RESUMO
Alzheimer's disease (AD) is a multi-faceted neurodegenerative disease. Thus, current therapeutic strategies require multitarget-drug combinations to treat or prevent the disease. At the present time, single drugs have proven to be inadequate in terms of addressing the multifactorial pathology of AD, and multitarget-directed drug design has not been successful. Based on these points of views, it is judged that combinatorial drug therapies that target several pathogenic factors may offer more attractive therapeutic options. Thus, we explored that the combination therapy with lower doses of cilostazol and aripiprazole with add-on donepezil (CAD) might have potential in the pathogenesis of AD. In the present study, we found the superior efficacies of donepezil add-on with combinatorial mixture of cilostazol plus aripiprazole in modulation of expression of AD-relevant genes: Aß accumulation, GSK-3ß, P300, acetylated tau, phosphorylated-tau levels, and activation of α-secretase/ADAM 10 through SIRT1 activation in the N2a Swe cells expressing human APP Swedish mutation (N2a Swe cells). We also assessed that CAD synergistically raised acetylcholine release and choline acetyltransferase (CHAT) expression that were declined by increased ß-amyloid level in the activated N2a Swe cells. Consequently, CAD treatment synergistically increased neurite elongation and improved cell viability through activations of PI3K, BDNF, ß-catenin and a7-nicotinic cholinergic receptors in neuronal cells in the presence of Aß1-42. This work endorses the possibility for efficient treatment of AD by supporting the synergistic therapeutic potential of donepezil add-on therapy in combination with lower doses of cilostazol and aripiprazole.
RESUMO
Cilostazol (an inhibitor of phosphodiesterase type III) has potent anti-inflammatory effects, and celecoxib (a COX-2 specific inhibitor) has been reported to improve the unsatisfactory profile of NSAIDs. This study investigated the synergistic anti-arthritic potential of a multitarget-based cotreatment, in which cilostazol was used as an add-on therapy for celecoxib, using the synovial fibroblasts of RA patients (RASFs). Increased COX-2 protein expression and PGE2 synthesis by LPS (1 µg/ml) were significantly and synergistically attenuated by cotreatment with 3 µM cilostazol and 30 µM celecoxib, whereas monotherapy with either cilostazol or celecoxib showed little effects. IL-10 mRNA levels in LPS-treated RASFs were moderately increased by pretreating cilostazol (1-10 µM) or celecoxib (10-50 µM) monotherapy, but 3 µM of cilostazol add-on for 30 µM celecoxib treatment synergistically increased IL-10 mRNA levels and IL-10 release to culture media. Cilostazol and celecoxib cotreatment similarly showed synergistic increase in SOCS3 mRNA levels. Accordingly, LPS-induced increases in IL-1ß and IL-6 mRNA and TNF-α release were significantly and synergistically diminished by cilostazol and celecoxib cotreatment. Moreover, synovial cell proliferation was significantly suppressed by cotreatment. Summarizing, cotreatment with cilostazol and celecoxib exhibited a synergistic increase in IL-10 production and SOCS3 expressions, thereby resulted in synergistic decreases in IL-1ß mRNA, IL-6 mRNA expression and TNF-α synthesis in association with synergistic decreases in COX-2 and PGE2 protein expression in the RA synovial fibroblasts. In conclusion, these observations suggest low concentrations of cilostazol and celecoxib cotreatment may ensure a synergistic anti-arthritic potential.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Celecoxib/farmacologia , Cilostazol/farmacologia , Citocinas/antagonistas & inibidores , Interleucina-10/biossíntese , Proteína 3 Supressora da Sinalização de Citocinas/biossíntese , Líquido Sinovial/imunologia , Artrite Reumatoide/imunologia , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Sinergismo Farmacológico , Fibroblastos/imunologia , Humanos , Interleucina-10/genética , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Líquido Sinovial/citologiaRESUMO
The aim of this study was to determine the effects and underlying mechanism of aripiprazole (APZ) augmentation for cilostazol (CLS)-treated post-ischemic stroke mice that were exposed to chronic mild stress (CMS). Compared to treatment with either APZ or CLS alone, the combined treatment resulted in a greater reduction in depressive behaviors, including anhedonia, despair-like behaviors, and memory impairments. This treatment also significantly reduced atrophic changes in the striatum, cortex, and midbrain of CMS-treated ischemic mice, and inhibited neuronal cell apoptosis, particularly in the striatum and the dentate gyrus of the hippocampus. Greater proliferation of neuronal progenitor cells was also observed in the ipsilateral striatum of the mice receiving combined treatment compared to mice receiving either drug alone. Phosphorylation of the cyclic adenosine monophosphate response element binding protein (CREB) was increased in the striatum, hippocampus, and midbrain of mice receiving combined treatment compared to treatment with either drug alone, particularly in the neurons of the striatum and hippocampus, and dopaminergic neurons of the midbrain. Our results suggest that APZ may augment the antidepressant effects of CLS via co-regulation of the CREB signaling pathway, resulting in the synergistic enhancement of their neuroprotective effects.
