it was involved in ischemic injury to endothelial cells. METHODS: EA.hy926 cell line, derived from human umbilical vein endothelial cells, was subjected to oxygen-glucose deprivation (OGD). The expression and distribution of CysLT(1) receptors were detected by immunofluorescent staining, immunogold labeling and immunoblotting analyses. Cell viability was evaluated using MTT reduction assay. Necrosis and apoptosis were determined by double fluorescent staining with propidium iodide and Hoechst 33342. RESULTS: CysLT(1) receptors were primarily distributed in the cytoplasm and nucleus in EA.hy926 cells, and few was found in the cell membrane. OGD induced the translocation of CysLT(1) receptors from the cytoplasm to the nucleus in a time-depen dent manner, with a peak reached at 6 h. OGD-induced nuclear translocation of CysLT(1) receptors was inhibited by pretreatment with the CysLT(1) receptor antagonist pranlukast (10 µmol/L), or by preincubation with NLS-pep, a peptide corresponding to the nuclear localization sequence of CysLT(1) receptor (10 µg/mL). However, zileuton, an inhibitor of 5-lipoxygenase that was a key enzyme in cysteinyl leukotriene generation, did not inhibit the nuclear translocation of CysLT(1) receptors. Moreover, preincubation with NLS-pep (0.4 µg/mL) significantly ameliorated OGD-induced cell viability reduction and necrosis. CONCLUSION: CysLT(1) receptors in endothelial cells translocate to the nucleus in a ligand-independent manner after ischemic insult in vitro, and it is involved in the ischemic injury.


Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , Receptores de Leucotrienos/metabolismo , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Técnicas de Cultura de Células , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Células Endoteliais/efeitos dos fármacos , Humanos , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Ligantes , Modelos Biológicos , Sinais de Localização Nuclear/farmacologia , Transporte Proteico
9.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 41(3): 259-66, 2012 05.
Artigo em Zh | MEDLINE | ID: mdl-22723160

RESUMO

OBJECTIVE: To determine the effect of montelukast, a cysteinyl leukotriene receptor 1 antagonist, on morphological changes in rat neurons after ischemic injury. METHODS: The in vivo ischemia injury was induced by oxygen-glucose deprivation (OGD) for 2 h and reperfusion (R) for 24 h (OGD/R) in rat neurons primary culture and mixed cortex culture. In the presence or absence of various concentrations of montelukast, neuron number, area of neuron, number of neuritis per neuron, branch number of primary neuritis and primary neurite length were determined for evaluating morphological changes in neurons. RESULTS: OGD/R significantly reduced neuron number, and altered neuron morphology. In cortical neuron cultures, montelukast (0.0001-1 µmol/L) attenuated OGD/R-induced reduction in neuron number, and inhibited OGD/R-induced increase in branch number of primary neuritis. In the mixed cultures, montelukast (0.0001-0.1 µmol/L) increased the primary neurite length, and reduced number of neuritis and branch number of primary neurite after OGD/R. CONCLUSION: Montelukast has a protective effect on ischemic injury in neurons.


Assuntos
Acetatos/farmacologia , Neurônios/patologia , Quinolinas/farmacologia , Animais , Animais Recém-Nascidos , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclopropanos , Glucose/farmacologia , Antagonistas de Leucotrienos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfetos
10.
Transl Psychiatry ; 12(1): 141, 2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379771

RESUMO

The cell adhesion molecule nectin3 and its presynaptic partner nectin1 have been linked to early-life stress-related cognitive disorders, but how the nectin1-nectin3 system contributes to stress-induced neuronal, circuit, and cognitive abnormalities remains to be studied. Here we show that in neonatally stressed male mice, temporal order and spatial working memories, which require the medial entorhinal cortex (MEC)-CA1 pathway, as well as the structural integrity of CA1 pyramidal neurons were markedly impaired in adulthood. These cognitive and structural abnormalities in stressed mice were associated with decreased nectin levels in entorhinal and hippocampal subregions, especially reduced nectin1 level in the MEC and nectin3 level in the CA1. Postnatal suppression of nectin1 but not nectin3 level in the MEC impaired spatial memory, whereas conditional inactivation of nectin1 from MEC excitatory neurons reproduced the adverse effects of early-life stress on MEC-dependent memories and neuronal plasticity in CA1. Our data suggest that early-life stress disrupts presynaptic nectin1-mediated interneuronal adhesion in the MEC-CA1 pathway, which may in turn contribute to stress-induced synaptic and cognitive deficits.


