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1.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
2.
J Neurosci ; 40(13): 2776-2788, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098904

RESUMO

Oligophrenin-1 (Ophn1) encodes a Rho GTPase activating protein whose mutations cause X-linked intellectual disability (XLID) in humans. Loss of function of Ophn1 leads to impairments in the maturation and function of excitatory and inhibitory synapses, causing deficits in synaptic structure, function and plasticity. Epilepsy is a frequent comorbidity in patients with Ophn1-dependent XLID, but the cellular bases of hyperexcitability are poorly understood. Here we report that male mice knock-out (KO) for Ophn1 display hippocampal epileptiform alterations, which are associated with changes in parvalbumin-, somatostatin- and neuropeptide Y-positive interneurons. Because loss of function of Ophn1 is related to enhanced activity of Rho-associated protein kinase (ROCK) and protein kinase A (PKA), we attempted to rescue Ophn1-dependent pathological phenotypes by treatment with the ROCK/PKA inhibitor fasudil. While acute administration of fasudil had no impact on seizure activity, seven weeks of treatment in adulthood were able to correct electrographic, neuroanatomical and synaptic alterations of Ophn1 deficient mice. These data demonstrate that hyperexcitability and the associated changes in GABAergic markers can be rescued at the adult stage in Ophn1-dependent XLID through ROCK/PKA inhibition.SIGNIFICANCE STATEMENT In this study we demonstrate enhanced seizure propensity and impairments in hippocampal GABAergic circuitry in Ophn1 mouse model of X-linked intellectual disability (XLID). Importantly, the enhanced susceptibility to seizures, accompanied by an alteration of GABAergic markers were rescued by Rho-associated protein kinase (ROCK)/protein kinase A (PKA) inhibitor fasudil, a drug already tested on humans. Because seizures can significantly impact the quality of life of XLID patients, the present data suggest a potential therapeutic pathway to correct alterations in GABAergic networks and dampen pathological hyperexcitability in adults with XLID.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Neurônios GABAérgicos/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Hipocampo/efeitos dos fármacos , Deficiência Intelectual/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Convulsões/fisiopatologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Neurônios GABAérgicos/fisiologia , Hipocampo/fisiopatologia , Deficiência Intelectual/genética , Camundongos , Camundongos Knockout , Convulsões/genética
3.
Chem Biol Interact ; 317: 108944, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935364

RESUMO

Bone mesenchymal stem cells (BMSCs) are a well-known donor graft source due to their potential for self-renewal and differentiation into multi-lineage cell types, including osteoblasts that are critical for fracture healing. Fasudil (FAS), a Rho kinase inhibitor, has been proven to induce the differentiation of bone marrow stem cells (BMSCs) into neuron-like cells. However, its role in the osteogenesis of BMSCs remain uncertain. Herein, we for the first time studied the effects of FAS on osteogenic differentiation in a mouse fracture model and further explored the involved mechanisms in mouse BMSCs. The results showed that FAS stimulated bone formation in the fracture mouse model. Additionally, at 30 µM, FAS significantly promotes alkaline phosphatase activity, mineralization, and the expression of osteogenic markers COL-1, RUNX2 and OCN in murine BMSCs. Blocking of P38 by SB202190 significantly reversed the effects of FAS, in vitro, suggesting that P38, but not ERK or JNK activation is required for FAS-induced osteogenesis. Collectively, our results indicate that FAS may be a promising agent for promoting fracture healing.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Feminino , Fraturas Ósseas , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Células-Tronco Mesenquimais , Camundongos , Osteogênese/efeitos dos fármacos , Fosforilação , Piridinas/farmacologia
4.
Braz J Med Biol Res ; 53(1): e8669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31859913

