RESUMO
Excessive reactive oxygen species (ROS) production plays a crucial role in causing various diseases, including inflammatory disorders. The activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling is implicated in stimulating inflammatory response and cytokines. Peroxiredoxin 2 (Prx2) is a 2-cysteine (Cys) peroxiredoxin capable of removing endogenous hydrogen peroxide (H2O2). PEP-1 peptide, a protein transduction domain, consists of three domains which are used to transduce exogenous therapeutic proteins into cells. The correlation between effectively transduced PEP-1-Prx2 and ROS-mediated inflammatory response is not clear. In the present study, we investigated the protective effects of cell permeable PEP-1-Prx2 on oxidative stress-induced inflammatory activity in Raw 264.7 cells and in a mouse ear edema model after exposure to lipopolysaccharides (LPS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Transduced PEP-1-Prx2 suppressed intracellular ROS accumulation and inhibited the activity of MAPKs and NF-κB signaling that led to the suppression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and cytokines in LPS-induced Raw 264.7 cells and TPA-induced mouse ear edema model. Given these results, we propose that PEP-1-Prx2 has therapeutic potential in the prevention of inflammatory disorders.
Assuntos
Cisteamina/análogos & derivados , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Cisteamina/metabolismo , Orelha/patologia , Edema/induzido quimicamente , Edema/metabolismo , Proteínas de Homeodomínio/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/genética , Óxido Nítrico , Peptídeos/genética , Piridinas/toxicidadeRESUMO
BACKGROUND: Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1-6) that can reduce hydrogen peroxide (H2O2) and organic hydroperoxides to water and alcohols. METHODS: We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis. RESULTS: Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H2O2-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H2O2-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage. CONCLUSIONS: These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE: PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.
Assuntos
Cisteamina/análogos & derivados , Proteínas de Homeodomínio/genética , Inflamação/tratamento farmacológico , Isquemia/tratamento farmacológico , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Animais , Apoptose/genética , Astrócitos/metabolismo , Astrócitos/patologia , Região CA1 Hipocampal/metabolismo , Cisteamina/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Inflamação/patologia , Isquemia/patologia , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios/citologia , Fármacos Neuroprotetores , Estresse Oxidativo/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H2O2)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H2O2-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator's expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.
Assuntos
4-Butirolactona/análogos & derivados , Anti-Inflamatórios/farmacologia , Dermatite de Contato/metabolismo , Macrófagos/imunologia , 4-Butirolactona/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Acetato de TetradecanoilforbolRESUMO
The DJ-1 gene is highly conserved in diverse species and DJ-1 is known as an anti-oxidative stress factor. In this study, we investigated the neuroprotective effects of DJ-1 against ischemic damage in the rabbit spinal cord. Tat-DJ-1 fusion proteins were constructed to facilitate the penetration of DJ-1 protein into the neurons. Tat-1-DJ-1 fusion protein was administered to the rabbit 30 min after ischemia/reperfusion, and transient spinal cord ischemia was induced by occlusion of the aorta at the subrenal region for 15 min. The administration of Tat-DJ-1 significantly improved the Tarlov score compared to that in the Tat (vehicle)-treated group at 24, 48 and 72 h after ischemia/reperfusion. At 72 h after ischemia/reperfusion, the number of cresyl violet-positive neurons was significantly increased in the Tat-DJ-1-treated group compared to that in the vehicle-treated group. Lipid peroxidation as judged from the malondialdehyde levels was significantly decreased in the Tat-DJ-1-treated group compared to that in the vehicle-treated group. In contrast, superoxide dismutase and catalase levels were significantly increased in the Tat-DJ-1-treated group compared to that in the vehicle-treated group. This result suggests that DJ-1 protects neurons from ischemic damage in the ventral horn of the spinal cord via its antioxidant effects.
