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1.
Toxicol Appl Pharmacol ; 307: 138-149, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27511913

RESUMO

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level. Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3ß through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3ß inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3ß signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant-negative MEK1 or wild-type GSK-3ß. Finally, our data demonstrate that nectandrin B has the ability to protect hepatocytes against oxidative injury through the activation of Nrf2/ARE pathway mediated by ERK phosphorylation and AMPK-dependent inactivation of GSK-3ß.


Assuntos
Hepatócitos/efeitos dos fármacos , Lignanas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glutationa/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Myristica , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , terc-Butil Hidroperóxido
2.
Pharmazie ; 70(11): 733-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26790190

RESUMO

Nonalcoholic fatty liver disease is recognized as the most commonly occurring chronic liver disease. Liver X receptor α (LXRα) and sterol regulatory element-binding protein (SREBP)-1c play a central role in de novo fatty acid synthesis. This study investigated pharmacological effects of nectandrin B, a lignan isolated from nutmeg extract, on hepatic lipogenesis stimulated by LXRα-SREBP-1c-mediated pathway and the possible molecular basis. The reporter gene assay revealed that nectandrin B completely represses LXRα activity enhanced by a synthetic LXRα ligand (T0901317) in HepG2 cells. The inhibitory effect was further supported by the suppression of mRNA expression of LXRα target genes, SREBP-1c and LXRα itself. Nectandrin B also inhibited the increase in SREBP-1c expression promoted by insulin plus high glucose, major contributors to hepatic lipid accumulation. LXRα-SREBP-1c-mediated induction of acetyl-CoA carboxylase 1 and fatty acid synthase, major genes for de novo lipogenesis, was suppressed by nectandrin B. Moreover, Oil Red O staining showed that nectandrin B notably attenuates LXRα-induced lipid accumulation. AMP-activated protein kinase (AMPK) inhibits the activities of LXRα and SREBP-1c. Nectandrin B strongly activated AMPK signaling in HepG2 cells. Taken together, the suppressive effects of nectandrin B on lipogenic gene expression and lipid accumulation in hepatocytes may be due to its inhibitory effect on the LXRα-SREBP-1c pathway presumably via AMPK activation. These results suggest the potential of nectandrin B as a therapeutic candidate for fatty liver disease.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Lignanas/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/antagonistas & inibidores , Fígado/metabolismo , Myristica/química , Acetil-CoA Carboxilase/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ácido Graxo Sintases/metabolismo , Fígado Gorduroso/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Receptores X do Fígado/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
3.
Data Brief ; 34: 106713, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33490333

RESUMO

The data presented in this article are related to a research paper on the modification of deformed nanostructure and mechanical performance of metastable high entropy alloys (HEAs) [1]. Fe50Mn25Cr15Co10 alloys with and without nitrogen were synthesized in a vacuum induction furnace using pure metals of 99.99% purity and FeCrN2 as nitrogen source. The nitrogen content was determined by Leco O/N-836 determinator for nitrogen-doped alloys. Transmission electron microscopy (TEM) were carried at 200 kV equipped with energy dispersive spectroscopy (EDS). Tensile testing was performed at room temperature. The strain rate jump tests were conducted by changing the strain rate between 10-3 and 10-2 s-1 to measure the strain rate sensitivity. The nanostructural evolutions by deformation including extended stacking faults (ESFs), ε-martensite and twins were examined using EBSD and TEM for the annealed samples and those strained to different strain levels. The role of partial dislocations on the formation of various PDIDs were analysed and the energies stored as deformed nanostructure (ESDN) after the PDID band formation were used to predict the evolution of various nanostructure with strain. The data and approach would provide a useful insight into the nanostructural evolution in metastable high entropy alloys.