Assuntos
Transtornos da Memória , Células Piramidais , Estresse Psicológico , Animais , Masculino , Camundongos , Hipocampo/metabolismo , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Células Piramidais/metabolismo , Memória Espacial/fisiologia , Nectinas , Adesão Celular
11.
J Cardiovasc Pharmacol ; 57(4): 479-88, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21283018

RESUMO

OBJECTIVE: Recently, we reported that pranlukast, an antagonist of cysteinyl leukotriene receptor 1, attenuates ischemic injury in endothelial cells by decreasing reactive oxygen species (ROS) production and inhibiting nuclear factor-κB activation in a leukotriene-independent manner. In this study, we investigated the effect of pranlukast on oxidative stress injury induced by hydrogen peroxide (H2O2) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. METHODS AND RESULTS: We found that H2O2 reduced cell viability and increased lactate dehydrogenase release in a concentration- and time-dependent manner. Necrosis was the main death mode, and the necrotic rate increased 32% after exposure to 220 µM H2O2 for 4 hours. Pretreatment with pranlukast significantly ameliorated the reduced viability and the increased lactate dehydrogenase release and necrosis after exposure to H2O2. We next examined the mechanisms underlying the antinecrotic effects of pranlukast. The results showed that pranlukast attenuated excessive ROS production and ameliorated the reduced superoxide dismuase and glutathione peroxidase activity in EA.hy926 cells exposed to H2O2. Pranlukast also inhibited the collapse of mitochondrial membrane potential (MMP) induced by H2O2. Inhibition of ROS production by N-acetyl-l-cysteine, a powerful antioxidant, reduced MMP collapse and necrosis. Inhibition of MMP collapse by cyclosporine A, a mitochondrial permeability transition inhibitor, attenuated necrosis but failed to reduce ROS production. In addition, we found no expression of 5-lipoxygenase in EA.hy926 cells and zileuton, a 5-lipoxygenase inhibitor, did not affect the cellular injury induced by H2O2. CONCLUSION: Pranlukast protects endothelial cells from H2O2-induced necrosis by inhibiting ROS-mediated collapse of mitochondrial membrane potential, and this is leukotriene-independent.


Assuntos
Cromonas/farmacologia , Antagonistas de Leucotrienos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Peróxido de Hidrogênio/administração & dosagem , Peróxido de Hidrogênio/toxicidade , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Necrose/tratamento farmacológico , Necrose/patologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
12.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(2): 145-9, 2011 03.
Artigo em Zh | MEDLINE | ID: mdl-21488209

RESUMO

OBJECTIVE: To evaluate the role of water channel AQP4 in NMDA-induced brain injury in mice. METHODS: In AQP4 gene knockout (AQP4(-/-)) mice, brain injury was induced by microinjection of NMDA into the cortex. The injured area was determined by toluidine blue staining, degenerated neurons were detected by Fluro-Jade B staining, and increased blood-brain barrier (BBB) permeability was evaluated by IgG immunostaining. RESULT: Compared with wild-type mice, AQP4(-/-) mice exhibited increased cortical lesion area, aggravated neuron degeneration, and increased BBB disruption after NMDA microinjection. CONCLUSION: AQP4 may play a protective role in NMDA-induced brain injury in mice.


Assuntos
Aquaporina 4/genética , Encéfalo/patologia , N-Metilaspartato/toxicidade , Animais , Aquaporina 4/fisiologia , Barreira Hematoencefálica/patologia , Encéfalo/efeitos dos fármacos , Camundongos , Camundongos Knockout
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(2): 123-30, 2011 03.
Artigo em Zh | MEDLINE | ID: mdl-21488206

RESUMO

OBJECTIVE: To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity. METHODS: The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 µg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity. RESULT: After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 µmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 µmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 µmol/L and 7.5 µmol/L. CONCLUSION: HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.


Assuntos
Células HEK293 , Receptores de Leucotrienos/genética , Avaliação Pré-Clínica de Medicamentos , Humanos , Antagonistas de Leucotrienos , Transfecção
14.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(2): 139-44, 2011 03.
Artigo em Zh | MEDLINE | ID: mdl-21488208

RESUMO

OBJECTIVE: To investigate the role of cysteinyl leukotriene (CysLT) receptors in the differentiation of rat glioma C6 cells. METHODS: Rat glioma C6 cells were treated with the agonist LTD(4), the CysLT(1) receptor antagonist montelukast and the differentiation inducer forskolin. Cell morphology and GFAP protein expression were determined after treatments. RESULT: Forskolin (10 µmol/L) induced morphological changes and GFAP protein expression (cell differentiation) in C6 cells, but LTD(4) (0.1-100 nmol/L) did not induce these changes. Montelukast (1 µmol/L) alone did not affect C6 cell differentiation, while it induced the differentiation when combined with the LTD(4) (100 nmol/L). CONCLUSION: The CysLT(2) receptor may modulate the differentiation of rat glioma C6 cells.