Assuntos
Proteínas Oncogênicas/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Isquemia do Cordão Espinal/prevenção & controle , Animais , Antioxidantes/farmacologia , Catalase/biossíntese , Membro Posterior/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Coelhos , Traumatismo por Reperfusão/fisiopatologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Superóxido Dismutase/biossínteseRESUMO
Oxidative stress initiates age-related reduction in hippocampal neurogenesis and the use of antioxidants has been proposed as an effective strategy to prevent or attenuate the reduction of neurogenesis in the hippocampus. In the present study, we investigated the effects of Cu,Zn-superoxide dismutase (SOD1) and/or peroxiredoxin-2 (PRX2) on cell proliferation and neuroblast differentiation in the dentate gyrus in a model of D-galactose-induced aging model. For this study, we constructed an expression vector, PEP-1, fused PEP-1 with SOD1 or PRX2, and generated PEP-1-SOD1 and PEP-1-PRX2 fusion protein. The aging model was induced by subcutaneous injection of D-galactose (100 mg/kg) to 6-week-old male mice for 10 weeks. PEP-1, PEP-1-SOD1 and/or PEP-1-PRX2 fusion protein was intraperitoneally administered to these mice at 13-week-old once a day for 3 weeks and sacrificed at 30 min after the last administrations. The administration of PEP-1-SOD1 and/or PEP-1-PRX2 significantly improved D-galactose-induced deficits on the escape latency, swimming speeds, platform crossings, spatial preference for the target quadrant in Morris water maze test. In addition, the administration of PEP-1-SOD1 and/or PEP-1-PRX2 ameliorated D-galactose-induced reductions of cell proliferation and neuroblast differentiation in the dentate gyrus and significantly reduced D-galactose-induced lipid peroxidation in the hippocampus. These effects were more prominent in the PEP-1-SOD1-treated group with PEP-1-PRX2. These results suggest that a SOD1 and/or PRX2 supplement to aged mice could improve the memory deficits, cell proliferation and neuroblast differentiation in the dentate gyrus of D-galactose induced aged mice by reducing lipid peroxidation.
Assuntos
Envelhecimento/efeitos dos fármacos , Hipocampo/fisiologia , Peroxirredoxinas/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Superóxido Dismutase/administração & dosagem , Animais , Antioxidantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisteamina/administração & dosagem , Cisteamina/análogos & derivados , Galactose/toxicidade , Hipocampo/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Camundongos , Neurogênese , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/administração & dosagem , Superóxido Dismutase-1RESUMO
Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.
Assuntos
Isquemia Encefálica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Proteínas Oncogênicas/administração & dosagem , Estresse Oxidativo , Proteínas Recombinantes de Fusão/administração & dosagem , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Animais , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gerbillinae , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/farmacocinética , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Camundongos , Fármacos Neuroprotetores/farmacocinética , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/farmacocinética , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Proteína Desglicase DJ-1 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacocinética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacocinéticaRESUMO
Parkinson's disease (PD) is caused by various factors such as reactive oxygen species (ROS), dysfunction of mitochondria, and aggregation of misfolded proteins, thereby leading to loss of dopaminergic (DA) neurons in the substantia nigra (SN) of the brain. Frataxin (FXN) is associated with iron homeostasis and biogenesis of iron-sulfur clusters in the electron transport chain complex. In this study, we investigated the potential of Tat-FXN to cross the blood-brain barrier (BBB) and protect DA neurons against oxidative stress in a mouse model of PD. Tat-FXN was effectively transduced into SH-SY5Y cells and blocked production of ROS and cleavage of DNA, significantly improving cell survival against 1-methyl-4-phenylpyridinium induced toxicity. In addition, Tat-FXN efficiently penetrated the BBB and exhibited a clear neuroprotective effect on tyrosine hydroxylase-specific DA neurons in the SN in a mice model of 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine-induced PD. Therefore, these results suggest that Tat-FXN may provide neuroprotective therapy for ROS related diseases including PD.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Produtos do Gene tat/genética , Proteínas de Ligação ao Ferro/metabolismo , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/patologia , Neurotoxinas/toxicidade , Aldeídos/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Proteínas de Ligação ao Ferro/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transdução Genética , FrataxinaRESUMO
BACKGROUND: Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult. METHODS: A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-MT-III protein. PEP-1-MT-III protein was purified using affinity chromatograph. Transduced PEP-1-MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry. RESULTS: Purified PEP-1-MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1-MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1-MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1-MT-III protein protects against oxidative stress induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE: Transduced PEP-1-MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1-MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Cisteamina/análogos & derivados , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Cisteamina/química , Cisteamina/farmacologia , Cisteamina/uso terapêutico , Gerbillinae , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Metalotioneína 3 , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/uso terapêutico , Neurônios/metabolismo , Neurônios/fisiologia , Estresse Oxidativo/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/uso terapêutico , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução Genética/métodosRESUMO
Parkinson's disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD.