RESUMO

This study aimed to investigate the therapeutic effect of fasudil on treating experimental autoimmune neuritis (EAN). Twenty-four EAN mice were randomly assigned to fasudil treatment (Fasudil group) or saline treatment (EAN model group) for 28 days. Clinical symptom score was evaluated every other day; inflammatory cell infiltration, demyelination, anti-myelin basic protein (MBP), inflammatory cytokines, inducible nitric oxide synthase (iNOS), and arginase-1 were detected in sciatic nerves at day 28. Th1, Th2, Th17, and Tregs proportions in splenocytes were detected at day 28. Clinical symptom score was found to be attenuated in the Fasudil group compared to the EAN model group from day 12 to day 28. Sciatic nerve inflammatory cell counts by HE staining and demyelination by luxol fast blue staining were both reduced, while MBP was increased in the Fasudil group compared to the EAN model group at day 28. Interferon γ (IFN-γ) and interleukin (IL)-17 were reduced, while IL-4 and IL-10 were elevated in the Fasudil group at day 28. Sciatic nerve M1 macrophages marker iNOS was decreased while M2 macrophages marker arginase-1 was increased in the Fasudil group at day 28. CD4+IFN-γ+ (Th1) and CD4+IL-17+ (Th17) cell proportions were both decreased, CD4+IL-4+ (Th2) cell proportion was similar, while CD25+FOXP3+ (Treg) cell proportion in splenocytes was increased in the Fasudil group. In summary, fasudil presented a good therapeutic effect for treating EAN by attenuating Th1/Th17 cells and promoting Tregs activation as well as M2 macrophages polarization.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Interferon gama/sangue , Interleucinas/sangue , Neurite Autoimune Experimental/tratamento farmacológico , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurite Autoimune Experimental/sangue , RNA Mitocondrial , Reação em Cadeia da Polimerase em Tempo Real , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Fatores de Tempo
5.
Int J Mol Sci ; 20(24)2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861195

RESUMO

Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α1-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the Emax and decreased the EC50 of PE-induced contractions indicating a hyperreactivity to α1-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α1-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P-S1P2-G12/13-ROCK signaling pathway appears to have a major influence on α1-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.


Assuntos
Lisofosfolipídeos/farmacologia , Receptores Adrenérgicos alfa 1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Animais , Sinergismo Farmacológico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenilefrina/farmacologia , Piridinas/farmacologia , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Quinases Associadas a rho/antagonistas & inibidores
6.
Int J Mol Sci ; 20(23)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771203

RESUMO

There are three critical pathways for the pathogenesis and progression of pulmonary arterial hypertension (PAH): the prostacyclin (prostaglandin I2) (PGI2), nitric oxide (NO), and endothelin pathways. The current approved drugs targeting these three pathways, including prostacyclin (PGI2), phosphodiesterase type-5 (PDE5) inhibitors, and endothelin receptor antagonists (ERAs), have been shown to be effective, however, PAH remains a severe clinical condition and the long-term survival of patients with PAH is still suboptimal. The full therapeutic abilities of available drugs are reduced by medication, patient non-compliance, and side effects. Nanoparticles are expected to address these problems by providing a novel drug delivery approach for the treatment of PAH. Drug-loaded nanoparticles for local delivery can optimize the efficacy and minimize the adverse effects of drugs. Prostacyclin (PGI2) analogue, PDE5 inhibitors, ERA, pitavastatin, imatinib, rapamycin, fasudil, and oligonucleotides-loaded nanoparticles have been reported to be effective in animal PAH models and in vitro studies. However, the efficacy and safety of nanoparticle mediated-drug delivery systems for PAH treatment in humans are unknown and further clinical studies are required to clarify these points.


