RESUMO
Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-ß isoforms, TGF-ß1 and TGF-ß2, and their combination. TGF-ß1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-ß1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.
Assuntos
Túnica Conjuntiva/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Movimento Celular , Células Cultivadas , Túnica Conjuntiva/citologia , Metilação de DNA , Células Epiteliais/citologia , Humanos , Immunoblotting , Imuno-Histoquímica , MicroRNAs/metabolismo , Regiões Promotoras GenéticasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythraea Rafn (CE) is used as a traditional medicinal plant in Serbia to treat different ailments due to its antidiabetic, antipyretic, antiflatulent and detoxification effects. AIM OF THE STUDY: Elucidation of the mechanisms that underlie the antioxidant and pro-survival effects of the CE extract (CEE) in beta-cells and pancreatic islets from streptozotocin (STZ)-treated diabetic rats. MATERIAL AND METHODS: Diabetes was induced in rats by multiple applications of low doses of STZ (40â¯mg/kg intraperitoneally (i.p.), for five consecutive days). CEE (100â¯mg/kg) was administered orally, in the pre-treated group for two weeks before diabetes induction, during the treatments with STZ and for four weeks after diabetes onset, and in the post-treatment group for four weeks after diabetes induction. The impact of CEE on diabetic islets was estimated by histological and immunohistochemical examination of the pancreas. Molecular mechanisms of the effects of CEE were also analyzed in insulinoma Rin-5F cells treated with STZ (12â¯mM) and CEE (0.25â¯mg/mL). Oxidative stress was evaluated by assessing the levels of DNA damage, lipid peroxidation, protein S-glutathionylation and enzymatic activities and expression of CAT, MnSOD, CuZnSOD, GPx and GR in beta-cells. The presence and activities of the redox-sensitive and islet-enriched regulatory proteins were also analyzed. RESULTS: Treatment with CEE ameliorated the insulin level and glycemic control in STZ-induced diabetic rats by improving the structural and functional properties of pancreatic islets through multiple routes of action. The disturbance of islet morphology and islet cell contents in diabetes was reduced by the CEE treatment and was associated with a protective effect of CEE on the levels of insulin, GLUT-2 and p-Akt in diabetic islets. The antioxidant effect of CEE on STZ-treated beta-cells was displayed as reduced DNA damage, lipid peroxidation, protein S-glutathionylation and alleviation of STZ-induced disruption in MnSOD, CuZnSOD and CAT enzyme activities. The oxidative stress-induced disturbance of the transcriptional regulation of CAT, MnSOD, CuZnSOD, GPx and GR enzymes in beta-cells was improved after the CEE treatment, and was observed as readjustment of the presence and activities of redox-sensitive NFκB-p65, FOXO3A, Sp1 and Nrf-2 transcription factors. The observed CEE-mediated induction of proliferative and pro-survival pathways and insulin expression/secretion after STZ-induced oxidative stress in beta-cells could be partially attributed to a fine-tuned modulation of the activities of pro-survival Akt, ERK and p38 kinases and islet-enriched Pdx-1 and MafA regulatory factors. CONCLUSIONS: The results of this study provide evidence that CEE improves the structural and functional properties of pancreatic beta-cells by correcting the endogenous antioxidant regulatory mechanisms and by promoting proliferative and pro-survival pathways in beta-cells.
Assuntos
Centaurium , Diabetes Mellitus Experimental/tratamento farmacológico , Células Secretoras de Insulina/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Linhagem Celular Tumoral , Dano ao DNA , Diabetes Mellitus Experimental/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Masculino , Estresse Oxidativo/efeitos dos fármacos , Componentes Aéreos da Planta , Ratos WistarRESUMO
Poly [ADP-ribose] polymerase 1 (PARP-1) has an inhibitory effect on C-X-C motif chemokine 12 gene (Cxcl12) transcription. We examined whether PARP-1 affects the epigenetic control of Cxcl12 expression by changing its DNA methylation pattern. We observed increased expression of Cxcl12 in PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-) in comparison to wild-type mouse embryonic fibroblasts (NIH3T3). In the Cxcl12 gene, a CpG island is present in the promoter, the 5' untranslated region (5' UTR), the first exon and in the first intron. The methylation state of Cxcl12 in each cell line was investigated by methylation-specific PCR (MSP) and high resolution melting analysis (HRM). Both methods revealed strong demethylation in PARP1-/- compared to NIH3T3 cells in all four DNA regions. Increased expression of the Ten-eleven translocation (Tet) genes in PARP1-/- cells indicated that TETs could be important factors in Cxcl12 demethylation in the absence of PARP-1, accounting for its increased expression. Our results showed that PARP-1 was a potential upstream player in (de)methylation events that modulated Cxcl12 expression.
