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1.
Immunobiology ; 223(11): 709-717, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30049418

RESUMO

Phosphoprotein enriched in astrocytes 15 (PEA15) plays a multi-functional role in neuronal cell survival, however the effects of PEA15 against inflammation have not been investigated yet. To examine the effects of PEP-1-PEA15 protein against lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 cells and in a 12-O-tetradecanoylphobol 13-acetate (TPA)-induced mouse model, we constructed and purified PEP-1-PEA15 protein, which can transduce into cells or tissues. PEP-1-PEA15 inhibited LPS-induced damage in cells including that caused by reactive oxygen species (ROS) production and DNA fragmentation. PEP-1-PEA15 also significantly suppressed activation of mitogen activated protein kinases (MAPKs), pro-inflammatory mediator proteins and various cytokines. In a TPA-induced mouse ear edema model, PEP-1-PEA15 significantly reduced ear weight and thickness as well as MAPK activation as well as the expression levels of COX-2, iNOS, IL-6, IL-1ß, and TNF-α. These results demonstrated that PEP-1-PEA15 showed anti-inflammatory effect in cells and animal model suggesting that this fusion protein protects cells or skin tissues from inflammatory response.


Assuntos
Cisteamina/análogos & derivados , Edema/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/imunologia , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Cisteamina/metabolismo , Citocinas/metabolismo , Fragmentação do DNA , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/imunologia
2.
Int J Mol Med ; 38(1): 217-24, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27222268

RESUMO

Antioxidant 1 (ATOX1) functions as an antioxidant against hydrogen peroxide and superoxide, and therefore may play a significant role in many human diseases, including diabetes mellitus (DM). In the present study, we examined the protective effects of Tat-ATOX1 protein on streptozotocin (STZ)-exposed pancreatic insulinoma cells (RINm5F) and in a mouse model of STZ-induced diabetes using western blot analysis, immunofluorescence staining and MTT assay, as well as histological and biochemical analysis. Purified Tat-ATOX1 protein was efficiently transduced into RINm5F cells in a dose- and time-dependent manner. Additionally, Tat-ATOX1 protein markedly inhibited reactive oxygen species (ROS) production, DNA damage and the activation of Akt and mitogen activated protein kinases (MAPKs) in STZ-exposed RINm5F cells. In addition, Tat-ATOX1 protein transduced into mice pancreatic tissues and significantly decreased blood glucose and hemoglobin A1c (HbA1c) levels as well as the body weight changes in a model of STZ-induced diabetes. These results indicate that transduced Tat-ATOX1 protein protects pancreatic ß-cells by inhibiting STZ-induced cellular toxicity in vitro and in vivo. Based on these findings, we suggest that Tat-ATOX1 protein has potential applications as a therapeutic agent for oxidative stress-induced diseases including DM.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Metalochaperonas/uso terapêutico , Pâncreas/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Produtos do Gene tat do Vírus da Imunodeficiência Humana/uso terapêutico , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Transporte de Cobre , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Chaperonas Moleculares , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
3.
Int J Mol Med ; 38(1): 225-35, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27221790

RESUMO

Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein 3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide (H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein (Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species (ROS), DNA fragmentation and reduced mitochondrial membrane potential (ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3 and -8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury.


Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Hipocampo/patologia , Proteínas Musculares/farmacologia , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Gerbillinae , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução Genética
4.
Toxicol Appl Pharmacol ; 286(2): 124-34, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25818598

RESUMO

Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.