4.
J Sex Med ; 7(10): 3385-95, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20233292

RESUMO

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) has been identified as an important fibrogenic cytokine associated with Peyronie's disease (PD). AIM: The aim of this study was to study the differential expression of the TGF-ß1 and Smad transcription factors in plaque tissue from PD patients and to determine the antifibrotic effect of SKI2162 (SK Chemicals, Seoul, South Korea), a novel small-molecule inhibitor of activin receptor-like kinase 5 (ALK5), a type I receptor of TGF-ß, in primary fibroblasts derived from human PD plaque. METHODS: Plaque tissue was isolated from five PD patients, and tunica albuginea tissue was obtained from four control patients. Plaque tissues from a patient with PD were used for primary fibroblast culture. Fibroblasts were pretreated with SKI2162 (10 µM) and then stimulated with TGF-ß1 (10ng/mL). MAIN OUTCOME MEASURES: The plaque or tunica albuginea tissue was stained with Masson's trichrome or antibody to TGF-ß1, phospho-Smad2 (P-Smad2), and P-Smad3. Protein was extracted from treated fibroblasts for Western blotting, and the membranes were probed with antibody to P-Smad2/Smad2, P-Smad3/Smad3, plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV. We also determined the inhibitory effect of SKI2162 on TGF-ß1-induced nuclear translocation of Smad2/3 in fibroblasts. RESULTS: The plaque tissue from PD patients showed higher TGF-ß1, P-Smad2, and P-Smad3 immunoreactivity than did the tunica albuginea tissue from control patients. SKI2162 not only blocked TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, but also inhibited the production of extracellular matrix markers in fibroblasts derived from human PD plaque. CONCLUSION: In light of the pivotal role of TGF-ß and Smads in the pathogenesis of PD, pharmacologic inhibition of ALK5 may represent a novel targeted approach to treating PD.


Assuntos
Induração Peniana/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adulto , Idoso , Western Blotting , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Induração Peniana/metabolismo , Pênis/efeitos dos fármacos , Pênis/metabolismo , Fosforilação , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
5.
J Sex Med ; 7(11): 3635-46, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20584113

RESUMO

INTRODUCTION: Patients with erectile dysfunction (ED) associated with type II diabetes often have impaired endothelial function and tend to respond poorly to oral phosphodiesterase type 5 inhibitors. Therefore, neovascularization is a promising strategy for curing diabetic ED. AIM: To determine the effectiveness of a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous angiogenesis and erectile function in a mouse model of type II diabetic ED. Methods. Sixteen-week-old male db/db mice (in which obesity and type II diabetes are caused by a mutation in the leptin receptor) and control C57BL/6J mice were used and divided into four groups (N=14 per group): age-matched controls; db/db mice receiving two successive intracavernous injections of phosphate-buffered saline (PBS) (days -3 and 0; 20 µL); db/db mice receiving a single intracavernous injection of COMP-Ang1 protein (day 0; 5.8 µg/20 µL); and db/db mice receiving two successive intracavernous injections of COMP-Ang1 protein (days -3 and 0; 5.8 µg/20 µL). MAIN OUTCOME MEASURES: Two weeks later, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with antibodies to platelet/endothelial cell adhesion molecule-1 (PECAM-1) (endothelial cell marker), phosphohistone H3 (PH3, a nuclear protein indicative of cell proliferation), phospho-endothelial nitric oxide synthase (eNOS), and eNOS. Penis specimens from a separate group of animals were used for cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) quantification. RESULTS: Local delivery of COMP-Ang1 protein significantly increased eNOS phosphorylation and cGMP and cAMP expression compared with that in the group treated with PBS. Repeated intracavernous injections of COMP-Ang1 protein completely restored erectile function and cavernous endothelial content through enhanced cavernous neoangiogenesis as evaluated by PECAM-1 and PH3 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, whereas a single injection of COMP-Ang1 protein elicited partial improvement. CONCLUSION: Cavernous neovascularization using recombinant Ang1 protein is a novel therapeutic strategy for the treatment of ED resulting from type II diabetes.