Assuntos
Antidepressivos/farmacologia , Aripiprazol/farmacologia , Estresse Psicológico , Acidente Vascular Cerebral/psicologia , Tetrazóis/farmacologia , Animais , Atrofia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cilostazol , Depressão/tratamento farmacológico , Modelos Animais de Doenças , Sinergismo Farmacológico , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Fenótipo , Fosforilação/efeitos dos fármacos , Estresse Psicológico/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologiaRESUMO
Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12-24h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP(+) giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.
RESUMO
High-mobility group box protein 1 (HMGB1), a nonhistone nuclear protein and a cytokine mediator, is implicated in the pathogenesis of rheumatoid arthritis (RA). Extracellular HMGB1 binds to its receptors and triggers downstream signal cascade leading to the perpetuation of synovitis and local tissue invasion. Here, we investigated a novel role of HMGB1 in regulating hypoxia-inducible factor (HIF)-1α to mediate angiogenesis in RA synovium. HIF-1α mRNA levels and activities in synovial fibroblasts from RA patients were enhanced by HMGB1. Pharmacological inhibition of TLR4 and NF-kappaB activation blocked the HMGB1-dependent upregulation of HIF-1α mRNA expression and its activity, suggesting the involvement of transcriptional regulation. HMGB1 stimulated expression of vascular endothelial growth factor (VEGF), and inhibition of HIF-1α attenuated HMGB1-induced VEGF. Conditioned media derived from HMGB1-stimulated synovial fibroblasts enhanced tube formation in human microvascular endothelial cells by upregulating HIF-1α. In the joint tissues of mice with collagen-induced arthritis, treatment with anti-HMGB1 neutralizing antibody prevented blood vessel formation in association with decreased expression of HIF-1α. These observations support the idea that increased HMGB1 induces an extension of inflamed synovium by accelerating angiogenesis in RA through enhancement of HIF-1α activation. Therefore, inhibition of HMGB1 could prove beneficial for the treatment of angiogenesis in RA.
Assuntos
Artrite Reumatoide/patologia , Proteína HMGB1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica/patologia , Membrana Sinovial/irrigação sanguínea , Animais , Anticorpos Neutralizantes/farmacologia , Artrite Experimental , Células Endoteliais/metabolismo , Ativação Enzimática , Feminino , Proteína HMGB1/antagonistas & inibidores , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno , Receptor 4 Toll-Like/antagonistas & inibidores , Transcrição Gênica , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
AIM: Increasing evidence suggests that probucol, a lipid-lowering agent with anti-oxidant activities, may be useful for the treatment of ischemic stroke with hyperlipidemia via reduction in cholesterol and neuroinflammation. In this study we examined whether probucol could protect against brain ischemic injury via anti-neuroinflammatory action in normal and hyperlipidemic mice. METHODS: Primary mouse microglia and murine BV2 microglia were exposed to lipopolysaccharide (LPS) for 3 h, and the release NO, PGE2, IL-1ß and IL-6, as well as the changes in NF-κB, MAPK and AP-1 signaling pathways were assessed. ApoE KO mice were fed a high-fat diet containing 0.004%, 0.02%, 0.1% (wt/wt) probucol for 10 weeks, whereas normal C57BL/6J mice received probucol (3, 10, 30 mg·kg(-1)·d(-1), po) for 4 d. Then all the mice were subjected to focal cerebral ischemia through middle cerebral artery occlusion (MCAO). The neurological deficits were scored 24 h after the surgery, and then brains were removed for measuring the cerebral infarct size and the production of pro-inflammatory mediators. RESULTS: In LPS-treated BV2 cells and primary microglial cells, pretreatment with probucol (1, 5, 10 µmol/L) dose-dependently inhibited the release of NO, PGE2, IL-1ß and IL-6, which occurred at the transcription levels. Furthermore, the inhibitory actions of probucol were associated with the downregulation of the NF-κB, MAPK and AP-1 signaling pathways. In the normal mice with MCAO, pre-administration of probucol dose-dependently decreased the infarct volume and improved neurological function. These effects were accompanied by the decreased production of pro-inflammatory mediators (iNOS, COX-2, IL-1, IL-6). In ApoE KO mice fed a high-fat diet, pre-administration of 0.1% probucol significantly reduced the infarct volume, improved the neurological deficits following MCAO, and decreased the total- and LDL-cholesterol levels. CONCLUSION: Probucol inhibits LPS-induced microglia activation and ameliorates cerebral ischemic injury in normal and hyperlipidemic mice via its anti-neuroinflammatory actions, suggesting that probucol has potential for the treatment of patients with or at risk for ischemic stroke and hyperlipidemia.