Assuntos
Nanopartículas/química , Hipertensão Arterial Pulmonar/tratamento farmacológico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Mesilato de Imatinib/uso terapêutico , Quinolinas/uso terapêutico
7.
J Pak Med Assoc ; 69(10): 1425-1430, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31622291

RESUMO

Objectives: To find interactions of the ligand with axonali nhibitors, and to check the optimum interactions of existing drugs as inhibitors for the protein that hinders the growth of injured neurons. METHODS: The study was conducted at Kongju National University, Korea from May 2016 to March 2017. It consisted of two parts. Molecular analysis and bioinformatics analysis. The study comprised a family of six with Charcot-Marie-Tooth phenotypes, recommended by a neurologist for molecular analysis on the clinical symptoms to find the mutations responsible for the disease. Blood samples were collected from each family member and total deoxyribonucleic acid was extracted and it was analysed for Reticulon 4 gene by sequencing the coding and intronic regions. However, a missense mutation was found on exon 2 of the gene in the proband and the whole family was subsequently analysed. Bioinformatics analysis and docking studies were carried out to investigate the potential behaviour of Reticulon 4 as therapeutic agent. Sequencing analysis was performed to find the pathoegenic variant responsible for Charcot-Marie-Tooth type 1. RESULTS: After checking pathogenicity of the mutation, Reticulon 4 gene was found to be not involved in Charcot- Marie-Tooth disease type 1. CONCLUSIONS: Reticulon 4 gene was not found to be involved in causing Charcot-Marie-Tooth disease type 1.


Assuntos
Axônios/metabolismo , Doença de Charcot-Marie-Tooth/genética , Proteínas Nogo/genética , Remielinização/genética , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/metabolismo , Adulto , Pré-Escolar , Simulação por Computador , Família , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Mutação de Sentido Incorreto , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Regeneração Nervosa/genética , Proteínas Nogo/metabolismo , Linhagem , Inibidores de Proteínas Quinases/metabolismo
8.
Med Sci Monit ; 25: 7605-7616, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31599230

RESUMO

BACKGROUND The aim of this study was to explore the impact of Ras homolog C/Rho-associated coiled-protein kinase (Rho/ROCK) signaling pathways intervention on biological characteristics of the human multiple myeloma cell lines RPMI-8226 and U266 cells, and to investigate the expression of RhoC, ROCK1, and ROCK2 in RPMI-8226 and U266 cells. MATERIAL AND METHODS RPMI8226 and U266 cell lines were treated by 5-aza-2-deoxycytidine (5-Aza-Dc), trichostatin A (TSA), RhoA inhibitor CCG-1423, Rac1 inhibitor NSC23766, and ROCK inhibitor fasudil. Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and clone formation. Cell apoptosis was examined by flow cytometry and TUNEL assay. The mRNA and protein expressions of RhoC, ROCK1, and ROCK2 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. RESULTS CCG-1423, NSC23766, and fasudil could significantly inhibit the proliferation of RPMI8226 and U266 cells. The inhibitory effect was dose- and time-dependent within a certain concentration range (P<0.05). After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 and U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05). Compared with the control group, the mRNA and protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 and U266 cells were significantly decreased with single 5-Aza-Dc or TSA treatment. However, the effects were obviously stronger after combined treatment of 5-Aza-CdR and TSA (P<0.05). CONCLUSIONS We found that 5-Aza-Dc and TSA can effectively decrease the mRNA and protein expressions of RhoC, ROCK1, and ROCK2. Furthermore, Rho and ROCK inhibitors significantly inhibit cell growth and induce cell apoptosis in the human multiple myeloma cell lines RPMI-8226 and U266.


Assuntos
Apoptose , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética
9.
PLoS One ; 14(10): e0223232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31581236