Assuntos
Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Proteínas Oncogênicas/genética , Estresse Oxidativo/fisiologia , Doença de Parkinson/genética , Animais , Antioxidantes/metabolismo , Morte Celular/genética , Morte Celular/fisiologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/metabolismo , Proteínas Oncogênicas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Peroxirredoxinas , Proteína Desglicase DJ-1 , Espécies Reativas de Oxigênio/metabolismo , Substância Negra/metabolismo , Substância Negra/fisiologia , Transdução Genética/métodosRESUMO
Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+) ) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP(+) -induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.
Assuntos
Cisteamina/análogos & derivados , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Doença de Parkinson Secundária/tratamento farmacológico , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Substância Negra/efeitos dos fármacos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisteamina/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Escherichia coli , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/metabolismo , Peptídeos/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-Rod , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
The protein transduction domains have been reported to have potential to deliver the exogenous molecules, including proteins, to living cells. However, poor transduction of proteins limits therapeutic application. In this study, we examined whether imipramine could stimulate the transduction efficiency of PEP-1 fused proteins into astrocytes. PEP-1-catalase (PEP-1- CAT) was transduced into astrocytes in a time- and dose-dependent manner, reducing cellular toxicity induced by H(2)O(2). Additionally, the group of PEP-1-CAT (+) imipramine showed enhancement of transduction efficiency and therefore increased cellular viability than that of PEP-1-CAT alone. In the gerbil ischemia models, PEP-1-CAT displayed significant neuroprotection in the CA1 region of the hippocampus. Interestingly, PEP-1-CAT (+) imipramine prevented neuronal cell death and lipid peroxidation more markedly than PEP-1-CAT alone. Therefore, our results suggest that imipramine can be used as a drug to enhance the transduction of PEP-1 fusion proteins to cells or animals and their efficacies against various disorders.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Catalase/uso terapêutico , Cisteamina/análogos & derivados , Imipramina/uso terapêutico , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Animais , Antidepressivos Tricíclicos/farmacologia , Antidepressivos Tricíclicos/uso terapêutico , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Catalase/genética , Catalase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cisteamina/farmacologia , Cisteamina/uso terapêutico , Gerbillinae , Imipramina/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução GenéticaRESUMO
AIMS: Cyclophilin A (CypA) is an immunophilin that acts as a receptor for the immunosuppressant drug cyclosporine A (CsA). CypA has emerged as a potential drug target for several inflammatory diseases, although the details of its mechanism are unclear. We examined the protective effects of CypA on inflammation in Raw 264.7 cells and animal models. MAIN METHODS: A human CypA gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-CypA protein. The protein expression level of cyclooxygenase-2 (COX-2) and cytokines was detected by Western blot, ELISA and mRNA level of COX-2 and cytokines were measured by RT-PCR. The nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) activation were analyzed by Western blot and electrophoretic mobility shift assay. Skin inflammation was detected with immunohistochemistry. KEY FINDINGS: Transduced PEP-1-CypA protein markedly inhibited lipopolysaccharide- and 12-O-tetradecanoyl phorbol-13-acetate-induced expression levels of COX-2 as well as pro-inflammatory cytokine levels in vitro and in vivo. Furthermore, transduced PEP-1-CypA protein resulted in a significant reduction in the activation of NF-kB and MAPK. SIGNIFICANCE: The results indicate that PEP-1-CypA inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK activation upon stimulation of inflammation in vitro and in vivo. PEP-1-CypA protein may potentially be used as a therapeutic agent against skin diseases-related inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Ciclofilina A/farmacologia , Inflamação/tratamento farmacológico , Peptídeo Hidrolases/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Western Blotting , Linhagem Celular , Ciclo-Oxigenase 2/biossíntese , Citocinas/biossíntese , Dinoprostona/biossíntese , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/biossíntese , NF-kappa B/biossínteseRESUMO
Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe(2+)), is up-regulated by several cellular stress and cell injuries, including inflammation, ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Edema/terapia , Heme Oxigenase-1/uso terapêutico , Inflamação/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Linhagem Celular , Modelos Animais de Doenças , Edema/tratamento farmacológico , Heme Oxigenase-1/genética , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Transdução GenéticaRESUMO
Immunophilin, FK506-binding protein 12 (FK506BP), is a receptor protein for the immunosuppressive drug FK506 by the FK506BP/FK506 complex. However, the precise function of FK506BP in inflammatory diseases remains unclear. Therefore, we examined the protective effects of FK506BP on atopic dermatitis (AD) in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced HaCaT cells and 2,4-dinitrofluorobenzene-induced AD-like dermatitis in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice using a cell-permeable PEP-1-FK506BP. Transduced PEP-1-FK506BP significantly inhibited the expression of cytokines, as well as the activation of NF-κB and mitogen-activated protein kinase (MAPK) in TNF-α/IFN-γ-induced HaCaT cells. Furthermore, topical application of PEP-1-FK506BP to NC/Nga mice markedly inhibited AD-like dermatitis as determined by a histological examination and assessment of serum IgE levels, as well as cytokines and chemokines. These results indicate that PEP-1-FK506BP inhibits NF-κB and MAPK activation in cells and AD-like skin lesions by reducing the expression levels of cytokines and chemokines, thus suggesting that PEP-1-FK506BP may be a potential therapeutic agent for AD.