Assuntos
Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Hiperlipidemias/complicações , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Probucol/farmacologia , Probucol/uso terapêutico , Animais , Apolipoproteínas E/genética , Isquemia Encefálica/patologia , Dieta Hiperlipídica , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Hiperlipidemias/metabolismo , Infarto/complicações , Infarto/tratamento farmacológico , Infarto/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microglia/citologia , Óxido Nítrico/metabolismo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacosRESUMO
The accumulation of plaques of ß-amyloid (Aß) peptides, a hallmark of Alzheimer's disease, results from the sequential cleavage of amyloid precursor protein (APP) by activation of ß- and γ-secretases. However, the production of Aß can be avoided by alternate cleavage of APP by α-and γ-secretases. We hypothesized that cilostazol attenuates Aß production by increasing a disintegrin and metalloproteinase 10 (ADAM10)/α-secretase activity via SIRT1-coupled retinoic acid receptor-ß (RARß) activation in N2a cells expressing human APP Swedish mutation (N2aSwe). To evoke endogenous Aß overproduction, the culture medium was switched from medium containing 10% fetal bovine serum (FBS) to medium containing 1% FBS, and cells were cultured for 3â¼24 hr. After depletion of FBS in media, N2aSwe cells showed increased accumulations of full-length APP (FL-APP) and Aß in a time-dependent manner (3-24 hr) in association with decreased ADAM10 protein expression. When pretreated with cilostazol (10-30 µM), FL-APP and Aß levels were significantly reduced, and ADAM10 and α-secretase activities were restored. Furthermore, the effect of cilostazol on ADAM10 expression was antagonized by pretreating Rp-cAMPS and sirtinol and by SIRT1-gene silencing. In the N2aSwe cells overexpressing the SIRT1 gene, ADAM10, and sAPPα levels were significantly elevated. In addition, like all-trans retinoic acid, cilostazol enhanced the protein expressions of RARß and ADAM10, and the cilostazol-stimulated ADAM10 elevation was significantly attenuated by LE135 (a RARß inhibitor), sirtinol, and RARß-gene silencing. In conclusion, cilostazol suppresses the accumulations of FL-APP and Aß by activating ADAM10 via the upregulation of SIRT1-coupled RARß.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Metaloproteinase 10 da Matriz/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Receptores do Ácido Retinoico/metabolismo , Sirtuína 1/metabolismo , Tetrazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Cilostazol , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Desintegrinas , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Naftóis/farmacologia , Neuroblastoma/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sirtuína 1/genética , Tionucleotídeos/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
ß-Amyloid (Aß) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3ß. Aß was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aß accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3ß P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 µM) and resveratrol (20 µM). Moreover, decreased expression of SIRT1 and its activity by Aß were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 µM, PKA inhibitor), TBCA (20 µM, inhibitor of CK2), and sirtinol (20 µM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.
