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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.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
J Pharmacol Exp Ther ; 369(1): 37-46, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30635471

RESUMO

Sustained ligand-activated preconditioning (SLP), induced with chronic opioid receptor (OR) agonism, enhances tolerance to ischemia/reperfusion injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) during the induction phase, and ß 2-adrenoceptor (ß 2-AR), Gs alpha subunit (Gα s), and protein kinase A (PKA) involvement in subsequent cardioprotection. Here, we tested for induction of a protective ß 2-AR/Gα s/PKA signaling axis with SLP to ascertain whether signaling changes were PI3K-dependent (by sustained cotreatment with wortmannin), and whether the downstream PKA target Rho kinase (ROCK) participates in subsequent cardioprotection (by acute treatment with fasudil). A protected phenotype was evident after 5 days of OR agonism (using morphine) in association with increased membrane versus reduced cytosolic levels of total and phosphorylated ß 2-ARs; increased membrane and cytosolic expression of 52 and 46 kDa Gα s isoforms, respectively; and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of ß 2-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol, and 6-[3-(dimethylamino)propionyl]-forskolin. Protection with SLP was not modified by ROCK inhibition, and changes in ß 2-AR, Gα s, and PKA expression appeared insensitive to PI3K inhibition, although 5 days of wortmannin alone exerted unexpected effects on signaling (also increasing membrane ß 2-AR and PKA expression/phosphorylation and Gα s levels). In summary, sustained OR agonism upregulates cardiac membrane ß 2-AR expression and phosphorylation in association with increased Gα s subtype levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears nonessential to OR upregulation of the ß 2-AR signal axis. This opioidergic remodeling of ß 2-AR signaling may explain ß 2-AR, Gα s, and PKA dependence of SLP protection.


Assuntos
Precondicionamento Isquêmico/métodos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Adenilil Ciclases/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
14.
J Cereb Blood Flow Metab ; 39(1): 58-73, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29135354

RESUMO

Depression after traumatic brain injury (TBI) is common but the mechanisms by which TBI causes depression are unknown. TBI decreases glutamate transporters GLT-1 and GLAST and allows extravasation of thrombin. We examined the effects of thrombin on transporter expression in primary hippocampal astrocytes. Application of a PAR-1 agonist caused down-regulation of GLT-1, which was prevented by inhibition of Rho kinase (ROCK). To confirm these mechanisms in vivo, we subjected mice to closed-skull TBI. Thrombin activity in the hippocampus increased one day following TBI. Seven days following TBI, expression of GLT-1 and GLAST was reduced in the hippocampus, and this was prevented by administration of the PAR-1 antagonist SCH79797. Inhibition of ROCK attenuated the decrease in GLT-1, but not GLAST, after TBI. We measured changes in glutamate levels in the hippocampus seven days after TBI using an implanted biosensor. Stress-induced glutamate levels were significantly increased following TBI and this was attenuated by treatment with the ROCK inhibitor fasudil. We quantified depressive behavior following TBI and found that inhibition of PAR-1 or ROCK decreased these behaviors. These results identify a novel mechanism by which TBI results in down-regulation of astrocyte glutamate transporters and implicate astrocyte and glutamate transporter dysfunction in depression following TBI.


Assuntos
Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/genética , Depressão/etiologia , Depressão/genética , Hipocampo/metabolismo , Trombina , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Comportamento Animal , Barreira Hematoencefálica/patologia , Depressão/psicologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Transportador 1 de Aminoácido Excitatório/biossíntese , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Masculino , Camundongos , Receptor PAR-1/genética , Proteínas Vesiculares de Transporte de Glutamato/genética , Quinases Associadas a rho/antagonistas & inibidores
15.
Toxicol Sci ; 168(1): 126-136, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30462329

