RESUMO
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by the absence of the protein dystrophin. Because oxidative stress contributes to the pathogenesis of DMD, we investigated if a green tea polyphenol blend (GTP) and its major polyphenol (-)-epigallocatechin gallate (EGCg), could protect muscle cell primary cultures from oxidative damage induced by hydrogen peroxide (H(2)O(2)) in the widely used mdx mouse model. On-line fluorimetric measurements using an H(2)O(2)-sensitive probe indicated that GTP and EGCg scavenged peroxide in a concentration-dependent manner. A 48 h exposure to EGCg increased glutathione content but did not alter the expression of proteins involved in membrane stabilization and repair. Pretreatment of dystrophic cultures with GTP or EGCg 48 h before exposure to H(2)O(2) improved cell survival. Normal cultures were protected by GTP but not by EGCg. 67LR, a receptor for EGCg, was seven times more abundant in dystrophic compared with normal cultures. Altogether our results demonstrate that GTP and EGCg protect muscle cells by scavenging H(2)O(2) and by improving the glutathione balance. In addition, the higher levels of 67LR in dystrophic muscle cells compared with normal ones likely contribute to EGCg-mediated survival.
Assuntos
Flavonoides/farmacologia , Glutationa/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Fenóis/farmacologia , Receptores de Laminina/metabolismo , Chá/química , Animais , Animais Recém-Nascidos , Catequina/análogos & derivados , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Polifenóis , Inibidores de Proteases/farmacologiaRESUMO
Advanced glycation endproducts (AGEs) and their precursor dicarbonyls are generally perceived as having adverse health effects. They are also considered to be initiators and promoters of disease and aging. However, proof for a causal relationship is lacking. On the other hand, it is known that AGEs and melanoidins possess beneficial properties, such as antioxidant and metal-chelating activities. Furthermore, some AGEs may stimulate the cellular detoxification system, generally known as the phase II drug metabolizing system. We show here that several reactive dicarbonyl intermediates have the capability to stimulate the cellular phase II detoxification systems in both a reporter cell line and primary rat hepatocytes. In addition, we demonstrate that dicarbonyls can attenuate the inflammatory signaling induced by tumor necrosis factor-alpha in a reporter cell system.
Assuntos
Anti-Inflamatórios/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Hepatócitos/fisiologia , Hidroquinonas/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Genes Reporter , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA/genética , RNA Mensageiro/genética , Ratos , Proteína de Ligação a TATA-Box/genéticaRESUMO
Probiotic bacteria have been shown to modulate the immune system of the gut and protect against infectious and inflammatory diseases of the gastro-intestinal tract. Ultraviolet radiation (UVR) is known to alter the cutaneous and systemic immune systems implicated in the development of skin tumors. In this study we investigated whether oral probiotics are able to modulate the immune system of the skin using hairless Skh:hr1 mice exposed to an acute dose of UVR. We show that nutritional supplementation with Lactobacillus johnsonii (La1) at 10(8) cfu/day for 10 days was able to protect against the UVR-induced suppression of contact hypersensitivity, the decreased epidermal Langerhans cell density and the increased IL-10 serum levels. In the absence of UV exposure, probiotic bacteria had no detectable effect on the immune system of the skin, thus acting only to re-establish skin homeostasis. In conclusion, our data demonstrate that ingested probiotic bacteria can maintain cutaneous immune capacity after UV exposure.
Assuntos
Probióticos/uso terapêutico , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Raios Ultravioleta , Animais , Dermatite de Contato/imunologia , Feminino , Homeostase/efeitos dos fármacos , Homeostase/efeitos da radiação , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/efeitos da radiação , Inflamação/fisiopatologia , Interleucina-10/sangue , Lactobacillus , Células de Langerhans/fisiologia , Camundongos , Camundongos Pelados , Camundongos Endogâmicos , Fenômenos Fisiológicos da Pele/efeitos da radiaçãoRESUMO
Cyclosporin A (CsA) generates superoxide in smooth muscle cells. Our earlier studies have demonstrated that the increase in the vasopressin type 1 receptor induced in vascular smooth muscle cells in the presence of CsA is probably due to superoxide (Krauskopf et al., J Biol Chem 278, 41685-41690, 2003). This increase in vasopressin receptor is likely at the base of increased vascular responsiveness to vasoconstrictor hormones and hypertension induced by CsA. Here, we demonstrate that CsA produces superoxide. In addition, our data show that superoxide generation does not originate from the major cellular superoxide generating systems NAD(P)H oxidase or xanthine oxidase. Our results suggest that the side effects of CsA could be diminished with the help of SOD mimetic drugs.
Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Aorta/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Etídio/análogos & derivados , Fluoresceínas , Sequestradores de Radicais Livres/farmacologia , Masculino , Miócitos de Músculo Liso/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genéticaRESUMO
BACKGROUND: Duchenne muscular dystrophy is a severe X-linked congenital disorder characterized by lethal muscle wasting caused by the absence of the structural protein dystrophin. OBJECTIVE: Because generation of reactive oxygen species appears to play an important role in the pathogenesis of this disease, we tested whether antioxidant green tea extract could diminish muscle necrosis in the mdx mouse dystrophy model. DESIGN: A diet supplemented with 0.01% or 0.05% green tea extract was fed to dams and neonates for 4 wk beginning on the day of birth. Muscle necrosis and regeneration were determined in stained cryosections of soleus and elongator digitorum longus muscles. Radical scavenging by green tea extract was determined in differentiated cultured C2C12 cells treated with tert-butylhydroperoxide, with the use of 2',7'-dichlorofluorescin diacetate as a radical detector. RESULTS: This feeding regimen significantly and dose-dependently reduced necrosis in the fast-twitch muscle elongator digitorum longus but at the doses tested had no effect on the slow-twitch soleus muscle. Green tea extract concentration-dependently decreased oxidative stress induced by tert-butylhydroperoxide treatment of cultured mouse C2C12 myotubes. The lower effective dose tested in mdx mice corresponds to approximately equal to 1.4 L (7 cups) green tea/d in humans. CONCLUSION: Green tea extract may improve muscle health by reducing or delaying necrosis in mdx mice by an antioxidant mechanism.