RESUMO

Hepatocellular carcinoma (HCC) is one of the most malignant cancers and the treatment options for this disease are limited and generally not effective. ROCK has been reported to be highly expressed in many cancer types and its inhibitor Fasudil has shown anti-cancer potential. However, its high toxicity and low solubility restrict its clinical application. Here, we report that Fasudil is effective against HCC and that a liposomal formulation (Lip-Fasudil) can enhance the anti-tumor effects of this drug both in vitro and in vivo. In vitro, Fasudil inhibited HCC cell growth with IC50 values of 0.025-0.04 µg/µL, with Lip-Fasudil showing slightly improved cytotoxicity with IC50 values of 0.02-0.025 µg/µL. Cellular mechanistic analysis indicated that Fasudil induced cell cycle arrest at the G2/M phase and that Lip-Fasudil enhanced this effect. Intriguingly, no apoptosis was detected in Fasudil- or Lip-Fasudil-treated HCC cells. In vivo, Fasudil inhibited the growth of HCC xenografts by 23% in nude mice. However, Lip-fasudil exerted anti-tumor effects (57% tumor inhibition) that were superior to those of Fasudil and similar to those of Topotecan (66%). In addition, Lip-fasudil resulted in an increased distribution of Fasudil in tumor tissues but a reduced distribution in normal organs. In conclusion, our results proved that Fasudil has the potential to be used for HCC treatment and that a liposomal formulation (Lip-Fasudil) could enhance anti-tumor efficacy and reduce systemic toxicity.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Lipossomos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
10.
Metab Brain Dis ; 34(6): 1787-1801, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31482248

RESUMO

Alzheimer's disease (AD), a chronic, progressive, neurodegenerative disorder, is the most common type of dementia. Beta amyloid (Aß) peptide aggregation and phosphorylated tau protein accumulation are considered as one of the causes for AD. Our previous studies have demonstrated the neuroprotective effect of the Rho kinase inhibitor fasudil, but the mechanism remains elucidated. In the present study, we examined the effects of fasudil on Aß1-42 aggregation and apoptosis and identified the intracellular signaling pathways involved in these actions in primary cultures of mouse hippocampal neurons. The results showed that fasudil increased neurite outgrowth (52.84%), decreased Aß burden (46.65%), Tau phosphorylation (96.84%), and ROCK-II expression. In addition, fasudil reversed Aß1-42-induced decreased expression of Bcl-2 and increases in caspase-3, cleaved-PARP, phospho-JNK(Thr183/Tyr185), and phospho-ASK1(Ser966). Further, fasudil decreased mitochondrial membrane potential and intracellular calcium overload in the neurons treated with Aß1-42. These results suggest that inhibition of Rho kinase by fasudil reverses Aß1-42-induced neuronal apoptosis via the ASK1/JNK signal pathway, calcium ions, and mitochondrial membrane potential. Fasudil could be a drug of choice for treatment of Alzheimer's disease.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Apoptose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Neurônios/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Peptídeos beta-Amiloides , Animais , Hipocampo/metabolismo , Camundongos , Neurônios/metabolismo , Fragmentos de Peptídeos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteínas tau/metabolismo
11.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500276

RESUMO

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: "In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43". In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1ß), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway.


Assuntos
Angiotensina II/efeitos adversos , Conexina 43/metabolismo , Nefropatias/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Creatinina/urina , Modelos Animais de Doenças , Regulação da Expressão Gênica , Nefropatias/induzido quimicamente , Nefropatias/tratamento farmacológico , Nefropatias/urina , Masculino , Estresse Oxidativo , Ratos , Transdução de Sinais , Fatores de Tempo
12.
Medicine (Baltimore) ; 98(35): e16885, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464917

RESUMO

BACKGROUND: This study aims to systematically assess the efficacy and safety of fasudil for the treatment of aneurysmal subarachnoid hemorrhage (ASH). METHODS: This study will include all of randomized controlled trials on the efficacy and safety of fasudil for the treatment of ASH. Ten electronic databases of PubMed, Embase, Cochrane Library, Google Scholar, Web of Science, Ovid, Cumulative Index to Nursing and Allied Health Literature, Allied and Complementary Medicine Database, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure will be searched from inception to the May 1, 2019 without language restrictions. We will also search gray literatures to avoid missing any other potential studies. Two authors will independently perform study selection, data extraction and management, and methodologic quality assessment. The primary outcome is limbs function. The secondary outcomes comprise of muscle strength, muscle tone, quality of life, and adverse events. RESULTS: This study will provide a comprehensive literature search on the current evidence of fasudil for the treatment of ASH from primary and secondary outcomes. CONCLUSION: The results of this study will present evidence to determine whether fasudil is an effective and safety treatment for patients with ASH. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42019136215.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Aneurisma Intracraniano/complicações , Hemorragia Subaracnóidea/tratamento farmacológico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/efeitos adversos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Projetos de Pesquisa/normas , Hemorragia Subaracnóidea/etiologia , Resultado do Tratamento
13.
Mol Pharmacol ; 96(3): 355-363, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31263019

RESUMO

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.