Assuntos
Queratina-2/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Sirtuína 1/biossíntese , Tauopatias/metabolismo , Tetrazóis/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Western Blotting , Linhagem Celular , Cilostazol , Imunofluorescência , Humanos , Camundongos , Neurônios/metabolismo , Transfecção , Proteínas tau/metabolismoRESUMO
Amyloid-ß peptide (Aß) deposits in the brain are critical in the neurotoxicity induced by Aß. This study elucidates the underlying signaling pathway by which cilostazol protects HT22 neuronal cells from Aß(1-40) (3-30 µM)-induced deterioration of cell proliferation, viability, and neurite elongation. Cilostazol rescued HT22 cells from the apoptotic cell death induced by Aß toxicity through the downregulation of phosphorylated p53 (Ser15), Bax, and caspase-3 and the upregulation of Bcl-2 expression, which improved neuronal cell proliferation and viability. Furthermore, Aß(1-40) suppressed both phosphorylated CK2α protein expression and CK2 activity in the cytosol; these were concentration dependently recovered by cilostazol (3-30 µM). Cilostazol significantly increased the levels of GSK-3ß phosphorylation at Ser9 and ß-catenin phosphorylation at Ser675 in the cytosol and nucleus. Cilostazol effects were reversed by KT5720 (1 µM, PKA inhibitor) and TBCA (40 µM, inhibitor of CK2) and CK2α knockdown by siRNA transfection. Likewise, Aß-stimulated GSK-3ß phosphorylation at Tyr 216 was decreased by cilostazol in the control but not in the CK2α siRNA-transfected cells. Furthermore, the Aß (10 µM)-induced suppression of neurite elongation was recovered by cilostazol; this recovery was attenuated by inhibitors such as KT5720 and TBCA and blocked by CK2α knockdown. In conclusion, increased cAMP-dependent protein kinase-linked CK2α activation underlies the pharmacological effects of cilostazol in downregulating p53 phosphorylation at Ser15 and upregulating GSK-3ß phosphorylation at Ser9/ß-catenin phosphorylation at Ser675, thereby suppressing Aß(1-40)-induced neurotoxicity and improving neurite elongation.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Caseína Quinase II/metabolismo , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Tetrazóis/farmacologia , Animais , Apoptose , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cilostazol , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Neuritos/metabolismo , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Alzheimer's disease (AD), which is characterized by progressive cognitive impairment, is the most common neurodegenerative disease. Here, we investigated the preventive effect of a phosphodiesterase III inhibitor, cilostazol against cognitive decline in AD mouse model. In vitro studies using N2a cells stably expressing human amyloid precursor protein Swedish mutation (N2aSwe) showed that cilostazol decreased the amyloid ß (Aß) levels in the conditioned medium and cell lysates. Cilostazol attenuated the expression of ApoE, which is responsible for Aß aggregation, in N2aSwe. Intracerebroventricular injection of Aß(25-35) in C57BL/6J mice resulted in increased immunoreactivity of Aß and p-Tau, and microglia activation in the brain. Oral administration of cilostazol for 2 weeks before Aß administration and once a day for 4 weeks post-surgery almost completely prevented the Aß-induced increases of Aß and p-Tau immunoreactivity, as well as CD11b immunoreactivity. However, post-treatment with cilostazol 4 weeks after Aß administration, when Aß was already accumulated, did not prevent the Aß-induced neuropathological responses. Furthermore, cilostazol did not affect the neprilysin and insulin degrading enzymes involved in the degradation of the Aß peptide, but decreased ApoE levels in Aß-injected brain. In addition, cilostazol significantly improved spatial learning and memory in Aß-injected mice. The findings suggest that a phosphodiesterase III inhibitor, cilostazol significantly decreased Aß accumulation and improved memory impairment induced by Aß(25-35). The beneficial effects of cilostazol might be explained by the reduction of Aß accumulation and tau phosphorylation, not through an increase in Aß degradation but via a significant decrease in ApoE-mediated Aß aggregation. Cilostazol may be the basis of a novel strategy for the therapy of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Inibidores da Fosfodiesterase 3/uso terapêutico , Tetrazóis/uso terapêutico , Doença de Alzheimer/complicações , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular Tumoral , Cilostazol , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/etiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/farmacologia , Fosforilação , Comportamento Espacial , Proteínas tau/metabolismoRESUMO
Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 µM) stimulated up-regulation of integrins ß1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 µM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 µM) and GGTI-298 (Rap1 inhibitor, 20 µM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 µM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin ß1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.
