RESUMO
Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose/genética , Insulina/metabolismo , Resistência à Insulina , Interleucina-1beta/metabolismo , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de SinaisRESUMO
Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.
Assuntos
Doença de Chagas/tratamento farmacológico , Modelos Animais de Doenças , Inflamação/prevenção & controle , Fígado/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Nitroimidazóis/farmacologia , Sepse/tratamento farmacológico , Tripanossomicidas/farmacologia , Animais , Antioxidantes/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Nitroimidazóis/uso terapêutico , Estresse Oxidativo , Receptor 4 Toll-Like/metabolismo , Tripanossomicidas/uso terapêuticoRESUMO
BACKGROUND: Ligaria cuneifolia (Lc) (R. et P.) Tiegh. (Loranthaceae) (Argentine mistletoe) is usually used in local folk medicine. OBJECTIVE: We studied the effect of treatment with the Lc proanthocyanidin-enriched fraction (PLc) in rats fed with Cho-enriched diet on plasma lipids levels, the hemorheological parameters, and biliary secretion. METHOD: Adult male Wistar rats were fed ad libitum with a Cho-enriched diet (Cho (97% purity) 8 g/kg of diet and corn oil 280 g/kg of diet) during 28 days. Then, were separated in six experimental groups (n=5 each one), which were injected ip every 24 h with: 1) saline solution (control group, C) and 2) PLc, 3 mg/100 g body weight (treated group, C+PLc), during 3, 7 and 10 days. Group C presented an increase in plasma levels of Cho and Triglycerides (TG), and also, accumulation of hepatic lipid droplets. Also, cell shape and their corresponding morphological index (MI) were altered too. RESULTS: The treatment with PLc at 3, 7 and 10 days produces a diminution in the plasma Cho, LDL-Cho and serum TG levels, accompanied by a diminution of the lipid accumulation in the liver. The rates of bile acid output in bile can explain the diminution of plasma Cho, evidencing that some of the enzymes involved in the cholesterol conversion into bile acids could be up regulated by the treatment with PLc, leading to the observed increase bile flow. PLc treatment leads to a diminution of plasma levels of Cho and TG. CONCLUSION: Essentially, the treatment with PLc, despite the duration produces a modification in hemorheological parameters approaching the values of the experimental group with standard diet. Plasma levels of Cho, LDL-Cho and TG represent selected markers to evaluate the effect of enriched extract from Ligaria cuneifolia. Further work is necessary to better evaluate the mechanisms by which PLc induces modifications in the lipids metabolism.
Assuntos
Anticolesterolemiantes/farmacologia , Colesterol na Dieta/sangue , Hipercolesterolemia/prevenção & controle , Loranthaceae , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Animais , Anticolesterolemiantes/isolamento & purificação , Biomarcadores/sangue , Modelos Animais de Doenças , Hipercolesterolemia/sangue , Hipercolesterolemia/etiologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Loranthaceae/química , Masculino , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Proantocianidinas/isolamento & purificação , Ratos Wistar , Fatores de Tempo , Triglicerídeos/sangueRESUMO
Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor-mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Gorduras na Dieta/efeitos adversos , Fígado Gorduroso/metabolismo , Resistência à Insulina/fisiologia , Fígado/enzimologia , Obesidade/metabolismo , Animais , Ciclo-Oxigenase 2/genética , Gorduras na Dieta/administração & dosagem , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Insulina/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
UNLABELLED: It was demonstrated that Ligaria cuneifolia (Lc) crude extract increased blood viscosity and decreased plasma cholesterol in rats. In the present study, we analyzed the Lc proanthocyanidin enriched fraction (PLc) to determine if it is capable of altering the hemorheological parameters while diminishing the plasma cholesterol. In vivo studies in adult male Wistar rats, randomized in three groups (nâ=â6 each one) were performed: 1. CONTROL: saline intraperitoneal (i.p.); 2. PLc 0.6âmg/100âg body weight (b.w.) i.p. and 3. PLc 3âmg/100âg b.w. i.p., every 24 hours during 3 days. IN VITRO STUDIES: with blood obtained by cardiac puncture, separated in aliquots and incubated with: 1. Saline solution (Control); 2. PLc 0.1âmg/mL, and 3. PLc 1.0âmg/mL, equivalent to doses in vivo experiments. The results demonstrated that in vivo PLc 0.6 and PLc 3 reduced plasma cholesterol (Cho) and LDL-Cho. Neither blood nor plasma viscosity was altered. Decrease of plasma cholesterol could be due to an increase of cholesterol and bile salts excretion leading to an increase of bile flow. In vitro experiments showed a direct interaction of PLc, at high concentration, with the erythrocyte membrane, inducing a switch from discocyte to stomatocyte. Only, PLc without hepatic metabolism produces hemorheological changes. Thus, PLc in vivo might be a pharmacological agent capable of decreasing plasma cholesterol.
