RESUMO
PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.
Assuntos
Ácido Ascórbico/farmacologia , Queimaduras/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Adulto , Araquidonato 12-Lipoxigenase/análise , Araquidonato 12-Lipoxigenase/efeitos dos fármacos , Queimaduras/tratamento farmacológico , Células Cultivadas , Estudos Transversais , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 1/efeitos dos fármacos , Oxidases Duais/análise , Oxidases Duais/efeitos dos fármacos , Feminino , Glutationa Peroxidase/análise , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Transferase/análise , Glutationa Transferase/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Peroxirredoxinas/análise , Peroxirredoxinas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Pele/efeitos dos fármacos , Pele/patologia , Estatísticas não Paramétricas , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Assuntos
Autofagia/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular , Citometria de Fluxo , Humanos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Assuntos
Humanos , Proteínas Quinases/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/metabolismo , Autofagia/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Eletrônica de Transmissão , Epitélio Pigmentado da Retina/citologia , Citometria de Fluxo , Proteínas de Membrana/metabolismoRESUMO
INTRODUCTION: In this study we investigated the effects of high-intensity resistance training (RT) on dexamethasone (DEX)-induced muscle atrophy in flexor hallucis longus (FHL), tibialis anterior (TA), and soleus (SOL) muscles. METHODS: Rats underwent either high-intensity RT or were kept sedentary. In the last 10 days they received either DEX (0.5 mg/kg/day, intraperitoneally) or saline. RESULTS: DEX reduced body weight (-21%), food intake (-28%), FHL and TA muscle mass (-20% and -18%, respectively), and increased muscle-specific ring finger 1 (MuRF-1) protein level (+37% and +45.5%). RT attenuated FHL muscle atrophy through a combination of low increase in MuRF-1 protein level (-3.5%) and significant increases in mammalian target of rapamycin (mTOR) (+63%) and p70S6K (+46% and +49% for control and DEX, respectively) protein levels. CONCLUSION: RT attenuated DEX-induced muscle atrophy through a combination of increases in mTOR and p70S6K protein levels and a low increase in MuRF-1 protein level.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Condicionamento Físico Animal/métodos , Treinamento Resistido/métodos , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Dexametasona/efeitos adversos , Comportamento Alimentar/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Skeletal muscle atrophy induced during sepsis syndrome produced by endotoxin in the form of LPS (lipopolysaccharide), is a pathological condition characterized by the loss of strength and muscle mass, an increase in MHC (myosin heavy chain) degradation, and an increase in the expression of atrogin-1 and MuRF-1 (muscle-specific RING-finger protein 1), two ubiquitin E3 ligases belonging to the ubiquitin-proteasome system. Ang-(1-7) [Angiotensin-(1-7)], through its Mas receptor, has beneficial effects in skeletal muscle. We evaluated in vivo the role of Ang-(1-7) and Mas receptor on the muscle wasting induced by LPS injection into C57BL/10J mice. In vitro studies were performed in murine C2C12 myotubes and isolated myofibres from EDL (extensor digitorum longus) muscle. In addition, the participation of p38 MAPK (mitogen-activated protein kinase) in the Ang-(1-7) effect on the LPS-induced muscle atrophy was evaluated. Our results show that Ang-(1-7) prevents the decrease in the diameter of myofibres and myotubes, the decrease in muscle strength, the diminution in MHC levels and the induction of atrogin-1 and MuRF-1 expression, all of which are induced by LPS. These effects were reversed by using A779, a Mas antagonist. Ang-(1-7) exerts these anti-atrophic effects at least in part by inhibiting the LPS-dependent activation of p38 MAPK both in vitro and in vivo. We have demonstrated for the first time that Ang-(1-7) counteracts the skeletal muscle atrophy induced by endotoxin through a mechanism dependent on the Mas receptor that involves a decrease in p38 MAPK phosphorylation. The present study indicates that Ang-(1-7) is a novel molecule with a potential therapeutic use to improve muscle wasting during endotoxin-induced sepsis syndrome.
Assuntos
Angiotensina I/farmacologia , Músculo Esquelético/metabolismo , Atrofia Muscular/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Análise de Variância , Animais , Células Cultivadas , Técnicas In Vitro , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/patologia , Distribuição Aleatória , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/complicações , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Vasodilatadores/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Long-term adaptation to resistance training is probably due to the cumulative molecular effects of each exercise session. Therefore, we studied in female Wistar rats the molecular effects of a chronic resistance training regimen (3 months) leading to skeletal muscle hypertrophy in the plantaris muscle. Our results demonstrated that muscle proteolytic genes MuRF-1 and Atrogin-1 were significantly decreased in the exercised group measured 24 h after the last resistance exercise session (41.64 and 61.19%, respectively; P < 0.05). Nonetheless, when measured at the same time point, 4EBP-1, GSK-3beta and eIF2Bepsilon mRNA levels and Akt, GSK-3beta and p70S6K protein levels (regulators of translation initiation) were not modified. Such data suggests that if gene transcription constitutes a control point in the protein synthesis pathway this regulation probably occurs in early adaptation periods or during extreme situations leading to skeletal muscle remodeling. However, proteolytic gene expression is modified even after a prolonged resistance training regimen leading to moderate skeletal muscle hypertrophy.