Assuntos
Angiopoietina-1/uso terapêutico , Diabetes Mellitus Tipo 2/patologia , Endotélio Vascular/efeitos dos fármacos , Disfunção Erétil/tratamento farmacológico , Ereção Peniana/efeitos dos fármacos , Pênis/efeitos dos fármacos , Angiopoietina-1/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , AMP Cíclico , GMP Cíclico , Disfunção Erétil/etiologia , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/uso terapêutico , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico
6.
J Sex Med ; 6(12): 3289-304, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19732306

RESUMO

INTRODUCTION: With the advent of genetically modified mice, it seems particularly advantageous to develop a mouse model of diabetic erectile dysfunction. AIM: To establish a mouse model of type I diabetes by implementation of either multiple low-dose streptozotocin (STZ) protocol or single high-dose STZ protocol and to evaluate morphologic alterations in the cavernous tissue and subsequent derangements in penile hemodynamics in vivo. METHODS: Eight-week-old C57BL/6J mice were divided into three groups: a control group, a group administered the multiple low-dose STZ protocol (50 mg/kg x 5 days), and a group administered the single high-dose STZ protocol (200 mg/kg). MAIN OUTCOME MEASURES: After 8 weeks, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with hydroethidine (in situ analysis of superoxide anion), TUNEL, or antibodies to nitrotyrosine (marker of peroxynitrite formation), PECAM-1, smooth muscle alpha-actin, and phospho-eNOS. Penis specimens from a separate group of animals were used for phospho-eNOS and eNOS western blot or cGMP determination. RESULTS: Erectile function was significantly less in diabetic groups than in control group. The generation of superoxide anion and nitrotyrosine and the number of apoptotic cells in both cavernous endothelial and smooth muscle cells were significantly higher in diabetic groups than in control group. Cavernous tissue phospho-eNOS and cGMP expression and the number of endothelial and smooth muscle cells were lower in diabetic groups than in control group. Both diabetic models resulted in similar structural and functional derangements in the corpus cavernosum; however, the mortality rate was higher in mice receiving single high-dose of STZ than in those receiving multiple low-doses. CONCLUSION: The mouse model of type I diabetes is useful and technically feasible for the study of the pathophysiologic mechanisms involved in diabetic erectile dysfunction.


Assuntos
Protocolos Clínicos , Diabetes Mellitus Experimental/complicações , Disfunção Erétil/etiologia , Disfunção Erétil/fisiopatologia , Pênis/patologia , Pênis/fisiopatologia , Animais , Diabetes Mellitus Tipo 1 , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Disfunção Erétil/diagnóstico , Estudos de Viabilidade , Hemodinâmica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/irrigação sanguínea , Músculo Liso/patologia , Músculo Liso/fisiopatologia , Pênis/irrigação sanguínea , Estreptozocina/administração & dosagem
7.
Transplantation ; 84(5): 634-8, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17876277

RESUMO

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation and matrix protein accumulation play important roles in the development and progression of chronic allograft vasculopathy. Mycophenolic acid (MPA) inhibits various types of mesenchymal cell proliferation and cellular reactive oxygen species (ROS) are involved in the anti-proliferative effect of MPA. In this study, we investigated the effects of MPA on oleic acid (OA)-induced VSMC proliferation and the role of ROS in this process. METHODS: Primary VSMCs from Sprague-Dawley rats were stimulated with 100 microM OA, with or without MPA (0.1- 10 microM) or 5 mM N-acetylcysteine (NAC) for one hour prior to the addition of OA. Cell proliferation was measured by methylthiazoletetrazolium (MTT) assays, proliferating cell nuclear antigen (PCNA) expression, and fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by fluorescence-activated cell scanning (FACS). RESULTS: OA (100 microM) increased cell proliferation, as measured by MTT (by 1.6-fold), PCNA expression, fibronectin secretion, and cellular ROS (by 1.6-fold). Treatment with MPA dose-dependently inhibited OA-induced VSMC proliferation, fibronectin secretion, and cellular ROS. Treatment with 5 mM NAC also inhibited OA-induced rat VSMC activation. CONCLUSIONS: These results suggest that MPA inhibits OA-induced VSMC proliferation and matrix protein synthesis partially by inhibiting cellular ROS.


Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Ácido Micofenólico/farmacologia , Ácido Oleico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Fibronectinas/metabolismo , Músculo Liso Vascular/citologia , Ratos , Ratos Sprague-Dawley
8.
Food Res Int ; 97: 340-346, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28578058

RESUMO

Cassia obtusifolia L. (Leguminosae) seeds are a well-known medicinal food in East Asia and are used to clear liver heat, sharpen vision, lubricate the intestines, and promote bowel movement. The aims of the present study were to identify the hepatoprotective components of C. obtusifolia seeds by bioactivity-guided isolation and to elucidate their mechanisms of action. Ten phenolic glycosides were isolated from the most active ethyl acetate fraction, and their chemical structures were elucidated by spectroscopic analyses. Among the isolated compounds, toralactone 9-O-gentiobioside (5) had the highest hepatoprotective efficacy against tert-butylhydroperoxide-induced cell death in HepG2 cells. Immunoblotting and real-time polymerase chain reaction analyses revealed that the hepatoprotective effects were exerted through nuclear factor erythroid-2-related factor 2 (Nrf2)-dependent antioxidative signaling. Together, these results provide insights into the effects of this medicinal plant as well as a basis for developing hepatoprotective agents as pharmaceuticals and/or nutraceuticals.


Assuntos
Cassia/química , Dissacarídeos/farmacologia , Extratos Vegetais/química , Substâncias Protetoras/farmacologia , Pironas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/efeitos dos fármacos , Sementes/química
9.
Diabetes ; 60(3): 969-80, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21270241

RESUMO

OBJECTIVE: Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. RESEARCH DESIGN AND METHODS: Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days -3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. RESULTS: Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47(phox) and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1-treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. CONCLUSIONS: These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction.


Assuntos
Angiopoietina-1/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Impotência Vasculogênica/tratamento farmacológico , Pênis/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Análise de Variância , Angiopoietina-1/metabolismo , Animais , Western Blotting , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Endotélio/metabolismo , Imuno-Histoquímica , Impotência Vasculogênica/etiologia , Impotência Vasculogênica/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo
10.
Pediatr Nephrol ; 24(4): 737-45, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19093139

RESUMO

The synthesis of extracellular matrix (ECM) in mesangial cells (MCs) plays important roles in the development and progression of renal diseases, including chronic allograft nephropathy. Mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase 2 (IMPDH2), suppresses MC proliferation and ECM synthesis. However, the exact inhibitory mechanism of MPA on MCs has not been clearly elucidated. In this study we compared the inhibitory effects of MPA and IMPDH2 reduction [by using small interfering RNA (siRNA)] on oleic acid (OA)-induced fibronectin secretion and cellular reactive oxygen species (ROS) in mouse MCs. Growth-arrested MCs were stimulated with OA in the presence or absence of MPA, IMPDH2 siRNA, N-acetylcysteine (NAC), transforming growth factor beta (TGF-beta) antibody or exogenous guanosine. Fibronectin secretion into the medium was examined by Western blot, dichlorodihydrofluorescein (DCF)-sensitive cellular ROS by fluorescence-activated cell scanning (FACS), TGF-beta levels in the media by enzyme-linked immunosorbent assay (ELISA). OA increased fibronectin secretion, TGF-beta and cellular ROS levels. A TGF-beta neutralizing antibody effectively suppressed OA-induced fibronectin secretion. NAC and MPA completely suppressed OA-induced fibronectin secretion and decreased the levels of TGF-beta and cellular ROS. However, IMPDH2 siRNA partly inhibited OA-induced MC activation. Exogenous guanosine successfully reversed the inhibitory effects of IMPDH2 siRNA on OA-induced MC activation. Pleiotropic inhibitory effect of MPA on OA-induced mouse MC activation was mediated via its antioxidant effect on cellular ROS production and partly via inhibition of IMPDH2 itself. Our results implicate ROS as an alternative therapeutic target for the prevention of hyperlipidemia-related glomerulopathy, chronic allograft nephropathy, and subsequent graft loss.