Assuntos
Cisteamina/análogos & derivados , Dermatite Atópica/tratamento farmacológico , Peptídeos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas de Ligação a Tacrolimo/uso terapêutico , Animais , Cisteamina/uso terapêutico , Dermatite Atópica/etiologia , Modelos Animais de Doenças , Imunoglobulina E/sangue , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Sistema de Sinalização das MAP Quinases , Camundongos , NF-kappa B/metabolismo , Peptídeos/genética , Peptídeos/fisiologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/fisiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
FK506 binding protein 12 (FK506BP) is an immunophilin that acts as a receptor for the immunosuppressant drug FK506. Although the precise action of FK506BP remains unclear, it has emerged as a potential drug target for several inflammatory diseases. This study investigated the protective effects of FK506BP on inflammation in vitro and in vivo using protein transduction. A cell-permeable expression vector PEP-1-FK506BP was constructed. Lipopolysaccharide (LPS)- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated Raw 264.7 cells and ICR mice were treated with PEP-1-FK506BP. The expression of inflammatory response enzymes and cytokines was analyzed by Western blot, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay. PEP-1-FK506BP efficiently transduced into Raw 264.7 cells and markedly inhibited the expression levels of cyclooxygenase-2 as well as pro-inflammatory cytokines. Furthermore, transduced PEP-1-FK506BP significantly reduced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the cells, whereas PEP-1-FK506BP reduced phosphorylation of p38 and extracellular signal-regulated kinase (ERK) in the animal models. These results indicate that PEP-1-FK506BP inhibits inflammatory response cytokines and enzymes by blocking NF-κB and MAPK including the phosphorylation of p38 and/or ERK MAPK in vitro and in vivo, suggesting that PEP-1-FK506BP may be a therapeutic agent against inflammatory skin diseases.
Assuntos
Imunossupressores/uso terapêutico , Macrófagos/efeitos dos fármacos , Dermatopatias/tratamento farmacológico , Proteínas de Ligação a Tacrolimo/metabolismo , Tacrolimo/uso terapêutico , Animais , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Inflamação , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Dermatopatias/imunologia , Proteínas de Ligação a Tacrolimo/administração & dosagem , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/imunologia , Transdução GenéticaRESUMO
Reactive oxygen species (ROS) actively contribute to the development of a number of human diseases including ischemia. In response to oxidative stress, frataxin has a significant ability to improve cell survival though its biological function is unclear in relation to ischemia. To explore frataxin's role in protecting against ischemic cell death, we constructed PEP-1-Frataxin cell-permeable fusion protein. In a dose- and time-dependent manner PEP-1-Frataxin rapidly transduced into astrocyte cells and protected them against oxidative stress-induced cell death. Further, using an animal model, immunohistochemical analysis revealed that PEP-1-Frataxin prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. These results demonstrate that transduced PEP-1-Frataxin protects against cell death in vitro and in vivo, suggesting that transduction of PEP-1-Frataxin could be useful as a therapeutic agent for various human diseases related to oxidative stress.