RESUMO

Methylmercury (MeHg) is an environmental neurotoxicant that induces neuropathological changes. In this study, we established chronic MeHg-intoxicated rats. These rats survived, and sustained MeHg-induced axonal degeneration, including the dorsal root nerve and the dorsal column of the spinal cord; these changes persisted 12 weeks after MeHg withdrawal. We demonstrated for the first time the restorative effect of Fasudil, a specific inhibitor of Rho-associated coiled coil-forming protein kinase, on axonal degeneration and corresponding neural dysfunction in the established chronic MeHg-intoxicated rats. To investigate the mechanism of this restorative effect, we focused on the expression of Rho protein families. This was supported by our previous study, which demonstrated that cotreatment with Fasudil prevented axonal degeneration by mitigating neurite extension/retraction incoordination caused by MeHg-induced suppression of Rac1 in vitro and in subacute MeHg-intoxicated rats. However, the mechanism of the restorative effect of Fasudil on axonal degeneration in chronic MeHg-intoxicated rats differed from MeHg-mediated neuritic extension/retraction incoordination. We found that the restorative effect of Fasudil was caused by the Fasudil-induced change of microglial phenotype, from proinflammatory to anti-inflammatory; moreover, Fasudil suppressed Rho-associated coiled coil-forming protein kinase activity. Treatment with Fasudil decreased the expression of proinflammatory factors, including tumor necrosis factor-α, inducible nitric oxide synthase, interleukin-1ß, and interleukin-6; furthermore, it inactivated the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. Additionally, Fasudil treatment was associated with increased levels of anti-inflammatory factors arginase-1 and interleukin-10. These results suggest that Rho-associated coiled coil-forming protein kinase inhibition may recover MeHg-mediated axonal degeneration and neural dysfunction in chronic MeHg intoxication.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Masculino , Compostos de Metilmercúrio/efeitos adversos , Compostos de Metilmercúrio/toxicidade , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/patologia , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
Neurobiol Dis ; 124: 520-530, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30593834

RESUMO

Adolescence represents a critical period of neurodevelopment, defined by structural and synaptic pruning within the prefrontal cortex. While characteristic of typical development, this structural instability may open a window of vulnerability to developing neuropsychiatric disorders, including depression. Thus, therapeutic interventions that support or expedite neural remodeling in adolescence may be advantageous. Here, we inhibited the neuronally-expressed cytoskeletal regulatory factor Rho-kinase (ROCK), focusing primarily on the clinically-viable ROCK inhibitor fasudil. ROCK inhibition had rapid antidepressant-like effects in adolescent mice, and its efficacy was comparable to ketamine and fluoxetine. It also modified levels of the antidepressant-related signaling factors, tropomyosin/tyrosine receptor kinase B and Akt, as well as the postsynaptic marker PSD-95, in the ventromedial prefrontal cortex (vmPFC). Meanwhile, adolescent-typical dendritic spine pruning on excitatory pyramidal neurons in the vmPFC was expedited. Further, vmPFC-specific shRNA-mediated reduction of ROCK2, the dominant ROCK isoform in the brain, had antidepressant-like consequences. We cautiously suggest that ROCK inhibitors may have therapeutic potential for adolescent-onset depression.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Antidepressivos/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/enzimologia , Inibidores de Proteínas Quinases/farmacologia
17.
Eur J Med Chem ; 161: 192-204, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30359818

RESUMO

Despite recent advance of therapeutic development, coronary artery disease (CAD) remains one of the major issues to public health. The use of genomics and systems biology approaches to inform drug discovery and development have offered the possibilities for new target identification and in silico drug repurposing. In this study, we propose a network-based, systems pharmacology framework for target identification and drug repurposing in pharmacologic treatment and chemoprevention of CAD. Specifically, we build in silico models by integrating known drug-target interactions, CAD genes derived from the genetic and genomic studies, and the human protein-protein interactome. We demonstrate that the proposed in silico models can successfully uncover approved drugs and novel natural products in potentially treating and preventing CAD. In case studies, we highlight several approved drugs (e.g., fasudil, parecoxib, and dexamethasone) or natural products (e.g., resveratrol, luteolin, daidzein and caffeic acid) with new mechanism-of-action in chemical intervention of CAD by network analysis. In summary, this study offers a powerful systems pharmacology approach for target identification and in silico drug repurposing on CAD.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Produtos Biológicos/farmacologia , Doença da Artéria Coronariana/tratamento farmacológico , Dexametasona/farmacologia , Isoxazóis/farmacologia , Biologia de Sistemas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Produtos Biológicos/química , Doença da Artéria Coronariana/genética , Dexametasona/química , Reposicionamento de Medicamentos , Humanos , Isoxazóis/química
18.
Eur J Pharmacol ; 843: 27-33, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445018