Assuntos
Quimiotaxia/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrina beta1/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Tetrazóis/farmacologia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , Células Cultivadas , Quimiotaxia/fisiologia , Cilostazol , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Recém-Nascido , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Tetrazóis/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Hypercholesterolemia may increase stroke risk by accelerating atherosclerosis, narrowing the luminal diameter in cerebral vessels, and disrupting both vascular endothelial and smooth muscle function. In the present study, we investigated the beneficial effects of combinatorial therapy with probucol and cilostazol on focal cerebral ischemia with hypercholesterolemia. Apolipoprotein E (ApoE) knockout (KO) mice were fed a high-fat diet with or without 0.5% probucol and/or 0.2% cilostazol for 10 weeks. Probucol alone and probucol and cilostazol significantly decreased total, low-density lipoprotein, and high-density lipoprotein cholesterol, whereas cilostazol did not affect the plasma cholesterol levels in ApoE KO mice. Administration of probucol alone and cilostazol alone significantly decreased atherosclerotic lesion area in the aorta, with a significant decrease evident using combinatorial administration. Middle cerebral artery occlusion resulted in significantly larger infarct volumes in ApoE KO mice fed 10 weeks of high-fat diet compared with those in ApoE KO mice fed a regular diet. The infarct volume was reduced significantly using probucol alone or cilostazol alone and even was reduced significantly by their combinatorial administration. Consistent with a larger infarct size, the combinatorial therapy prominently improved neurological function. The combinatorial administration increased cerebral blood flow during ischemia. Expression of endothelial nitric oxide synthase and adiponectin in the cortex were decreased by high-fat diet but were elevated by combinatorial treatment. Adiponectin expression colocalized within the cerebral vascular endothelium. The data suggest that the combination of probucol and cilostazol prevents cerebrovascular damage in focal cerebral ischemic mice with hypercholesterolemia by up-regulation of endothelial nitric oxide synthase and adiponectin.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Hipercolesterolemia/tratamento farmacológico , Probucol/administração & dosagem , Tetrazóis/administração & dosagem , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Cilostazol , Quimioterapia Combinada , Hipercolesterolemia/complicações , Hipercolesterolemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
OBJECTIVE: To assess the effects of cilostazol in inhibiting proliferation and enhancing apoptosis in synovial cells from patients with rheumatoid arthritis (RA). METHODS: Synovial cell proliferation was measured by MTT assay. The expression of NF-kappaB, IkappaBalpha, Bcl-2, Bax, heme oxygenase 1 (HO-1), and Nrf2 was determined by Western blotting. RESULTS: Cilostazol suppressed synovial cell proliferation by arresting the G(2)/M phases of the cell cycle, and this was reversed by KT5720, an inhibitor of protein kinase A. Cilostazol increased the number of TUNEL-positive cells, with increased cytochrome c release and apoptosis-inducing factor translocation as well as increased caspase 3 activation. Cilostazol (10 microM) and cobalt protoporphyrin IX (CoPP) increased HO-1 messenger RNA and protein expression. These effects were suppressed by zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor. Cilostazol and CoPP significantly increased IkappaBalpha in the cytosol and decreased NF-kappaB p65 expression in the nucleus. Increased expression of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 induced by lipopolysaccharide was attenuated by cilostazol and CoPP, and this was reversed by ZnPP. In mice with collagen-induced arthritis treated with cilostazol (10 and 30 mg/kg/day), paw thickness was decreased with increased apoptotic cells in the joints. In synovial cells transfected with small interfering RNA (siRNA) targeting HO-1, cilostazol did not suppress expression of TNFalpha, IL-1beta, and IL-6, in contrast to findings with negative control cells. Cilostazol- and CoPP-induced HO-1 expression was diminished in cells transfected with Nrf2 siRNA. CONCLUSION: Cilostazol suppressed proliferation of synovial cells from RA patients by enhancing apoptosis, and also inhibited cytokine production via mediation of cAMP-dependent protein kinase activation-coupled Nrf2-linked HO-1 expression.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Citocinas/biossíntese , Heme Oxigenase-1/biossíntese , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Membrana Sinovial/efeitos dos fármacos , Tetrazóis/farmacologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Western Blotting , Divisão Celular/efeitos dos fármacos , Cilostazol , Indução Enzimática , Camundongos , Camundongos Endogâmicos DBA , Membrana Sinovial/citologiaRESUMO
BACKGROUND: Helicobacter pylori CagA dysregulates cell signaling pathways and leads to targeted transcriptional up-regulation of genes implicated in gastric cell injury. The aim of this study was to determine the effects of rebamipide on CagA-induced effects on gastric epithelial cells. We investigated the effects of rebamipide treatment (pre- or post-treatment before or after CagA transfection) on CagA-induced gastric cell injury. METHOD: We evaluated the morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization by confocal microscopy, IL-8 production by ELISA, and NF-κB activation by luciferase assay in AGS gastric epithelial cells and MDCK cells. RESULTS: Transfection of CagA into gastric epithelial cells induced morphologic changes (hummingbird phenotype), ZO-1 mislocalization, and IL-8 production in gastric epithelial cells. Pre-treatment with rebamipide inhibits CagA-induced effects on gastric epithelial cells, including morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization, IL-8 production, and NF-κB activity. CONCLUSIONS: These results suggest that rebamipide might have a potential role in the protection of H. pylori CagA-induced effects on gastric epithelial cells.