Assuntos
Colesterol/sangue , Hemorreologia/efeitos dos fármacos , Loranthaceae/química , Extratos Vegetais/química , Folhas de Planta/química , Animais , Viscosidade Sanguínea/efeitos dos fármacos , Técnicas In Vitro , Masculino , Extratos Vegetais/farmacologia , Proantocianidinas , Ratos , Ratos WistarRESUMO
Interferon-α2b (IFN-α2b) reduces proliferation and increases apoptosis in hepatocellular carcinoma cells by decreasing ß-catenin/TCF4/Smads interaction. Forkhead box O-class 3a (FoxO3a) participates in proliferation and apoptosis and interacts with ß-catenin and Smads. FoxO3a is inhibited by Akt, IκB kinase ß (IKKß), and extracellular-signal-regulated kinase (Erk), which promote FoxO3a sequestration in the cytosol, and accumulates in the nucleus upon phosphorylation by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38 MAPK). We analyzed FoxO3a subcellular localization, the participating kinases, FoxO3a/ß-catenin/Smads association, and FoxO3a target gene expression in IFN-α2b-stimulated HepG2/C3A and Huh7 cells. Total FoxO3a and Akt-phosphorylated FoxO3a levels decreased in the cytosol, whereas total FoxO3a levels increased in the nucleus upon IFN-α2b stimulus. IFN-α2b reduced Akt, IKKß, and Erk activation, and increased JNK and p38 MAPK activation. p38 MAPK inhibition blocked IFN-α2b-induced FoxO3a nuclear localization. IFN-α2b enhanced FoxO3a association with ß-catenin and Smad2/3/7. Two-step coimmunoprecipitation experiments suggest that these proteins coexist in the same complex. The expression of several FoxO3a target genes increased with IFN-α2b. FoxO3a knockdown prevented the induction of these genes, suggesting that FoxO3a acts as mediator of IFN-α2b action. Results suggest a ß-catenin/Smads switch from TCF4 to FoxO3a. Such events would contribute to the IFN-α2b-mediated effects on cellular proliferation and apoptosis. These results demonstrate new mechanisms for IFN-α action, showing the importance of its application in antitumorigenic therapies.
Assuntos
Carcinoma Hepatocelular/terapia , Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Imunoterapia/métodos , Interferon-alfa/farmacologia , Proteínas Smad/metabolismo , beta Catenina/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinoma Hepatocelular/imunologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
We previously found that mitochondrial aquaporin-8 (mtAQP8) channels facilitate mitochondrial H2O2 release in human hepatoma HepG2 cells and that their knockdown causes oxidant-induced mitochondrial dysfunction and loss of viability. Here, we studied whether apoptosis or necrosis is involved as the mode of cell death. We confirmed that siRNA-induced mtAQP8 knockdown significantly decreased HepG2 viability by MTT assay, LDH leakage, and trypan blue exclusion test. Analysis of mitochondrial proapoptotic Bax-to-antiapoptotic BclXL ratio, mitochondrial cytochrome c release and caspase-3 activation showed no alterations in mtAQP8-knockdown cells. This indicates a primary mechanism of cell death other than the intrinsic mitochondrial apoptotic pathway. Thus, nuclear staining with DAPI did not reveal any increase of apoptotic features, i.e. chromatin condensation or nuclear fragmentation. Flow cytometry studies after double cell staining with annexin V and propidium iodide confirmed lack of apoptosis and suggested necrosis as the primary mechanism of death in mtAQP8-knockdown HepG2 cells. Necrosis was further supported by the increased nuclear delocalization and extracellular release of the High Mobility Group Box 1 protein. The knockdown of mtAQP8 in another human hepatoma-derived cell line, i.e. HuH-7 cells, also induced necrotic but not apoptotic death. Our data suggest that mtAQP8 knockdown induces necrotic cell death in human neoplastic hepatic cells, a finding that might be relevant to therapeutic strategies against hepatoma cells.