Assuntos
Quimiocina CXCL12/genética , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Epigênese Genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Proto-Oncogênicas/genética , Regiões 5' não Traduzidas , Animais , Quimiocina CXCL12/metabolismo , Ilhas de CpG , DNA/genética , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Éxons , Íntrons , Camundongos , Camundongos Knockout , Células NIH 3T3 , Poli(ADP-Ribose) Polimerase-1/deficiência , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.
Assuntos
Diabetes Mellitus Experimental/complicações , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Hepatopatias/complicações , Receptor 4 Toll-Like/metabolismo , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Haptoglobinas/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Interleucina-6/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Quinases/metabolismo , Piruvatos/farmacologia , Ratos Wistar , Estreptozocina , Fator de Necrose Tumoral alfa/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythraea Rafn (CE) is a traditional medicinal herb in Serbia with antidiabetic, digestive, antipyretic and antiflatulent effects AIM OF THE STUDY: To investigate the potential protective effects of the methanol extract of the aerial parts of CE against glyco-oxidative stress in red blood cells (RBCs) in rats with experimentally induced diabetes. MATERIAL AND METHODS: Diabetes was induced in Wistar rats by intraperitoneal (i.p.) injection of multiple low-dose streptozotocin (STZ) (40mg/kg, for five consecutive days), with the 1st day after the last STZ injection taken as the day of diabetes onset. The methanol extract of CE (100mg/kg) was administered orally and daily, two weeks before the first STZ injection, during the 5-day treatment with STZ, and for four weeks after the STZ injections (pre-treated group) or for four weeks after diabetes onset (post-treated group). The effect of CE extract administration on the redox status of RBCs was evaluated by assessing lipid peroxidation, the ratio of reduced/oxidized glutathione (GSH/GSSG), the level of S-glutathionylated proteins (GSSP) and the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in RBCs four weeks after diabetes onset. The major biochemical parameters of diabetes, protein glycation/glycosylation of erythrocytes and parameters which correlate with their aggregation and deformability were also evaluated. RESULTS: Daily application of CE extract to STZ-induced diabetic rats provided important antidiabetic effects, observed in both pre-treated and post-treated groups of diabetic rats as elevated serum insulin concentration, reduction of blood glucose and glycated hemoglobin concentrations and an improved lipid profile. Antioxidant effects of CE extract were detected in RBCs of diabetic rats and observed as decreased lipid peroxidation and ameliorated oxidative damage as a result of increased SOD, CAT and GR activities, an improved GSH/GSSG ratio and reduced GSSP levels. Moreover, the CE extract protected RBC proteins from hyperglycemia-induced damage by reducing non-enzymatic glycation and enzymatic glycosylation processes. CE extract was more effective when applied before diabetes induction (pre-treated group). CONCLUSIONS: The results of this study show that the Centaurium erythraea methanol extract protects RBCs in diabetic animals from oxidative damage. They provide additional support for the application of this traditionally used plant in diabetes management.
Assuntos
Centaurium/química , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Eritrócitos/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Animais , Antioxidantes/metabolismo , Glicemia/metabolismo , Flavonoides/análise , Sequestradores de Radicais Livres/farmacologia , Glutationa/metabolismo , Hipoglicemiantes/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Metanol , Fenóis/análise , Extratos Vegetais/farmacologia , Ratos , Ratos WistarRESUMO
Due to intrinsically low levels of antioxidant enzyme expression and activity, insulin producing pancreatic ß-cells are particularly susceptible to free radical attack. In diabetes mellitus, which is accompanied by high levels of oxidative stress, this feature of ß-cells significantly contributes to their damage and dysfunction. In light of the documented pro-survival effect of chemokine C-X-C Ligand 12 (CXCL12) on pancreatic ß-cells, we examined its potential role in antioxidant protection. We report that CXCL12 overexpression enhanced the resistance of rat insulinoma (Rin-5F) and primary pancreatic islet cells to hydrogen peroxide (H2O2). CXCL12 lowered the levels of DNA damage and lipid peroxidation and preserved insulin expression. This effect was mediated through an increase in catalase (CAT) activity. By activating downstream p38, Akt and ERK kinases, CXCL12 facilitated Nrf2 nuclear translocation and enhanced its binding to the CAT gene promoter, inducing constitutive CAT expression and activity that was essential for protecting ß-cells from H2O2.