Assuntos
Inibidores Enzimáticos/farmacologia , Fibronectinas/efeitos dos fármacos , IMP Desidrogenase/antagonistas & inibidores , Células Mesangiais/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Ácido Oleico/farmacologia , Acetilcisteína/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Antagonismo de Drogas , Fibronectinas/genética , Fibronectinas/metabolismo , Guanosina/farmacologia , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Transgênicos , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/imunologia
11.
J Am Soc Nephrol ; 16(3): 667-75, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15677311

RESUMO

Epithelial-mesenchymal transition (EMT) plays an important role in renal tubulointerstitial fibrosis and TGF-beta1 is the key inducer of EMT. Phosphorylation of Smad proteins and/or mitogen-activated protein kinases (MAPK) is required for TGF-beta1-induced EMT. Because reactive oxygen species (ROS) are involved in TGF-beta1 signaling and are upstream signaling molecules to MAPK, this study examined the role of ROS in TGF-beta1-induced MAPK activation and EMT in rat proximal tubular epithelial cells. Growth-arrested and synchronized NRK-52E cells were stimulated with TGF-beta1 (0.2 to 20 ng/ml) or H(2)O(2) (1 to 500 microM) in the presence or absence of antioxidants (N-acetylcysteine or catalase), inhibitors of NADPH oxidase (diphenyleneiodonium and apocynin), mitochondrial electron transfer chain subunit I (rotenone), and MAPK (PD 98059, an MEK [MAP kinase/ERK kinase] inhibitor, or p38 MAPK inhibitor) for up to 96 h. TGF-beta1 increased dichlorofluorescein-sensitive cellular ROS, phosphorylated Smad 2, p38 MAPK, extracellular signal-regulated kinases (ERK)1/2, alpha-smooth muscle actin (alpha-SMA) expression, and fibronectin secretion and decreased E-cadherin expression. Antioxidants effectively inhibited TGF-beta1-induced cellular ROS, phosphorylation of Smad 2, p38 MAPK, and ERK, and EMT. H(2)O(2) reproduced all of the effects of TGF-beta1 with the exception of Smad 2 phosphorylation. Chemical inhibition of ERK but not p38 MAPK inhibited TGF-beta1-induced Smad 2 phosphorylation, and both MAPK inhibitors inhibited TGF-beta1- and H(2)O(2)-induced EMT. Diphenyleneiodonium, apocynin, and rotenone also significantly inhibited TGF-beta1-induced ROS. Thus, this data suggest that ROS play an important role in TGF-beta1-induced EMT primarily through activation of MAPK and subsequently through ERK-directed activation of Smad pathway in proximal tubular epithelial cells.


Assuntos
Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nefrite Intersticial/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Actinas/metabolismo , Animais , Antioxidantes/farmacologia , Caderinas/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Mesoderma/citologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Nefrite Intersticial/metabolismo , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Proteína Smad2 , Transativadores/metabolismo , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Kidney Int ; 65(4): 1170-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15086456

RESUMO

BACKGROUND: We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC. METHODS: The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H(2)O(2)) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism. RESULTS: D-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H(2)O(2) or H(2)O(2) continuously generated by glucose oxidase up-regulated fibronectin expression [corrected]. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose-and H(2)O(2)-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion. CONCLUSION: The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions.


Assuntos
Fibronectinas/metabolismo , Glucose/administração & dosagem , Peritônio/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Antioxidantes/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , NADPH Oxidases/fisiologia , Peritônio/citologia
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