Assuntos
Mutação Puntual , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Trifosfato de Adenosina/metabolismo , Sítio Alostérico , Amidas/química , Amidas/farmacologia , Sítios de Ligação , Humanos , Imidazóis/química , Imidazóis/farmacologia , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Antagonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/genética , Corantes de Rosanilina/química , Corantes de Rosanilina/farmacologia , Tiazóis/química , Tiazóis/farmacologia
14.
Food Funct ; 10(7): 4071-4080, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31225563

RESUMO

This study was conducted to discover the effectiveness of dietary peptides (γ-[Glu](n=1,2)-Phe/-Met/-Val) as stimulators of cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) secretion. The kokumi-active γ-[Glu](n=1,2)-Phe/-Met/-Val at concentrations of 2.5-10 mM would trigger the release of CCK and GLP-1 by activating the calcium-sensing receptor (CaSR). The CaSR-mediated Ca2+/CaM/CaMK pathway was proposed in γ-[Glu](n=1,2)-Phe/-Met/-Val-induced CCK and GLP-1 secretion based on the following results: the exposure to γ-Glu-Phe increased the protein expression level (western blot analysis), the intracellular calcium ([Ca2+]i) mobilization in response to γ-[Glu](n=1,2)-Phe/-Met/-Val was strongly enhanced, and the inhibitors of signaling pathway proteins (NPS-2143, BAPTA-AM, and KN62) abolished partially γ-[Glu](n=1,2)-Phe/-Met/-Val-induced CCK and GLP-1 secretion.


Assuntos
Colecistocinina/metabolismo , Dipeptídeos/farmacologia , Hormônios Gastrointestinais/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Humanos , Naftalenos
15.
Graefes Arch Clin Exp Ophthalmol ; 257(8): 1699-1708, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152312

RESUMO

PURPOSE: To investigate the influence of the selective Rho-kinase (ROCK) inhibitor, fasudil, on the mRNA level of proinflammatory factors and the retinal vascular development in mice with oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice underwent standard protocol for OIR induction from postnatal days 7 to 12. Subsequently, they received a daily intraperitoneal injection of fasudil or sodium chloride from P12 to P16. Analyses were performed using vascular staining on retinal flat mounts, RNA expression by qPCR, and immunohistochemistry on paraffin sections. RESULTS: On retinal flat mounts, the proportion of avascular area and tuft formation did not differ between the fasudil and NaCl group. Immunohistochemical staining revealed a less intense staining with inflammatory markers after fasudil. Nevertheless, there were no differences on RNA level between the two groups. CONCLUSIONS: In conclusion, our findings support that daily systemic application of fasudil does not decrease retinal neovascularization in rodents with oxygen-induced retinopathy. The results of our study together with the controversial results on the effects of different ROCK inhibitors from the literature makes it apparent that effects of ROCK inhibition are more complex, and further studies are necessary to analyze its potential therapeutic effects.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Doenças Retinianas/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Retina/efeitos dos fármacos , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/enzimologia , Resultado do Tratamento , Quinases Associadas a rho/metabolismo
16.
Congenit Heart Dis ; 14(4): 645-650, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31166081