Assuntos
Catalase/metabolismo , Quimiocina CXCL12/farmacologia , Citoproteção/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos Wistar , Fatores de Transcrição/metabolismoRESUMO
Diabetes is characterized by a deficit in the number of functional pancreatic ß-cells. Understanding the mechanisms that stimulate neogenesis of ß-cells should contribute to improved maintenance of ß-cell mass. Chemokine CXCL12 has recently become established as a novel ß-cell growth factor, however the mechanisms controlling its expression require clarification. We investigated the proteins involved in the transcriptional regulation of the rat ß-cell CXCL12 gene (Cxcl12). Using the electrophoretic mobility shift assay and chromatin immunoprecipitation, we established the in vitro and in vivo binding of C/EBPß, C/EBPα, STAT3, p53, FOXO3a, and HMG I/Y to the Cxcl12 promoter. Co-immunoprecipitation experiments revealed protein-protein interactions between YY1 and PARP-1, FOXO3a and PARP-1, Sp1 and PARP-1, p53 and PARP-1, C/EBPß and PARP-1, YY1 and p53, YY1 and FOXO3a, p53 and FOXO3a, Sp1 and FOXO3a, C/EBPß and FOXO3a, C/EBPα and FOXO3a, Sp1 and STAT3. Our data lay the foundation for research into the interplay of signaling pathways that determine the ß-cell Cxcl12 expression profile.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Quimiocina CXCL12/genética , Células Secretoras de Insulina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição STAT3/genética , Ativação TranscricionalRESUMO
The diabetes prevention paradigm envisages the application of strategies that support the maintenance of appropriate ß-cell numbers. Herein we show that overexpression of CXC chemokine ligand12 (CXCL12) considerably improves the viability of isolated rat Langerhans islet cells and Rin-5F pancreatic ß-cells after hydrogen peroxide treatment. In rat islets and wt cells hydrogen peroxide treatment induced necrotic cell death that was mediated by the rapid and extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1). In contrast, CXCL12-overexpressing cells were protected from necrotic cell death as a result of significantly reduced PARP-1 activity. CXCL12 downstream signalling through Akt kinase was responsible for the reduction of PARP-1 activity which switched cell death from necrosis to apoptosis, providing increased protection to cells from oxidative stress. Our results offer a novel aspect of the CXCL12-mediated improvement of ß-cell viability which is based on its antinecrotic action through modulation of PARP-1 activity.
Assuntos
Quimiocina CXCL12/metabolismo , Células Secretoras de Insulina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Quimiocina CXCL12/genética , Peróxido de Hidrogênio/efeitos adversos , Peróxido de Hidrogênio/farmacologia , Células Secretoras de Insulina/patologia , Masculino , Necrose , Oxidantes/efeitos adversos , Oxidantes/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional interplay of these proteins could finely balance Cxcl12 transcription.