Assuntos
Alanina/análogos & derivados , Antígenos de Bactérias/toxicidade , Antioxidantes/farmacologia , Proteínas de Bactérias/toxicidade , Células Epiteliais/efeitos dos fármacos , Quinolonas/farmacologia , Estômago/citologia , Alanina/farmacologia , Animais , Antiulcerosos/farmacologia , Linhagem Celular , Cães , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estômago/efeitos dos fármacos , Fatores de Tempo , Proteína da Zônula de Oclusão-1RESUMO
DA-1241 is a novel small molecule G protein-coupled receptor 119 (GPR119) agonist in early clinical development for type 2 diabetic patients. This study aimed to elucidate the pharmacological characteristics of DA-1241 for its hypoglycemic action. DA-1241 potently and selectively activated GPR119 with enhanced maximum efficacy. DA-1241 increased intracellular cAMP in HIT-T15 insulinoma cells (EC50, 14.7 nM) and increased insulin secretion (EC50, 22.3 nM) in association with enhanced human insulin promoter activity. Accordingly, postprandial plasma insulin levels were increased in mice after single oral administration of DA-1241. Postprandial glucose excursion was significantly reduced by single oral administration of DA-1241 in wild-type mice but not in GPR119 knockout mice. GLP-1 secretion was increased by DA-1241 treatment in mice. Thus, upon combined sitagliptin and DA-1241 treatment in high-fat diet/streptozotocin (HFD/STZ)-induced diabetic mice, plasma active GLP-1 levels were synergistically increased. Accordingly, blood glucose and triglyceride levels were significantly lowered both by DA-1241 and sitagliptin alone and in combination. Immunohistochemical analysis revealed that ß-cell mass with reduced PDX1 levels in the islets from HFD/STZ diabetic mice was significantly preserved by DA-1241, whereas increased glucagon and BiP levels were significantly suppressed. In HIT-T15 insulinoma cells subjected to ER stress, decreased cell viability was significantly rescued by treatment with DA-1241. Additionally, increased apoptosis was largely attenuated by DA-1241 by inhibiting BiP and CHOP expression through suppression of p38 MAPK. In conclusion, these studies provide evidence that DA-1241 can be a promising antidiabetic drug by potentially preserving pancreatic functions through suppressing ER stress and increasing PDX1 expression.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Hipoglicemiantes/farmacologia , Oxidiazóis/farmacologia , Pâncreas/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Transativadores/metabolismo , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Linhagem Celular Tumoral , Cricetinae , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Proteínas de Homeodomínio/genética , Insulina/sangue , Masculino , Camundongos Endogâmicos ICR , Camundongos Knockout , Oxidiazóis/uso terapêutico , Pâncreas/metabolismo , Pâncreas/patologia , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Estreptozocina , Transativadores/genética , Triglicerídeos/sangue , Regulação para CimaRESUMO
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-gamma is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-gamma. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-gamma was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-gamma transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-gamma transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-gamma, suggesting post-translational activation of PPAR-gamma might be critical step for adipogenic gene expression.