Assuntos
Apoptose , Aquaporinas/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Mitocôndrias/metabolismo , Aquaporinas/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/metabolismo , NecroseRESUMO
SCOPE: Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G1 and S phases, accumulation in G2, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION: The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.
Assuntos
Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Quercetina/farmacologia , Animais , Proteína Quinase CDC2/metabolismo , Divisão Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , PPAR alfa/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: FoxO3a, a member of the FOXO family of transcription factors, is expressed in adult liver and modulates the expression of genes involved in apoptosis. FoxO3a is post-translationally regulated, negatively by PI3K/Akt and MAPK/Erk and positively by oxidative stress/JNK pathways. In previous works, we have demonstrated that interferon-α2b (IFN-α2b) induces apoptosis of hepatic preneoplastic foci through the production of reactive oxygen species (ROS). AIMS: To investigate the post-translational signal events triggered by the oxidative stress induced by IFN-α2b and the modulation of FoxO3a transcriptional activity during these events in rat preneoplastic liver. METHODS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis. A group of animals received IFN-α2b and another group received IFN-α2b and ascorbic acid (ASC), by intraperitoneal injection. Lipid peroxidation, immunohistochemistry, immunoblotting, co-immunoprecipitation and sqRT-PCR assays were performed to explore the role of ROS, JNK, Akt, Erk, FoxO3a, ß-catenin and PUMA in the IFN-α2b-mediated apoptotic mechanism. RESULTS: In vivo IFN-α2b treatment induced endogenous production of ROS which activated JNK. IFN-α2b blocked the activation of Akt and Erk, avoiding FoxO3a activity repression. Activated JNK was responsible for the nuclear translocation and transcriptional activity of FoxO3a which positively modulated the expression of PUMA, a proapoptotic player. In addition, nuclear FoxO3a competed for the nuclear ß-catenin associated to TCF, inhibiting the canonical Wnt signalling pathway. CONCLUSIONS: The data presented here propose a model in which in vivo IFN-α2b treatment induces nuclear translocation and transcriptional activity of FoxO3a, triggering the mitochondrial apoptotic pathway in hepatic preneoplastic foci.
Assuntos
Apoptose/genética , Carcinogênese/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína Forkhead Box O3 , Regulação da Expressão Gênica/genética , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Interferon alfa-2 , Interferon-alfa/farmacologia , Peroxidação de Lipídeos , Masculino , Modelos Biológicos , Processamento de Proteína Pós-Traducional/genética , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.
Assuntos
Apoptose , Ciclo-Oxigenase 2/metabolismo , Hiperglicemia/metabolismo , Fígado/metabolismo , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , Citocromos c/biossíntese , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Estreptozocina , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteína X Associada a bcl-2/biossíntese , Proteína de Morte Celular Associada a bcl/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
The liver has a remarkable ability to regenerate in response to surgical removal or chemical insult. The mechanisms regulating regenerative processes are complex, and incompletely understood. A large number genes, which are not normally expressed in the quiescent liver, are activated. Immediately after partial hepatectomy (PH) (1-6 h), nitric oxide (NO) is synthesized by liver parenchymal and nonparenchymal cells from L-arginine, via induction of the inducible form of nitric oxide synthase (iNOS). NO is a highly reactive molecule, known to be involved in diverse biological processes in nearly all aspects of life. Liver regeneration is a major area within the field of NO research. Our review describes several processes that have been suggested to be modulated by the NO released following PH, including proliferation, apoptosis and angiogenesis in the remnant tissue. Because iNOS up regulation has such profound physiologic effects, its regulation is strictly controlled. The up regulation of iNOS after PH and the subsequent production of NO induce positive effects on the regulation of early stages of the regenerative process. However, overproduction (> 100%) can have detrimental effects, including apoptosis. Thus, the iNOS induction after PH is necessary, and enough to allow for the normal regenerative process.