Assuntos
Quimiocina CXCL12/genética , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transcrição Gênica , Fator de Transcrição YY1/metabolismo , Animais , Metabolismo Basal/efeitos dos fármacos , Metabolismo Basal/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerase-1 , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos , Estreptozocina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The present study aimed to investigate the effects of the treatment with a-lipoic acid (LA), a naturally occurring compound possessing antioxidant activity, on liver oxidant stress in a rat model of streptozotocin (STZ)-induced diabetes by examining potential mechanistic points that influence changes in the expression of antioxidant enzymes such as catalase (CAT) and CuZn/Mn superoxide dismutase(s) (SOD). LA was administered for 4 weeks by daily intraperitoneal injections (10 mg/kg) to STZ-induced diabetic rats, starting from the last STZ treatment. LA administration practically normalised the activities of the indicators of hepatocellular injury, alanine and aspartate aminotransferases, and lowered oxidative stress, as observed by the thiobarbituric acid-reactive substance assay, restored the reduced glutathione:glutathione disulphide ratio and increased the protein sulfhydryl group content. The lower level of DNA damage detected by the comet assay revealed that LA reduced cytotoxic signalling, exerting a hepatoprotective effect. The LA-treated diabetic rats displayed restored specific enzymatic activities of CAT, CuZnSOD and MnSOD. Quantitative real-time PCR analysis showed that LA restored CAT gene expression to its physiological level and increased CuZnSOD gene expression, but the gene expression of MnSOD remained at the diabetic level. Although the amounts of CAT and CuZnSOD protein expression returned to the control levels, the protein expression of MnSOD was elevated. These results suggested that LA administration affected CAT and CuZnSOD expression mainly at the transcriptional level, and MnSOD expression at the post-transcriptional level. The observed LA-promoted decrease in the O-GlcNAcylation of extracellular signal-regulated kinase, protein 38 kinase, NF-kB, CCAAT/enhancer-binding protein and the antioxidative enzymes themselves in diabetic rats suggests that the regulatory mechanisms that supported the changes in antioxidative enzyme expression were also influenced by post-translational mechanisms.
Assuntos
Antioxidantes/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Fígado/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ácido Tióctico/uso terapêutico , Aminoacilação , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Fator de Ligação a CCAAT , Catalase/metabolismo , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Fígado/metabolismo , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Oxirredução , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Ácido Tióctico/farmacologia , Transaminases/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
PURPOSE: The combined hyperglycemia lowering and antioxidant actions of α-lipoic acid (LA) contribute to its usefulness in preventing renal injury and other diabetic complications. The precise mechanisms by which LA alters diabetic oxidative renal injury are not known. We hypothesized that LA through its hypoglycemic effect lowers O-GlcNAcylation which influences the expression and activities of antioxidant enzymes which assume important roles in preventing diabetes-induced oxidative renal injury. METHODS: An experimental model of diabetes was induced in rats by the administration of 40 mg/kg streptozotocin (STZ) intraperitoneally (i.p.) for five consecutive days. LA was applied at a dose of 10 mg/kg i.p. for 4 weeks, starting from the last day of STZ administration. RESULTS: An improved glycemic status of LA-treated diabetic rats was accompanied by a significant suppression of oxidative stress and a reduction of oxidative damage of lipids, proteins and DNA. LA treatment normalized CuZn-superoxide dismutase (SOD) and catalase activities in renal tissue of diabetic rats. These changes were allied with upregulated gene expression and lower levels of O-GlcNA glycosylation. The accompanying increase in MnSOD activity was only linked with upregulated gene expression. The observed antioxidant enzyme gene regulation was accompanied by nuclear translocation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), enhanced expression of heat shock proteins (HSPs) and by reduction in O-GlcNAcylation of HSP90, HSP70, and extracellular regulated kinase and p38. CONCLUSION: α-Lipoic acid administration activates a coordinated cytoprotective response against diabetes-induced oxidative injury in kidney tissue through an O-GlcNAc-dependent mechanism.
Assuntos
Acetilglucosamina/metabolismo , Antioxidantes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Rim/efeitos dos fármacos , Ácido Tióctico/farmacologia , Animais , Glicemia/metabolismo , Catalase/metabolismo , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Glutationa/metabolismo , Glicosilação , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Hiperglicemia/tratamento farmacológico , Rim/enzimologia , Nefropatias/prevenção & controle , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais , Estreptozocina , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP ß. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/ß, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPß, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPß acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.