Assuntos
Antioxidantes/uso terapêutico , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/prevenção & controle , Extratos Vegetais/uso terapêutico , Espécies Reativas de Oxigênio , Chá , Animais , Células Cultivadas , Dieta , Camundongos , Músculo Esquelético/efeitos dos fármacos , NecroseRESUMO
Duchenne muscular dystrophy arises due to the lack of the cytoskeletal protein dystrophin. In Duchenne muscular dystrophy muscle, the lack of dystrophin is accompanied by alterations in the dystrophin-glycoprotein complex. We and others have found that the absence of dystrophin in cells of the Duchenne muscular dystrophy animal model, the mdx mouse, leads to elevated Ca(2+) influx and cytosolic Ca(2+) concentrations when exposed to stress. We have also shown that alpha-methylprednisolone, the only drug used successfully in the therapy of Duchenne muscular dystrophy, and creatine lowered cytosolic Ca(2+) levels in mdx myotubes. It is likely that chronic elevation of [Ca(2+)] in the cytosol in response to stress is an initiating event for apoptosis and/or necrosis in Duchenne muscular dystrophy or mdx muscle and that alterations in mitochondrial function and metabolism are involved. Other cellular signalling pathways (e.g. nitric oxide) might also be affected.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Distrofina/deficiência , Metilprednisolona/farmacologia , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/metabolismo , Animais , Apoptose , Creatina/farmacologia , Citoplasma/metabolismo , Glucocorticoides/farmacologia , Camundongos , Camundongos Endogâmicos mdx , Mitocôndrias/metabolismo , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/metabolismo , Necrose , Transdução de Sinais/efeitos dos fármacosRESUMO
1. Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). 2. The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. 3. We show that CsA (1 -- 10 microM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. 4. Using cytochrome c as substrate, we show that CsA (10 microM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5. CsA (10 microM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6. CsA (10 microM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.
Assuntos
Ciclosporina/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Imunossupressores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Aorta/citologia , Células Cultivadas , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Inibidores Enzimáticos/farmacologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Radicais Livres/metabolismo , Humanos , Técnicas In Vitro , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos WKY , Ratos Sprague-DawleyRESUMO
SCOPE: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. METHODS AND RESULTS: In our study, we used glycolaldehyde-modified ß-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. CONCLUSION: Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.
Assuntos
Acetaldeído/análogos & derivados , Produtos Finais de Glicação Avançada/metabolismo , Inflamação/metabolismo , Lactoglobulinas/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Acetaldeído/química , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Detergentes/química , Endotoxinas/isolamento & purificação , Endotoxinas/farmacologia , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/isolamento & purificação , Humanos , Lactoglobulinas/química , Lactoglobulinas/isolamento & purificação , Octoxinol , Polietilenoglicóis/química , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Reprodutibilidade dos TestesRESUMO
Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.
Assuntos
Inflamação/etiologia , Lactoglobulinas/metabolismo , Lisina/análogos & derivados , Receptores Imunológicos/metabolismo , Albumina Sérica/metabolismo , Linhagem Celular , Células Epiteliais , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Glicosilação , Glioxilatos/química , Humanos , Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Lactoglobulinas/química , Lisina/química , RNA Mensageiro/análise , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Albumina Sérica/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
Duchenne muscular dystrophy is a frequent muscular disorder caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that contributes to the stabilization of muscle fiber membrane during muscle activity. Affected individuals show progressive muscle wasting that generally causes death by age 30. In this study, the dystrophic mdx(5Cv) mouse model was used to investigate the effects of green tea extract, its major component (-)-epigallocatechin gallate, and pentoxifylline on dystrophic muscle quality and function. Three-week-old mdx(5Cv) mice were fed for either 1 or 5 wk a control chow or a chow containing the test substances. Histological examination showed a delay in necrosis of the extensor digitorum longus muscle in treated mice. Mechanical properties of triceps surae muscles were recorded while the mice were under deep anesthesia. Phasic and tetanic tensions of treated mice were increased, reaching values close to those of normal mice. The phasic-to-tetanic tension ratio was corrected. Finally, muscles from treated mice exhibited 30-50% more residual force in a fatigue assay. These results demonstrate that diet supplementation of dystrophic mdx(5Cv) mice with green tea extract or (-)-epigallocatechin gallate protected muscle against the first massive wave of necrosis and stimulated muscle adaptation toward a stronger and more resistant phenotype.
Assuntos
Antioxidantes/farmacologia , Camellia sinensis/química , Catequina/análogos & derivados , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/metabolismo , Extratos Vegetais/farmacologia , Preparações de Plantas/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , Catequina/farmacologia , Catequina/uso terapêutico , Dieta , Modelos Animais de Doenças , Sequestradores de Radicais Livres/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/patologia , Pentoxifilina/farmacologia , Fitoterapia , Extratos Vegetais/uso terapêutico , Preparações de Plantas/uso terapêuticoRESUMO
Superoxide is known to affect vascular physiology in several ways and has also been recognized to contribute significantly to vascular physiopathology. Here we discuss the emerging role of superoxide as an essential signaling molecule in normal physiology.
Assuntos
Transdução de Sinais/fisiologia , Superóxidos/metabolismo , Animais , Divisão Celular/fisiologia , HumanosRESUMO
Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.