Assuntos
Regeneração Hepática , Fígado/metabolismo , Óxido Nítrico/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Hepatectomia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/cirurgia , Regeneração Hepática/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.
Assuntos
Aquaporinas/genética , Aquaporinas/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Potencial da Membrana Mitocondrial/genética , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Ciclosporina/farmacologia , Células Hep G2 , Humanos , Peróxido de Hidrogênio/metabolismo , L-Lactato Desidrogenase/metabolismo , Microscopia de Fluorescência , Oxidantes/metabolismo , Permeabilidade , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Sais de Tetrazólio , Tiazóis , Azul TripanoRESUMO
Wnt/ß-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated ß-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-ß1 (TGF-ß1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/ß-catenin and TGF-ß1/Smads pathways is the association of Smads with ß-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-ß1 treatments on Wnt/ß-catenin pathway, Smads proteins levels, ß-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-ß1 attenuated Wnt/ß-catenin signal by decreasing ß-catenin and Frizzled7 receptor proteins contents and the interaction of ß-catenin with TCF4. Truncated ß-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates ß-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-ß1 proved to be effective as modulators of Wnt/ß-catenin pathway in HCC cell lines holding both wild-type and truncated ß-catenin. Since the inhibition of ß-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-ß1 could be useful as potential treatments in patients with HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Interferon-alfa/farmacologia , Neoplasias Hepáticas/metabolismo , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Interferon alfa-2 , Neoplasias Hepáticas/patologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fator de Transcrição 4 , Fatores de Transcrição/fisiologia , Proteína Wnt3A/farmacologiaRESUMO
Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Regeneração Hepática/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Óxidos N-Cíclicos/farmacologia , Desferroxamina/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Modelos Animais de Doenças , Hepatectomia , Insulina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiopatologia , Prognóstico , Ratos , Ratos Wistar , Marcadores de Spin , EstreptozocinaRESUMO
We analyzed the contribution of TNF-α intracellular pathway in the development of apoptosis in the liver of streptozotocin-induced diabetic rats. In liver tissue, diabetes promoted a significant increase of TNF-α/TNF-R1, and led to the activation of caspase-8, of nuclear factor kappa B (NFκB), and JNK signaling pathways. The activation of NFκB led to an induction of iNOS and consequent increase in NO production. As a consequence of such changes a significant increase of caspase-3 activity and of apoptotic index were observed in the liver of diabetic animals. Importantly, the treatment in vivo of diabetic rats with etanercept (TNF-α blocking antibody) or aminoguanidine (selective iNOS inhibitor) significantly attenuated the induction of apoptosis by reduction of caspase-3 activity. Overall, we demonstrated that in the diabetes enhances TNF-α in the liver, which may be a fundamental key leading to apoptotic cell death, through activation of caspase-8, NFκB and JNK pathways.
Assuntos
Apoptose , Diabetes Mellitus Tipo 1/metabolismo , Fígado/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 8/metabolismo , Inibidores de Caspase , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/patologia , Espectroscopia de Ressonância de Spin Eletrônica , Etanercepte , Guanidinas/farmacologia , Imunoglobulina G/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/patologia , Masculino , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Wistar , Receptores do Fator de Necrose Tumoral , Transdução de Sinais , EstreptozocinaRESUMO
Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP. Our results show that BZL reduces leukocytes in peripheral blood accompanied by an increase in peritoneal macrophages 24h after CLP. In the liver of these septic mice, BZL decreased expression of mRNA and protein for TNF-α and NOS-2 by inhibition of NF-κB and MAPK (p-38 and ERK). The body of evidence suggests that the immunomodulatory effects of BZL could act selectively, as it is able to decrease the systemic inflammatory reaction and the hepatic response but it can increase the number of cells in the site of infection.