Assuntos
Reação de Fase Aguda/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Restrição Calórica , Haptoglobinas/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição RelA/metabolismo , Reação de Fase Aguda/induzido quimicamente , Animais , Western Blotting , Cromatografia de Afinidade , Imunoprecipitação , Masculino , Ratos , Receptor Cross-Talk/imunologia , Estatísticas não Paramétricas , Terebintina/administração & dosagem , Terebintina/toxicidadeRESUMO
To examine the protective potential of the Cotinus coggygria Scop. methanol extract, Wistar rats were treated with the hepatotoxic compound pyrogallol, which possesses a potent ability to generate free radicals and induce oxidative stress. The ability of the extract to counteract the oxidative stress was examined in rats that were injected with the extract intraperitoneally (500 mg·(kg body weight)(-1)) either 2 or 12 h before the pyrogallol treatment. The extract possesses a reducing activity in vitro and an ability to chelate the ferrous ion both in vivo and in vitro. Application of the extract prior to pyrogallol treatment led to a decrease in the levels of thiobarbituric acid-reactive substances, aspartate aminotransferase, and alanine aminotransferase, increased activities of antioxidant enzymes and attenuation of DNA damage, as well as increased Akt activity and inhibition of NF-κB protein expression. Treatment with the extract 12 h prior to pyrogallol administration was more effective in suppressing pyrogallol-induced oxidative damage than the 2 h pretreatment. Extract administration promoted an increase in acute phase reactants haptoglobin and α(2)-macroglobulin that was short of a full-fledged acute phase response. Administration of the extract considerably improved the markers of oxidative stress, thus revealing a potential hepatoprotective activity. Our results suggest that Akt activation, NF-κB inhibition, and induction of the acute phase play important roles in mediating hepatic protection by the extract. The greater effectiveness of the 12 h pretreatment with extract points to the important role that preconditioning assumes in improving resistance to subsequent exposure to oxidative stress.
Assuntos
Anacardiaceae , Antioxidantes/farmacologia , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pirogalol/toxicidade , Animais , Catalase/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/enzimologia , Masculino , NF-kappa B/metabolismo , Oxirredução/efeitos dos fármacos , Caules de Planta , Substâncias Protetoras/farmacologia , Pirogalol/farmacologia , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
Previously, we showed that administration of the acute-phase protein α(2)-macroglobulin (α(2)M) to rats before total-body irradiation with 6.7 Gy (LD(50/30)) of X-rays provides the same level of radioprotection as amifostine. Here, we compare the cytoprotective effects of α(2)M and amifostine on rat liver. The potential of the liver to replenish cells destroyed by ionizing radiation was assessed by immunoblot analysis with antibody to proliferating cell nuclear antigen (PCNA). After irradiation, in unprotected rats PCNA decreased 6-fold from the basal level. In rats pretreated with either α(2)M or amifostine, PCNA was increased throughout a 4 week follow-up period, indicating that hepatocyte proliferation was unaffected. Since PCNA is an important component of the repair machinery, its increased expression was accompanied by significantly lower DNA damage in α(2)M- and amifostine-treated rats. At 2 weeks after irradiation, the Comet assay revealed a 15-fold increase in DNA damage in unprotected rats, while in α(2)M- and amifostine-treated rats we observed 3- and 4-fold rise in damage, respectively. The improved protection to DNA damage was supported by elevated activity of the antioxidant systems. Compared to untreated rats, pretreatments with α(2)M and amifostine led to similar increases in levels of the inflammatory cytokine IL-6 and the redox-sensitive transcription factor NFκB, promoting upregulation of MnSOD, the major component of the cell's antioxidant axis, and subsequent increases in Mn/CuZnSOD and catalase enzymatic activities. The results show that α(2)M induces protein factors whose interplay underlies radioprotection and support the idea that α(2)M is the central effector of natural radioprotection in the rat.
Assuntos
Citoproteção/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/efeitos da radiação , Irradiação Corporal Total , alfa-Macroglobulinas/administração & dosagem , alfa-Macroglobulinas/farmacologia , Amifostina/farmacologia , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos da radiação , Interleucina-6/sangue , Fígado/citologia , Fígado/metabolismo , Doses de Radiação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms of cryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die by necrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenic temperature revealed the activation of endonuclease p23 and its de novo association with the nuclear matrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumed order underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activation. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity, were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed a presumed p23 activation factor. We observed that the activation of p23 was the result of a proteolytic event in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear proteins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed. Although both processes induced p23 activation, the incubation experiments generated proteolytic hallmarks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavage products and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possess the potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cytosol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of cell death.