Assuntos
Adipócitos/fisiologia , Adipogenia/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Transcricional , Células 3T3-L1 , Adipócitos/metabolismo , Animais , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Inativação Gênica , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Cilostazol is known to be a specific type III phosphodiesterase inhibitor, which promotes increased intracellular cAMP levels. We assessed the effect of cilostazol on production of angioneurins and chemokines and recruitment of new endothelial cells for vasculogenesis in a mouse model of transient forebrain ischemia. Pyramidal cell loss was prominently evident 3-28 days postischemia, which was markedly ameliorated by cilostazol treatment. Expression of angioneurins, including endothelial nitric oxide synthase, vascular endothelial growth factor, and brain-derived neurotrophic factor, was up-regulated by cilostazol treatment in the postischemic hippocampus. Cilostazol also increased Sca-1/vascular endothelial growth factor receptor-2 positive cells in the bone marrow and circulating peripheral blood and the number of stromal cell-derived factor-1alpha-positive cells in the molecular layer of the hippocampus, which colocalized with CD31. CXCR4 chemokine receptors were up-regulated by cilostazol in mouse bone marrow-derived endothelial progenitor cells, suggesting that cilostazol may be important in targeting or homing in of bone marrow-derived stem cells to areas of injured tissues. CD31-positive cells were colocalized with almost all bromodeoxyuridine-positive cells in the molecular layer, indicating stimulation of endothelial cell proliferation by cilostazol. These data suggest that cilostazol markedly enhances neovascularization in the hippocampus CA1 area in a mouse model of transient forebrain ischemia, providing a beneficial interface in which both bone marrow-derived endothelial progenitor cells and angioneurins influence neurogenesis in injured tissue. (c) 2010 Wiley-Liss, Inc.
Assuntos
Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Tetrazóis/farmacologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/patologia , Região CA1 Hipocampal/fisiopatologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cilostazol , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Hipocampo/patologia , Hipocampo/fisiopatologia , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/patologia , Prosencéfalo/fisiopatologiaRESUMO
In a previous study, cilostazol promoted differentiation of 3T3-L1 fibroblasts into adipocytes and improved insulin sensitivity by stimulating peroxisome proliferator-activated receptor (PPAR) gamma transcription. This study evaluated the in vivo efficacy of cilostazol to protect a db/db mouse model of type 2 diabetes against altered metabolic abnormalities and proinflammatory markers via activation of PPARgamma transcription. Eight-week-old db/db mice were treated with cilostazol or rosiglitazone for 12 days. Cilostazol significantly decreased plasma glucose and triglyceride levels, as did rosiglitazone, a PPARgamma agonist. Elevated plasma insulin and resistin levels were significantly decreased by cilostazol, and decreased adiponectin mRNA expression was elevated along with increased plasma adiponectin. Cilostazol significantly increased both adipocyte fatty acid binding protein and fatty acid transport protein-1 mRNA expressions with increased glucose transport 4 in the adipose tissue. Cilostazol and rosiglitazone significantly suppressed proinflammatory markers (superoxide, tumor necrosis factor-alpha, and vascular cell adhesion molecule-1) in the carotid artery of db/db mice. In an in vitro study with 3T3-L1 fibroblasts, cilostazol significantly increased PPARgamma transcription activity, as did rosiglitazone. The transcription activity stimulated by cilostazol was attenuated by KT5720 [(9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo [3,4-I][1,6]-benzodiazocine-10-carboxylic acid hexyl ester], a cAMP-dependent protein kinase inhibitor, and GW9662 (2-chloro-5-nitrobenzanilide), an antagonist of PPARgamma activity, indicative of implication of the phosphatidylinositol 3-kinase/Akt signal pathway. These results suggest that cilostazol may improve insulin sensitivity along with anti-inflammatory effects in type 2 diabetic patients via activation of both cAMP-dependent protein kinase and PPARgamma transcription.