Assuntos
Ceco/patologia , Fígado/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Nitroimidazóis/uso terapêutico , Sepse/tratamento farmacológico , Sepse/enzimologia , Animais , Ceco/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Contagem de Leucócitos , Ligadura , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitroimidazóis/farmacologia , Punções , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sepse/fisiopatologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
UNLABELLED: We tested the in vivo and the in vitro effects of both Ligaria cuneifolia catechin- and quercetin-enriched fractions on erythrocyte shape and deformability, and on plasma cholesterol level. For in vivo studies, adult male Wistar rats were randomized in three experimental groups which received intraperitoneally, once a day, 3 days: CONTROL: saline solution (C; n = 6); catechin from L. cuneifolia, 0.60 mg/100 g body weight (CLc; n = 6), or quercetin from L. cuneifolia, 2.3 mg/100 g body weight (QLc; n = 6). For in vitro studies, blood samples obtained from male Wistar rats were divided into three fractions, which were incubated with saline solution (C), catechin (CLc; n = 5) and quercetin (QLc; n = 5), in a concentration equivalent to 0.60 mg/100 g body weight, and 2.3 mg/100 g body weight, respectively. CLc significantly reduced the rigidity index due to a diminished mean concentration volume. QLc induced erythrocyte rigidization (less deformability), thus increasing blood viscosity. Neither of the two treatments produced any changes in plasmatic or biliary excretion of cholesterol. Opposite results were observed in rigidity index with CLc and QLc. In vitro studies showed an interaction of both CLc and QLc with the erythrocyte membrane, which induced changes in the erythrocyte shape from discocyte to stomatocyte.
Assuntos
Viscosidade Sanguínea/efeitos dos fármacos , Catequina/farmacologia , Colesterol/sangue , Loranthaceae/química , Extratos Vegetais/farmacologia , Quercetina/farmacologia , Animais , Catequina/química , Forma Celular/efeitos dos fármacos , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemorreologia/efeitos dos fármacos , Masculino , Quercetina/química , Ratos , Ratos WistarRESUMO
In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.
Assuntos
Apoptose , Radical Hidroxila/metabolismo , Hiperglicemia/metabolismo , Insulina/metabolismo , Fígado/citologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Citocromos c/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Hiperglicemia/genética , Hiperglicemia/fisiopatologia , Fígado/metabolismo , Masculino , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
Wnt/beta-catenin/T cell factor (TCF) pathway is activated in several types of human cancers, promoting cell growth and proliferation. Forkhead box containing protein class O (FOXO) transcription factors compete with TCF for beta-catenin binding, particularly under cellular oxidative stress conditions. Contrary to beta-catenin/TCF, beta-catenin/FOXO promotes the transcription of genes involved in cell cycle arrest and apoptosis. We have previously demonstrated that in vivo interferon-alpha2b (IFN-alpha2b) administration induces apoptosis in preneoplastic livers, a mechanism mediated by reactive oxygen species (ROS) and transforming growth factor-beta(1) (TGF-beta(1)). This study was aimed to assess the status of the Wnt/beta-catenin/TCF pathway in a very early stage of rat hepatocarcinogenesis and to further evaluate the effects of in vivo IFN-alpha2b treatment on it. We demonstrated that the Wnt/beta-catenin/TCF pathway is activated in preneoplastic rat livers. More important, in vivo IFN-alpha2b treatment inhibits Wnt/beta-catenin/TCF pathway and promotes programed cell death possibly providing a link with FOXO pathway.
Assuntos
Interferon-alfa/farmacologia , Neoplasias Hepáticas Experimentais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Interferon alfa-2 , Masculino , Ratos , Ratos Wistar , Proteínas RecombinantesRESUMO
Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.