RESUMO

Improper cervical function may lead premature or late-term birth. The RhoA/Rho-kinase (ROCK) signalling pathway takes part in cellular functions including smooth muscle contraction. No information is available about the cervical expression of the RhoA/ROCK system during pregnancy. Our aim was to detect the mRNA and protein expression of ROCK enzymes in rat cervices and to evaluate the effects of RhoA/ROCK inhibitors on cervical resistance. The mRNA and protein expressions of RhoA, ROCK I and II were measured in non-pregnant, pregnant and postpartum rat cervices and during parturition by Real-time qPCR and Western blot. The cervical resistance modifying effects of RhoA (simvastatin) and ROCK (fasudil, Y-27632) (10-6M) were investigated in tissue bath experiments. RhoA mRNA was increased on post-partum day 3, while the RhoA protein expression was decreased near and during parturition. ROCK I mRNA and protein expressions were fluctuating with a decrease in protein expression during parturition. ROCK II mRNA and protein expressions were sharply reduced during parturition. Simvastatin increased the cervical resistance on pregnancy days 20 and 22 while Y-27632 and fasudil reduced the resistance on pregnancy days 20. The decrease in RhoA/ROCK expression near parturition may take part in cervical ripening, especially in the final processes leading to delivery. ROCK inhibitors might be potential drug candidates to treat insufficient cervical ripening late-term pregnancies. The effect of simvastatin possibly due to its unique smooth muscle contracting activity in pregnant cervix. Compounds with simvastatin-like action might be new drug candidates for preterm cervical ripening.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Maturidade Cervical/efeitos dos fármacos , Colo do Útero/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Piridinas/farmacologia , Sinvastatina/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Maturidade Cervical/fisiologia , Colo do Útero/fisiologia , Feminino , Técnicas In Vitro , Contração Muscular/fisiologia , Gravidez , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia
19.
Int J Cancer ; 144(9): 2227-2238, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30374974

RESUMO

Vasculogenic mimicry (VM) is a special vascular pattern in malignant tumors, which is composed of highly aggressive tumor cells. This tumor cell-mediated blood supply pattern is closely associated with a poor prognosis in cancer patients. The interaction of axon guidance factor Sema4D and its high affinity receptor plexinB1 could activate small GTPase RhoA and its downstream ROCKs; this process has an active role in the migration of endothelial cells and tumor angiogenesis. Here, we have begun to uncover the role of this pathway in VM formation in non-small cell lung cancer (NSCLC). First, we confirmed this special form of vasculature in NSCLC tissues and found the existence of VM channels in tumor tissues was correlated with Sema4D expression. Further, we found that inhibition of Sema4D in the human NSCLC cells H1299 and HCC827 reduces VM formation both in vitro and in vivo. Moreover, we demonstrated that downregulating the expression of plexinB1 by siRNA expressing vectors and inhibiting the RhoA/ROCK signaling pathway using fasudil can reduce VM formation of H1299 and HCC827 cells. Finally, we found that suppression of Sema4D leads to less stress fibers and depleted the motility of H1299 and HCC827 cells. Collectively, our study implicates Sema4D plays an important role in the process of VM formation in NSCLC through activating the RhoA/ROCK pathway and regulating tumor cell plasticity and migration. Modulation of the Sema4D/plexinB1 and downstream RhoA/ROCK pathway may prevent the tumor blood supply through the VM pattern, which may eventually halt growth and metastasis of NSCLC.


Assuntos
Antígenos CD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Neovascularização Patológica/patologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Células A549 , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/genética , Semaforinas/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Clin Hemorheol Microcirc ; 71(1): 3-8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29660902

RESUMO

OBJECTIVE: This study aims to study the effect of Rho kinase inhibitor fasudil on the expression endothelin-1 (ET-1) and nitric oxide (NO) in rats with hypoxic pulmonary hypertension (HPH). METHODS: Twenty-four male SD rats were randomly divided into three groups: control group, model group (HPH group) and HPH+fasudil group. The rat HPH model was established by intermittent hypoxia (IH) at atmospheric pressure. Mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), ET-1 and NO levels, and pulmonary vascular structural changes were observed in all groups. RESULTS: MPAP, RVHI and ET-1 levels were significantly higher in HPH group than in control group, while NO was significantly lower than in control group. In addition, mPAP, RVHI and ET-1 were significantly lower in the HPH+fasudil group than in the HPH group. In the HPH group, ET-1 level was significantly and positively correlated with mPAP and RVHI, NO was negatively correlated with mPAP and RVHI levels, and ET-1 level was significantly and negatively correlated with NO level. In the HPH group, pulmonary arteriolar walls were generally thickened, and lumen stenosis was obvious; while after fasudil treatment, pulmonary arteriolar wall thickening and stenosis degree were significantly reduced. CONCLUSION: Fasudil can significantly reduce ET-l level and increase NO level in HPH rats, suppressing the development of pulmonary arterial hypertension.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Endotelina-1/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Óxido Nítrico/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Modelos Animais de Doenças , Hipertensão Pulmonar/patologia , Masculino , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley
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