Assuntos
Oxirredutases do Álcool/farmacologia , Anti-Inflamatórios/farmacologia , Edema/tratamento farmacológico , Produtos do Gene tat/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Animais , Orelha Externa/patologia , Edema/induzido quimicamente , Edema/patologia , Ativação Enzimática/efeitos dos fármacos , Lipopolissacarídeos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Frações Subcelulares/efeitos dos fármacos , Acetato de Tetradecanoilforbol
5.
J Cell Mol Med ; 19(6): 1333-45, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25781353

RESUMO

Oxidative stress-induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat-Atox1 and examined the roles of Tat-Atox1 in oxidative stress-induced hippocampal HT-22 cell death and an ischaemic injury animal model. Tat-Atox1 effectively transduced into HT-22 cells and it protected cells against the effects of hydrogen peroxide (H2O2)-induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat-Atox1 regulated cellular survival signalling such as p53, Bad/Bcl-2, Akt and mitogen-activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat-Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat-Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat-Atox1 protects against oxidative stress-induced HT-22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat-Atox1 has potential as a therapeutic agent for the treatment of oxidative stress-induced ischaemic damage.


Assuntos
Apoptose/efeitos dos fármacos , Isquemia/prevenção & controle , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Transporte de Cobre , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Hipocampo/citologia , Humanos , Isquemia/fisiopatologia , Metalochaperonas/genética , Metalochaperonas/metabolismo , Metalochaperonas/farmacologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Chaperonas Moleculares , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Prosencéfalo/irrigação sanguínea , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Free Radic Biol Med ; 69: 181-96, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24440593

RESUMO

Human carbonyl reductase 1 (CBR1) is a member of the NADPH-dependent short-chain dehydrogenase/reductase superfamily that is known to play an important role in neuronal cell survival via its antioxidant function. Oxidative stress is one of the major causes of degenerative disorders including ischemia. However, the role CBR1 plays with regard to ischemic injury is as yet poorly understood. Protein transduction domains such as PEP-1 are well known and now commonly used to deliver therapeutic proteins into cells. In this study, we prepared PEP-1-CBR1 protein and examined whether it protects against oxidative-stress-induced neuronal cell damage. PEP-1-CBR1 protein was efficiently transduced into hippocampal neuronal HT-22 cells and protected against hydrogen peroxide (H2O2)-induced neuronal cell death. Transduced PEP-1-CBR1 protein drastically inhibited H2O2-induced reactive oxygen species production, the oxidation of intracellular macromolecules, and the activation of mitogen-activated protein kinases, as well as cellular apoptosis. Furthermore, we demonstrated that transduced PEP-1-CBR1 protein markedly protected against neuronal cell death in the CA1 region of the hippocampus resulting from ischemic injury in an animal model. In addition, PEP-1-CBR1 protein drastically reduced activation of glial cells and lipid peroxidation in an animal model. These results indicate that PEP-1-CBR1 protein significantly protects against oxidative-stress-induced neuronal cell death in vitro and in vivo. Therefore, we suggest that PEP-1-CBR1 protein may be a therapeutic agent for the treatment of ischemic injuries as well as oxidative-stress-induced cell damage and death.


Assuntos
Oxirredutases do Álcool/genética , Isquemia/genética , Estresse Oxidativo , Proteínas Recombinantes de Fusão/genética , Oxirredutases do Álcool/metabolismo , Antioxidantes/metabolismo , Apoptose/genética , Sobrevivência Celular/genética , Cisteamina/análogos & derivados , Cisteamina/metabolismo , Hipocampo/metabolismo , Humanos , Isquemia/etiologia , Isquemia/patologia , Neurônios/patologia , Peptídeos/genética , Peptídeos/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/metabolismo
7.
Free Radic Biol Med ; 63: 432-45, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23770196

RESUMO

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1-SIRT2 protein. Purified PEP-1-SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H2O2)-induced cell death and cytotoxicity. Also, transduced PEP-1-SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1-SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1-SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1-SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1-SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.


Assuntos
Inflamação/metabolismo , Estresse Oxidativo , Peptídeos/metabolismo , Sirtuína 2/metabolismo , Animais , Antioxidantes/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Peróxido de Hidrogênio/toxicidade , Inflamação/tratamento farmacológico , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/genética , Ésteres de Forbol/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 2/administração & dosagem , Sirtuína 2/genética , Pele/efeitos dos fármacos , Pele/crescimento & desenvolvimento
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