RESUMO

OBJECTIVE: The optimal dose of Fasudil is still controversial in congenital heart disease accompanied with severe pulmonary hypertension (CHD-PAH). This study aimed to compare acute hemodynamic changes after different doses of Fasudil in 60 consecutive adult patients with CHD-PAH. DESIGN: Prospective randomized controlled trial. SETTING: Tertiary cardiology center. PATIENTS: Adult patients with CHD-PAH. INTERVENTIONS: Patients were randomized to Fasudil 30 or 60 mg. OUTCOME MEASURES: The hemodynamic parameters were measured at baseline and after 30 minutes of Fasudil through right cardiac catheterization. Blood gas results were obtained from the pulmonary artery, right ventricle, right atrium, superior and inferior vena cava, and femoral artery. Pulmonary vascular resistance (PVR) and systemic arterial resistance (SVR) were calculated. RESULTS: The changes in systolic pulmonary artery pressure (sPAP) (-13.1% vs -9.3%, P < .05), diastolic PAP (dPAP) (-17.6% vs -14.5%, P < .05), mean PAP (mPAP) (-12.4% vs -8.5%, P < .05), and PVR (-35.8% vs -22.2%, P < .05) were more pronounced in the 60-mg group than in the 30-mg group. All patients had no obvious adverse reactions related to peripheral blood pressure. CONCLUSIONS: Fasudil could improve the hemodynamics of patients with CHD-PAH, especially with the 60-mg dose. There were no serious adverse reactions.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Pressão Sanguínea/fisiologia , Cardiopatias Congênitas/tratamento farmacológico , Hipertensão Pulmonar/tratamento farmacológico , Resistência Vascular/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , Adolescente , Adulto , Pressão Sanguínea/efeitos dos fármacos , Cateterismo Cardíaco , Relação Dose-Resposta a Droga , Feminino , Seguimentos , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/fisiopatologia , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/fisiopatologia , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Inibidores de Proteínas Quinases/administração & dosagem , Resultado do Tratamento , Resistência Vascular/fisiologia , Adulto Jovem
17.
Clin Immunol ; 203: 142-153, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31078707

RESUMO

Aberrant ROCK activation has been found in patients with several autoimmune diseases, but the role of ROCK in myasthenia gravis (MG) has not yet been clearly investigated. Here, we demonstrated that ROCK activity was significantly higher in peripheral blood mononuclear cells (PBMCs) from MG patients. ROCK inhibitor Fasudil down-regulated the proportions of Th1 and Th17 cells in PBMCs of MG patients in vitro. Intraperitoneal injection of Fasudil ameliorated the severity of experimental autoimmune myasthenia gravis (EAMG) rats and restored the balance of Th1/Th2/Th17/Treg subsets. Furthermore, Fasudil inhibited the proliferation of antigen-specific Th1 and Th17 cells, and inhibited CD4 + T cells differentiated into Th1 and Th17 through decreasing phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through increasing phosphorylated Stat5. We conclude that dysregulated ROCK activity may be involved in the pathogenic immune response of MG and inhibition of ROCK activity might serve as a novel treatment strategy for MG.


Assuntos
Miastenia Gravis/imunologia , Fator de Transcrição STAT5/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Homeostase , Humanos , Fosforilação , Ratos , Ratos Endogâmicos Lew , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Quinases Associadas a rho/antagonistas & inibidores
18.
Basic Clin Pharmacol Toxicol ; 125(2): 152-165, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30916885

RESUMO

Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1 /Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1 /Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood-brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte-myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo-OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down-regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Encéfalo/efeitos dos fármacos , Intoxicação por Monóxido de Carbono/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Encéfalo/patologia , Monóxido de Carbono/toxicidade , Intoxicação por Monóxido de Carbono/etiologia , Intoxicação por Monóxido de Carbono/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Proteínas da Mielina/metabolismo , Proteínas Nogo/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Ratos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
19.
Toxicol Appl Pharmacol ; 369: 60-72, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30831131