Assuntos
Glioma/patologia , Antagonistas de Leucotrienos/farmacologia , Receptores de Leucotrienos/agonistas , Acetatos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colforsina/farmacologia , Ciclopropanos , Cisteína , Glioma/metabolismo , Leucotrieno D4/farmacologia , Leucotrienos , Quinolinas/farmacologia , Ratos , Sulfetos
15.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(2): 131-8, 2011 03.
Artigo em Zh | MEDLINE | ID: mdl-21488207

RESUMO

OBJECTIVE: To prepare and identify a polyclonal antibody (pAb) against (mouse) cysteinyl leukotriene receptor 1 (CysLT(1)) and to investigate the changes of CysLT(1) receptor expression in BV2 microglial cells after rotenone treatment. METHODS: Rabbits were immunized with KLH-coupled CysLT(1) peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by ELISA method, and the specificity of the pAb was tested by antigen blockade. After BV2 cells were treated with rotenone (0.01-1 µmol/L) for 24 h, the expression of CysLT(1) was determined by immunostaining, Western blotting and RT-PCR. RESULT: The pAb showed a titer of 1/32728, and was not cross-reacted with antigens of CysLT(2) receptor and GPR17. Immunostaining, Western blotting and RT-PCR analysis showed the expression of CysLT(1) receptor in BV2 microglia. Rotenone at 1µmol/L significantly induced an increased expression of CysLT(1) receptor. CONCLUSION: The prepared CysLT(1) receptor polyclonal antibody has a high titer and high specificity to meet testing requirements of Western blotting and immunostaining; CysLT(1) is associated with rotenone-induced injury of BV2 microglial cells.


Assuntos
Microglia/metabolismo , Receptores de Leucotrienos/metabolismo , Rotenona/farmacologia , Animais , Células Cultivadas , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Coelhos , Receptores de Leucotrienos/imunologia
16.
Acta Pharmacol Sin ; 31(2): 137-44, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20139896

RESUMO

AIM: To determine whether the flavonoid baicalin attenuates oxygen-glucose deprivation (OGD)-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase (5-LOX) activation in PC12 cells. METHODS: The effects of baicalin and the 5-LOX inhibitor zileuton on the changes induced by OGD/recovery or H(2)O(2) (an exogenous reactive oxygen species [ROS]) in green fluorescent protein-5-LOX-transfected PC12 cells were compared. RESULTS: Both baicalin and zileuton attenuated OGD/recovery- and H(2)O(2)-induced injury and inhibited OGD/recovery-induced production of 5-LOX metabolites (cysteinyl leukotrienes) in a concentration-dependent manner. However, baicalin did not reduce baseline cysteinyl leukotriene levels. Baicalin also reduced OGD/recovery-induced ROS production and inhibited 5-LOX translocation to the nuclear envelope and p38 phosphorylation induced by OGD/recovery and H(2)O(2). In contrast, zileuton did not show these effects. CONCLUSION: Baicalin can inhibit 5-LOX activation after ischemic injury, which may partly result from inhibition of the ROS/p38 mitogen-activated protein kinase pathway.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Flavonoides/farmacologia , Glucose/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Animais , Ativação Enzimática , Peróxido de Hidrogênio/metabolismo , Células PC12 , Fosforilação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Sheng Li Xue Bao ; 62(2): 101-8, 2010 Apr 25.
Artigo em Zh | MEDLINE | ID: mdl-20401443