Assuntos
Cisteamina/análogos & derivados , Proteínas de Ligação ao Ferro/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Região CA1 Hipocampal/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Corantes , Cisteamina/farmacologia , Relação Dose-Resposta a Droga , Vetores Genéticos , Gerbillinae , Humanos , Proteínas de Ligação ao Ferro/biossíntese , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/patologia , Microscopia de Fluorescência , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Sais de Tetrazólio , Tiazóis , Transdução Genética , FrataxinaRESUMO
Though protein transduction domains (PTDs) are well known for the delivery of exogenous therapeutic proteins into living cells, the overall low efficiency of transduction is a serious obstacle. We investigated the effect of bog blueberry anthocyanins (BBA) on protein transduction efficiency and found that BBA enhanced the transduction efficiencies of Tat-SOD fusion protein into HeLa cells and mice skin. The enzymatic activities in the cells and skin tissue in the presence of BBA were markedly increased compared to controls. Further, BBA did not demonstrate any cell toxicity at various concentrations. Although the mechanism is not fully understood, we suggest that BBA might alter the conformation of the membrane, which would indicate that BBA can be used as a protein transduction enhancer for the efficient delivery of therapeutic proteins for a variety of disorders.
Assuntos
Antocianinas/farmacologia , Mirtilos Azuis (Planta)/química , Transdução Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Antocianinas/toxicidade , HIV-1 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos ICR , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Pele/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Reactive oxygen species (ROS) have been implicated in the pathogenesis of ischemic brain injury. Sensitive to apoptosis gene (SAG) is a RING-finger protein that exhibits antioxidant activity against a variety of redox reagents. However, the protective effect of SAG in brain ischemic injury is unclear. Here, we investigated the protective effects of a Tat-SAG fusion protein against cell death and ischemic insult. When Tat-SAG fusion protein was added to the culture medium of astrocytes, it rapidly entered the cells and protected them against oxidative stress-induced cell death. Immunohistochemical analysis revealed that, when Tat-SAG fusion protein was intraperitoneally injected into gerbils, wild-type Tat-SAG prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. In addition, wild-type Tat-SAG fusion protein decreased lipid peroxidation in the brain compared with mutant Tat-SAG- or vehicle-treated animals. Our results demonstrate that Tat-SAG fusion protein is a tool for the treatment of ischemic insult and it can be used in protein therapy for various disorders related to ROS, including stroke.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Produtos do Gene tat/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Citoproteção , Produtos do Gene tat/genética , Gerbillinae , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Estresse Oxidativo , Engenharia de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Antioxidant enzymes are considered to have beneficial effects against various diseases mediated by reactive oxygen species (ROS). Ischemia is characterized by both oxidative stress and changes in the antioxidant defense system. Catalase (CAT) and superoxide dismutase (SOD) are major antioxidant enzymes by which cells counteract the deleterious effects of ROS. To investigate the protective effects of CAT, we constructed PEP-1-CAT cell-permeative expression vectors. When PEP-1-CAT fusion proteins were added to the culture medium of neuronal cells, they rapidly entered the cells and protected them against oxidative stress-induced neuronal cell death. Immunohistochemical analysis revealed that PEP-1-CAT prevented neuronal cell death in the hippocampus induced by transient forebrain ischemia. Moreover, we showed that the protective effect of PEP-1-CAT was observed in neuronal cells treated with PEP-1-SOD. Therefore, we suggest that transduced PEP-1-CAT and PEP-1-SOD fusion proteins could be useful as therapeutic agents for various human diseases related to oxidative stress, including stroke.
Assuntos
Catalase/metabolismo , Cisteamina/análogos & derivados , Ataque Isquêmico Transitório/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Catalase/genética , Catalase/isolamento & purificação , Cisteamina/isolamento & purificação , Cisteamina/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Ataque Isquêmico Transitório/patologia , Peroxidação de Lipídeos , Estresse Oxidativo , Peptídeos/genética , Peptídeos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Arginine deiminase (ADI), an arginine-degrading enzyme, has anti-proliferative and anti-tumor activities and is capable of inhibiting the production of nitric oxide (NO). Modulation of nitric oxide (NO) production is considered a promising approach for the treatment of various diseases including cancer, inflammation and neuronal disorders. In this study, an ADI gene was fused with an HIV-1 Tat peptide in a bacterial expression vector to produce an genetic in-frame Tat-ADI fusion protein. When added exogenously to the culture media, the expressed and purified Tat-ADI fusion proteins were efficiently transduced into macrophage Raw 264.7 cells in a time- and dose-dependent manner. Furthermore, transduced Tat-ADI fusion proteins markedly increased cell viability in cells treated with lipopolysaccharide (LPS). This increase in viability was mediated by an inhibition of NO production. These results suggest that this Tat-ADI fusion protein can be used in protein therapies of NO-related disorders such as cancer, inflammation and neuronal diseases.