RESUMO

Hypoxic pulmonary vasoconstriction (HPV) can be modulated by Rho/Rho kinase signaling, which can alter HPV vascular function via regulating myosin light chain phosphorylation, in a manner generally believed to be Ca2+-independent. We hypothesized that the RhoA/ROCK signaling pathway also can regulate HPV vascular function via a Ca2+-dependent mechanism, signaling through the functional transient receptor potential canonical (TRPC) channels. In this study, male BALB/c mice were exposed to normoxic or 10% oxygen (hypoxic) conditions for six weeks, after which systolic pressure and right ventricular hypertrophy were assessed. Transient intracellular calcium was monitored using a fluorescence imaging system. Muscle tension was measured with a contractile force recording system, and protein expression was assessed by immunoblotting. We found that the expressions of RhoA and ROCK were increased in mouse pulmonary arteries (PAs) under conditions of chronic hypoxia. Inhibition of the RhoA/ROCK signaling pathway prevented the development of hypoxic pulmonary hypertension (HPH), as evidenced by significantly reduced PA remodeling and pulmonary vasoconstriction. Immunoblotting results revealed that inhibition of the RhoA/ROCK signaling pathway significantly decreased the expression of HIF-1α. Knockdown of HIF-1α down-regulated the expression and function of the TRPC1 and TRPC6 channels in PASMCs under conditions of hypoxia. Contraction of the PAs and a Ca2+ influx into PASMCs through either receptor- or store-operated Ca2+ channels were also increased after hypoxia. However, RhoA/ROCK inhibitors markedly attenuated these changes. These results indicate that inhibition of the RhoA/ROCK signaling pathway ameliorates HPH via HIF-1α-dependent functional TRPCs.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Arterial/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Hipertensão Arterial Pulmonar/prevenção & controle , Piridinas/farmacologia , Canais de Cátion TRPC/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Sinalização do Cálcio , Linhagem Celular , Modelos Animais de Doenças , Hipóxia/complicações , Hipóxia/enzimologia , Hipóxia/fisiopatologia , Masculino , Camundongos Endogâmicos BALB C , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/enzimologia , Hipertensão Arterial Pulmonar/enzimologia , Hipertensão Arterial Pulmonar/etiologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/fisiopatologia , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética
20.
Stroke ; 50(3): 738-744, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30744543

RESUMO

Background and Purpose- Previously, murine models Krit1 +/- Msh2 -/ - and Ccm2 +/ - Trp53 -/ - showed a reduction or no effect on cerebral cavernous malformation (CCM) burden and favorable effects on lesional hemorrhage by the robust Rock (Rho-associated protein kinase) inhibitor fasudil and by simvastatin (a weak pleiotropic inhibitor of Rock). Herein, we concurrently investigated treatment of the more aggressive Pdcd10/Ccm3 model with fasudil, simvastatin, and higher dose atorvastatin to determined effectiveness of Rock inhibition. Methods- The murine models, Pdcd10 +/ - Trp53 -/ - and Pdcd10 +/ - Msh2 -/ -, were contemporaneously treated from weaning to 5 months of age with fasudil (100 mg/kg per day in drinking water, n=9), simvastatin (40 mg/kg per day in chow, n=11), atorvastatin (80 mg/kg per day in chow, n=10), or with placebo (n=16). We assessed CCM volume in mouse brains by microcomputed tomography. Lesion burden was calculated as lesion volume normalized to total brain volume. We analyzed chronic hemorrhage in CCM lesions by quantitative intensity of Perls staining in brain sections. Results- The Pdcd10 +/ - Trp53 -/ - /Msh2 -/ - models showed a mean CCM lesion burden per mouse reduction from 0.0091 in placebos to 0.0042 ( P=0.027) by fasudil, and to 0.0047 ( P=0.025) by atorvastatin treatment, but was not changed significantly by simvastatin. Hemorrhage intensity per brain was commensurately decreased by Rock inhibition. Conclusions- These results support the exploration of proof of concept effect of high-dose atorvastatin on human CCM disease for potential therapeutic testing.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Hemangioma Cavernoso do Sistema Nervoso Central/tratamento farmacológico , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/genética , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Proteínas Reguladoras de Apoptose , Atorvastatina/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hemorragias Intracranianas/diagnóstico por imagem , Proteína KRIT1/genética , Camundongos , Camundongos Knockout , Sinvastatina/uso terapêutico , Microtomografia por Raio-X
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