Assuntos
Apoptose/fisiologia , Endonucleases/metabolismo , Ativação Enzimática/fisiologia , Congelamento/efeitos adversos , Fígado/enzimologia , Animais , Western Blotting , Núcleo Celular/metabolismo , Ensaio Cometa , Criocirurgia , Eletroforese em Gel de Poliacrilamida , Laminina/metabolismo , Fígado/patologia , Masculino , Necrose , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos WistarRESUMO
The organophosphorus compounds soman and paraoxon induce the acute-phase (AP) response. All phases of the AP response, from macrophage activation and stimulation of glucocorticoid secretion to AP protein expression appear to be under the control of similar molecular mechanisms to those during the turpentine-induced AP response. The AP protein content in the circulation 24 h after either soman, paraoxon or turpentine administration was injury-specific. Both soman and paraoxon poisoning were characterized by significantly increased synthesis of alpha(1)-acid glycoprotein (AGP) that displayed an immunomodulatory effect in vitro. This result suggests that after organophosphate poisoning AGP participates in vivo in a negative feedback mechanism that prevents over-activity of the immune system.
Assuntos
Fatores Imunológicos/imunologia , Orosomucoide/imunologia , Paraoxon/toxicidade , Soman/toxicidade , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/imunologia , Animais , Células Cultivadas , Corticosterona/sangue , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Orosomucoide/genética , Orosomucoide/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Terebintina/toxicidadeRESUMO
The synthesis of alpha-2-macroglobulin (alpha(2)M) is low in adult rat liver and elevated in fetal liver. During the acute-phase (AP) response it becomes significantly increased in both adult and fetal liver. In this work, the cross talk of STAT3 and NF-kappaB transcription factors during alpha(2)M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF-kappaB with their active equivalents, the 86 and 91 kDa isoforms and p65-subunit, respectively, in the nuclear extract and nuclear matrix. Different partitioning dynamics of the transcription factors were observed. At the level of protein-DNA interactions, studied by alpha(2)M promoter affinity chromatography, it was established that different ratios of promoter-binding STAT3 isoforms participated in elevated hepatic transcription in the basal state fetus and the AP-adult, but only the 91 kDa isoform in the AP-fetus. Unchanged levels of DNA-bound p65 in the control and AP-fetus suggest that it participated in constitutive transcription. The promoter-binding of p65 observed in the AP-adult suggests that it was involved in transcriptional stimulation of alpha(2)M expression. The selective enrichment of the AP-adult nuclear matrix with promoter-binding STAT3 disclosed the importance of this association in the induction of transcription. Protein-protein interactions were examined by co-immunoprecipitation. Interactions between the 86 kDa STAT3 isoform and p65 that were observed in the control and AP-fetus and of both the 86 and 91 kDa STAT3 isoforms with p65 in the AP-adult, suggest that protein-protein interactions were functionally connected to increased transcription. We concluded that alpha(2)M gene expression is driven by developmental- and AP-related mechanisms that rely on STAT3/NF-kappaB interplay.
Assuntos
Reação de Fase Aguda , Regulação da Expressão Gênica , Fígado , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição RelA/metabolismo , alfa-Macroglobulinas/metabolismo , Animais , Sequência de Bases , Núcleo Celular/química , Núcleo Celular/metabolismo , Feminino , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , alfa-Macroglobulinas/genéticaRESUMO
A functional interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co-localizes with lamin B under physiological conditions, where PARP-1 only has basal activity. After DNA damage and the associated activation of PARP-1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B-PARP-1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP-functions in damage and its fate in apoptosis.
Assuntos
Núcleo Celular/ultraestrutura , Lamina Tipo B/metabolismo , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Raios gama , Imuno-Histoquímica/métodos , Camundongos , Poli(ADP-Ribose) Polimerase-1RESUMO
The ability of dimethyl sulfoxide (DMSO) to induce the acute-phase (AP) response was examined. Injection of DMSO to laboratory rats caused a rapid doubling of the plasma corticosterone concentration 2 h after treatment. The elevated corticosterone concentration promoted the synthesis of mRNAs for several AP reactants. At 24 h after DMSO administration the relative serum concentration of cysteine-proteinase inhibitor (CPI) increased about 710%, alpha1-acid glycoprotein (AGP) 630%, alpha1-macroglobulin (MG) 510%, gamma fibrinogen (Fb) 420%, haptoglobin (Hp) 280%, whereas the relative concentration of albumin, a "negative" AP reactant, decreased to 93%. The extent and kinetics of the corticosterone increase and the general increase of AP reactant mRNAs and protein serum concentrations after DMSO administration corresponded to the overall changes observed during the turpentine-induced AP response. On the basis of these findings it was concluded that DMSO was capable of promoting the AP response in rats.