RESUMEN
BACKGROUND: Ischemic stroke is one of the leading causes of mortality worldwide. The available treatments are not effective. Phosphodiesterase 9A (PDE9A) is an intracellular cyclic guanosine monophosphate (cGMP) hydrolase considered to be a promising therapeutic target for brain diseases. This study explored neuroprotective effects and the underlying mechanism of LW33, a novel PDE9A inhibitor, on ischemic stroke in vitro and in vivo. METHODS: A middle cerebral artery occlusion (MCAO) model was established in adult male Sprague-Dawley rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in human SH-SY5Y cells to mimic ischemia-reperfusion injury in vitro. RESULTS: LW33 increased cell viability, reduced lactate dehydrogenase activity, and OGD/R-induced apoptosis of SH-SY5Y cells. The protective effects of LW33 against stroke occurred in the recovery phase. LW33 administration significantly reduced cerebral infarction volume in MCAO rats, without causing significant deformation or necrosis of neurons in the cortex. LW33 also improved learning and cognitive dysfunction and reduced other pathological changes in MCAO rats in the recovery period. Moreover, LW33 stimulated the cGMP/PKG/CREB pathway and up-regulated the expression of the apoptosis-related proteins, and this effect was reversed by KT5823 treatment. CONCLUSION: LW33 inhibited cell apoptosis and promoted neuronal repair to alleviate OGD/R and MCAO induced pathological alterations via the cGMP/PKG/CREB pathway, indicating that LW33 may be a promising therapeutic target for ischemic stroke.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Fármacos Neuroprotectores , Daño por Reperfusión , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevención & control , GMP Cíclico , Glucosa/farmacología , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Oxígeno/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Transducción de SeñalRESUMEN
We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPAR gamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ(2)-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ(2), but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ(2) was partly, but not completely, blocked by PPAR gamma antagonist (GW9662) suggesting the 15d-PGJ(2) exerted its effect by PPAR gamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ(2) in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Condrosarcoma/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Condrosarcoma/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
OBJECTIVE: To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis. METHODS: Autoantibody-mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6-7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16INK4a and p21(WAF1/Cip1) were examined by real-time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16INK4a and p21(WAF1/Cip1) were determined by chromatin immunoprecipitation assay. RESULTS: A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor alpha and interleukin-1beta in the synovial tissues of mice with AMA. FK228 inhibited the in vitro proliferation of RASFs in a dose-dependent manner. Treatment of cells with FK228 induced the expression of p16INK4a and up-regulated the expression of p21(WAF1/Cip1). These effects of FK228 on p16INK4a and p21(WAF1/Cip1) were related to the acetylation of the promoter region of the genes. CONCLUSION: Our findings strongly suggest that systemic administration of HDA inhibitors may represent a novel therapeutic target in RA by means of cell cycle arrest in RASFs via induction of p16INK4a expression and increase in p21(WAF1/Cip1) expression.
Asunto(s)
Artritis Experimental/patología , Artritis Reumatoide/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Ciclinas/análisis , Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Membrana Sinovial/patología , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Western Blotting , Proteínas de Ciclo Celular/análisis , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Depsipéptidos/administración & dosificación , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Fibroblastos/patología , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos DBA , Reacción en Cadena de la PolimerasaRESUMEN
Vascular endothelial growth factor (VEGF)-mediated angiogenesis is essential for bone formation. However, the effect of VEGF on osteoblastic cells during osteoblastogenesis is still controversial. The aim of this study was to clarify the relationship between osteoblastic cells derived from human mesenchymal stem cells (MSCs) and VEGF in the early stage of osteoblastic differentiation. Continuous dexamethasone treatment with a low concentration stimulated osteoblastogenesis of MSCs and the expression of VEGF121 mRNA. The VEGF secretion from osteoblastic cells also increased along with osteoblastogenesis. Neuropilin-1, which mainly binds VEGF165, was detected at all stages during early osteoblastogenesis, but VEGF receptor-1 and -2 were not detected on RT-PCR analyses. In this study, VEGF had no direct effect on the proliferation of osteoblastic cells. However, the secreted VEGF in the conditioned medium of osteoblastic cells exhibited high angiogenic power as to endothelial cell proliferation. Our findings indicated that VEGF121 principally acts as the main angiogenic factor in the early stage of human osteoblastogenesis. The present study also demonstrated the differential expression of VEGF121 during osteoblastogenesis. The increase of VEGF in the early stage might be a useful marker of induction of bone formation due to human MSCs.