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , PPAR gama/biossíntese , Inibidores de Fosfodiesterase/farmacologia , Tetrazóis/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Molécula 1 de Adesão de Célula Vascular/biossíntese , Células 3T3-L1 , Adiponectina/sangue , Adiponectina/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Biomarcadores/análise , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Cilostazol , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/biossíntese , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/uso terapêutico , Receptores para Leptina/deficiência , Tetrazóis/administração & dosagem , Tetrazóis/uso terapêutico , Ativação Transcricional/efeitos dos fármacos , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Cilostazol exerts potent anti-inflammatory effects and celecoxib, a COX-2 specific inhibitor, improves the unsatisfactory profile of NSAIDs. It was aimed to assess the anti-arthritic potential of celecoxib add-on for cilostazol therapy in collagen induced arthritis (CIA), and to elucidate the implication of interleukin (IL)-10 in the action of cilostazol and celecoxib cotreatment. Cotreatment of RAW 264.7 cells with 10⯵M cilostazol and 0.3⯵M celecoxib synergistically suppressed RANKL-induced increases in RANK mRNA and protein levels. When cultured in the presence of RANKL for 5â¯days, RANKL-stimulated expressions of osteoclastogenic genes (OSCAR, DC-STAMP, and cathepsin K mRNA) and the expression of RANK mRNA were markedly elevated. Furthermore, these gene expressions, including that of RANK, were significantly suppressed by cotreatment with cilostazol (10⯵M) and celecoxib (0.3⯵M). In addition, this co-treatment strongly down-regulated RANKL-induced NFATc1 protein and TRAP activity (key osteoclastogenic factors), and these down-regulations were significantly prevented by pretreating cells with IL-10 neutralizing antibody. Furthermore, increased osteoclast formation and extensive resorption pit formation by bone marrow-derived monocytes obtained from C57BL/6 mice cultured in the presence of M-CSF/RANKL were markedly suppressed by cilostazol and celecoxib cotreatment. Consequently, hindlimb paw thicknesses in DBA/1J CIA mice were significantly reduced by cilostazol (10â¯mg/kg/d) and celecoxib (5â¯mg/kg/d) cotreatment. These results were accompanied by synergistic suppression of cartilage depletion and bone erosion and reductions in arthritis scores in the CIA mice. In conclusion, serum IL-10 levels in these mice were markedly increased by cilostazol and celecoxib cotreatment, whereas elevated serum IL-1ß levels were markedly reduced. Cotreatment with low-dose cilostazol and celecoxib may ensure the synergistic anti-arthritic potential.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Celecoxib/uso terapêutico , Cilostazol/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Catepsina K/genética , Celecoxib/farmacologia , Cilostazol/farmacologia , Citocinas/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas do Tecido Nervoso/genética , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Células RAW 264.7 , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptores de Superfície Celular/genéticaRESUMO
Cerebrovascular dysfunction is crucially associated with cognitive impairment and a high prevalence of psychotic symptoms in the vascular dementia characterized by oxidative stress and multifactorial neurodegeneration. In this study, the significant decrease in BDNF expression in HT22â¯cells due to H2O2 (0.25â¯mM) was little affected by either aripiprazole (1⯵M) or cilostazol (1⯵M) alone, but significantly increased by cotreatment with both drugs. Even in the presence of H2O2, P-CK2α (Tyr 255), nuclear P-CREB (Ser 133), and nuclear P-ß-catenin (Ser 675) levels were significantly increased in a synergistic manner by aripiprazole plus cilostazol cotreatment. Aripiprazole and cilostazol cotreatment synergistically increased P-GSK-3ß (Ser 9) level. Nrf2/HO-1 expression was significantly elevated time- and concentration-dependently by either aripiprazole or cilostazol. In line with these, concurrent treatment with aripiprazole (1⯵M) plus cilostazol (1⯵M) significantly increased Nrf2 and HO-1 expression in a synergistic manner, accompanying with increased ARE luciferase activity, while each drug monotherapy showed little effects. Consequently, this cotreatment synergistically ameliorated the attenuated neurite outgrowth induced by H2O2 in the HT22â¯cells, and these were inhibited by K252A (inhibitor of BDNF receptor), TBCA (CK2 inhibitor), imatinib (ß-catenin inhibitor) and ZnPP (inhibitor of HO-1), indicating that BDNF, P-CK2α, ß-catenin and HO-1 activation are implicated in the enhanced neurite outgrowth. This study highlights that cotreatment with low concentrations of aripiprazole and cilostazol synergistically elicits neuroprotective effects by overcoming oxidative stress-evoked neurotoxicity associated with increased neurite outgrowth, providing a rationale for the use of this combinatorial treatment in vascular dementia.