RESUMO

The aim of the present study is to investigate the role of nordihydroguaiaretic acid (NDGA) on inflammatory cells accumulation after focal cerebral ischemia and the underlying mechanism. Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion (MCAO) followed by 72 h of reperfusion. NDGA (5 and 10 mg/kg) was administered intraperitoneally 30 min, 2, 24, 48 h after reperfusion, respectively. The brain injuries were observed by neurological and histological examination. Endogenous IgG exudation, neutrophils and macrophages/microglia accumulation, and intercellular adhesion molecule-1 (ICAM-1) protein expression were determined by immunohistochemistry 72 h after reperfusion. ICAM-1 mRNA was determined by RT-PCR 72 h after reperfusion. The catalysates of 5-lipoxygenase (5-LOX), leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs), were evaluated by ELISA 3 h after reperfusion. The results showed that NDGA ameliorated neurological dysfunction, decreased infarct volume, and inhibited endogenous IgG exudation, neutrophils infiltration, ICAM-1 mRNA and protein expression 72 h after reperfusion. Moreover, NDGA reduced the levels of LTB4 and CysLTs 3 h after reperfusion. However, NDGA did not reduce the accumulation of macrophages/microglia 72 h after reperfusion. These results suggest that NDGA decreases neutrophil infiltration in the subacute phase of focal cerebral ischemia via inhibiting 5-LOX activation.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Isquemia Encefálica/fisiopatologia , Inflamação/fisiopatologia , Inibidores de Lipoxigenase/farmacologia , Masoprocol/farmacologia , Neutrófilos/efeitos dos fármacos , Animais , Isquemia Encefálica/complicações , Imunoglobulina G/imunologia , Inflamação/etiologia , Inflamação/prevenção & controle , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucotrieno B4/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/prevenção & controle
18.
J Neurosci Res ; 87(4): 991-1001, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18951527

RESUMO

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Hipóxia Celular , Glucose/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Antioxidantes/farmacologia , Antipirina/análogos & derivados , Antipirina/farmacologia , Araquidonato 5-Lipoxigenase/genética , Ácidos Cafeicos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Edaravone , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Leucotrienos/metabolismo , Inibidores de Lipoxigenase , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/antagonistas & inibidores , Células PC12 , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
19.
J Cardiovasc Pharmacol ; 53(1): 77-85, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19129732

RESUMO

The anti-inflammatory effects of pranlukast, an antagonist of cysteinyl leukotriene receptor 1, may be rendered not only by antileukotriene activity but also by other pharmacological activities. Previous studies indicate that pranlukast reduces ischemic tissue injury partially through decreasing vascular permeability, but its effect on ischemic injury in endothelial cells is not known. Thus, in this study, we investigated the effect of pranlukast on ischemia-like injury induced by oxygen-glucose deprivation (OGD) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. We found that cell viability was reduced, lactate dehydrogenase release was increased 4-8 hours after OGD, and necrosis was induced 8 hours after OGD. Production of reactive oxygen species (ROS) increased by 211%, 176%, and 128%, respectively, 0.5, 1, and 2 hours after OGD. Nuclear factor-kappaB (NF-kappaB) was translocated to the nuclei 4-8 hours after OGD. Pranlukast ameliorated the reduced viability, the increased lactate dehydrogenase release, and necrosis after OGD. It also reduced ROS production and inhibited NF-kappaB nuclear translocation after OGD. The ROS scavenger, edaravone, inhibited OGD-induced nuclear translocation of NF-kappaB as well. Edaravone and pyrrolidine dithiocarbamate (a specific NF-kappaB inhibitor) protected endothelial cells from the OGD-induced injury. However, zileuton, a 5-lipoxygenase inhibitor, did not affect the cell injury, ROS production, and NF-kappaB nuclear translocation after OGD. The exogenous leukotriene D4 did not induce cell injury, ROS production, and NF-kappaB translocation. Thus, we conclude that pranlukast protects endothelial cells from ischemia-like injury via decreasing ROS production and inhibiting NF-kappaB activation, which is leukotriene independent.


Assuntos
NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/farmacologia , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromonas , Células Endoteliais/metabolismo , Glucose/genética , Glucose/metabolismo , Glucose/farmacologia , Humanos , Isquemia/genética , Isquemia/metabolismo , Antagonistas de Leucotrienos/metabolismo , Antagonistas de Leucotrienos/farmacologia , Leucotrieno D4/genética , Leucotrieno D4/metabolismo , Leucotrieno D4/farmacologia , Leucotrienos/genética , Leucotrienos/metabolismo , Leucotrienos/farmacologia , NF-kappa B/genética , Necrose/genética , Necrose/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia
20.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 38(6): 584-90, 2009 11.
Artigo em Zh | MEDLINE | ID: mdl-20014483

RESUMO

OBJECTIVE: To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells. METHODS: Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4. RESULT: RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells. CONCLUSION: The eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.


Assuntos
Vetores Genéticos/genética , Receptores Acoplados a Proteínas G/biossíntese , Transfecção , Animais , Sequência de Bases , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA