RESUMO
There is no known treatment for fatty liver, a ubiquitous cause of chronic liver disease. However, because it is associated with hyperinsulinemia and insulin-resistance, insulin-sensitizing agents might be beneficial. To evaluate this possibility, insulin-resistant ob/ob mice with fatty livers were treated with metformin, an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease, reversing hepatomegaly, steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states.
Assuntos
Fígado Gorduroso/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Obesidade , Fator de Necrose Tumoral alfa/biossíntese , Trifosfato de Adenosina/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Ingestão de Energia/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Hepatomegalia/tratamento farmacológico , Homeostase/efeitos dos fármacos , Resistência à Insulina , Lipídeos/biossíntese , Camundongos , Camundongos Obesos , Transaminases/efeitos dos fármacosRESUMO
AIMS: To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. METHODS AND RESULTS: A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni(2+) affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC(50) values of rHHPC were 277.5 +/- 25.8 microg ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 microg ml(-1) against peroxyl radicals. CONCLUSIONS: Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. SIGNIFICANCE AND IMPACT OF THE STUDY: A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.
Assuntos
Ficocianina/biossíntese , Spirulina/metabolismo , Proteínas de Bactérias/química , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Radical Hidroxila/metabolismo , Liases/química , Mutagênese Insercional , Ficocianina/química , Ficocianina/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Espectrometria de Fluorescência , Spirulina/genética , Synechocystis/genética , Synechocystis/metabolismoAssuntos
Estomatite Aftosa , Estudos de Casos e Controles , Hemorragia , Humanos , Pescoço , FaringeRESUMO
It is not known whether obesity increases the risk for hepatocellular carcinoma (HCC) simply because it promotes cirrhosis, a general risk factor for HCC, or via some other mechanism that operates independently of cirrhosis. If the latter occurs, then hepatocyte hyperplasia, an early event during the neoplastic process, might begin before liver cirrhosis develops. Genetically obese, leptin-deficient ob/ob mice are models for nonalcoholic fatty liver disease (NAFLD), a type of liver disease that is strongly associated with obesity and type 2 diabetes. Similar to obese, diabetic patients, ob/ob mice have an increased incidence of HCC. However, unlike humans with NAFLD, they rarely, if ever, develop cirrhosis spontaneously. To determine whether the noncirrhotic livers of ob/ob mice with NAFLD exhibit hepatocyte hyperplasia, parameters of proliferation and apoptosis were compared in adult ob/ob mice and their healthy litter mates. Adult ob/ob mice have an increase in liver mass relative to body mass. This hepatomegaly cannot be explained solely by lipid accumulation and is accompanied by significant increases in hepatocyte proliferative activity (as evidenced by increased Erk activation, cell-cycle related gene expression, bromodeoxyuridine incorporation, and hepatic DNA content) with concomitant inhibition of hepatocyte apoptosis (as evidenced by decreased numbers of apoptotic hepatocytes, induction of several antiapoptotic mechanisms, and decreased activation of procaspase 3). Thus, liver hyperplasia is evident at the earliest stage of NAFLD in ob/ob mice, which supports the concept that obesity-related metabolic abnormalities, rather than cirrhosis, initiate the hepatic neoplastic process during obesity.
Assuntos
Fígado Gorduroso/patologia , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Obesidade/complicações , Lesões Pré-Cancerosas/patologia , Animais , Apoptose/fisiologia , Caspase 3 , Caspases/metabolismo , Divisão Celular/fisiologia , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/etiologia , Expressão Gênica , Genes cdc/fisiologia , Hepatomegalia , Hiperplasia/etiologia , Fígado/enzimologia , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/etiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/enzimologia , Obesidade/patologia , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/etiologiaRESUMO
Five patients developed retinal detachments within one month of undergoing neodymium-YAG laser posterior capsulotomy. In four patients rhegmatogenous retinal detachments developed in association with typical aphakic breaks; in the fifth patient a previously stable extramacular traction detachment extended into the fovea. In all five patients scleral buckling or vitrectomy successfully reattached the retina and visual acuity improved. None of the findings associated with the detachments could be definitely attributed to the YAG laser. These included the lack of structural or positional changes in the vitreous as well as the absence of retinal damage. We were unable to learn the specific settings used for each laser but were told that the minimum energy levels needed to produce a capsulotomy were used.
Assuntos
Extração de Catarata , Lasers/efeitos adversos , Descolamento Retiniano/etiologia , Idoso , Extração de Catarata/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neodímio/efeitos adversos , Complicações Pós-Operatórias , Descolamento Retiniano/fisiopatologia , Recurvamento da Esclera , Acuidade VisualRESUMO
AIM: To study the pathogenetic effects of salted pork (SP) (a special food in Zhuanghe City, a region of northern China that is a high-risk area for stomach cancer) on stomach cancer, and a provide scientific basis for the primary prevention of stomach cancer in this high-risk region. METHODS: This study consisted of three distinct parts. The first part involved a study of SP mutagenicity and employed both the Ames test and micronuclei assay using V79 cells. The second part included a study of SP's effect on the gastric mucosa of residents in the Zhuanghe area who had consumed SP for more than ten years. Additionally, these studies involved an analysis of the dose effect relationship between SP and pathological changes in gastric mucosa, with a total of 300 cases analyzed. The third part of this study involved an observation of the mucosal lesions from experimental dogs by both gastroscopy and mucosal biopsy. Six healthy male dogs were selected, three were fed with SP, and the others served as controls. RESULTS: This study revealed that SP extract could mutate Salmonella typhimurium TA98 and induce an increase in both the micro nuclei rate (MNR) and micro nuclei cell rate (MNCR) of V79 at a dose range of 20-80 µL/mL. There were significant dose-effect relations between SP and either MNR or MNCR. Pathological changes in the gastric mucosa of local residents who had consumed SP were significantly different from those of the control group. In people who had consumed SP for ten years, mucosal lesions were found that contained evidence of necrosis and erosion; In those who consumed SP for ten-20 years, both hyperplasia and dysplasia were seen in addition to the above lesions. In individuals who had consumed SP for 20-30 years, severe dysplasia and malignant changes were found. Furthermore, SP had damaging effect on the gastric mucosa of dogs that were fed SP. The mucosal lesions became more severe with increased feeding time. CONCLUSION: SP is a strong mutagen and long-term SP exposure may result in repeated gastric mucosal damage and repair, ultimately leading to severe dysplasia and malignancy.
RESUMO
We have isolated a conditionally transformed liver progenitor cell line with phenotypic similarities to both hepatoblasts (bipotent embryonic liver cells that give rise to hepatocytes and intrahepatic biliary epithelial cells) and liver epithelial cells (primitive hepatic cells isolated from adult livers capable of generating both hepatocytic and biliary lineages). Cell line L2039 was derived from E14 fetal mouse liver after transformation with temperature-sensitive SV-40 large T antigen. At 33 degrees C, these cells have an epithelial morphology with a high nucleocytoplasmic ratio and express both hepatocytic and biliary genes, including albumin, alpha-fetoprotein, glutamine synthetase, insulinlike growth factor II receptor, fibronectin and laminin, and cytokeratins 8 and 19, a set of markers characteristic for hepatoblasts. The presence of cytokeratin 14, vimentin, and several oval-cell antigens link cell line L2039 to nonparenchymal liver epithelial cell populations thought to contain progenitor cells. Serum-free, hormonally defined media conditions and extracellular matrix requirements were determined for growth and differentiation of this cell line. During culture on type IV collagen at 39 degrees C, L2039 cells cease dividing and demonstrate hepatocytic differentiation with the assumption of a hepatocytelike morphology and glucocorticoid-dependent regulation of liver-specific genes, including albumin, alpha-fetoprotein, phosphoenolpyruvate carboxykinase, and liver-enriched transcription factors. The number of albumin-positive cells increases during culture at 39 degrees C, indicating that L2039 cells convert from a prehepatocytic to a hepatocytic phenotype. Under conditions specific for hepatocytic differentiation, C/EBPs were expressed and differentially regulated, with C/EBPbeta and C/EBPdelta upregulated early and C/EBPalpha only slightly expressed after 7 d, indicating that C/EBPalpha may not be a crucial factor in commitment to the hepatocytic phenotype.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Animais , Divisão Celular , Linhagem Celular Transformada , Fígado/embriologia , Camundongos , Células-Tronco , TemperaturaRESUMO
Gastric biopsies in 690 subjects from the high and low risk areas of gastric cancer were examined for identification of Cp in the gastric mucosa by Warthin-Starry, Gimenez and Gram's stains. The result showed that the positive rate was 60-62% in the high risk area whereas it was only 12.6% in the low risk area in Liaoning province. 81-85% of the positive subjects had active chronic gastritis including chronic superficial and atrophic gastritis. The result indicates a close correlation between the active chronic gastritis and Cp infection. Therefore, control of the Cp infection in the gastric mucosa is very important for lowering the incidence of chronic gastritis, a well known precursor of gastric cancer.
Assuntos
Infecções por Campylobacter/epidemiologia , Mucosa Gástrica/microbiologia , Gastrite/epidemiologia , Neoplasias Gástricas/etiologia , Adolescente , Adulto , Idoso , Campylobacter/isolamento & purificação , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/patologia , China/epidemiologia , Feminino , Mucosa Gástrica/patologia , Gastrite/microbiologia , Gastrite/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
An infrared sensor was inserted at the film plane of a fundus camera. The signal was visualized on an oscilloscope. In this manner we measured infrared reflectance from the surface of the fundus. The purpose was to characterize choroidal malignant melanomas more reliably than is done with infrared color translation photography. Control lesions were choroidal nevi, metastatic tumors, and disciform macular degenerations. Correlations were made with radioactive phosphorus (32P) uptake, fluorescein angiography, and histopathologic findings. Several cases are presented, one in which this new method of infrared detection was the first diagnostic test to detect the spread of a choroidal melanoma. The simplicity of this technique and its increased accuracy justify the needed further refinements.
Assuntos
Neoplasias da Coroide/diagnóstico , Fundo de Olho , Raios Infravermelhos , Melanoma/diagnóstico , Adulto , Idoso , Neoplasias da Coroide/patologia , Feminino , Angiofluoresceinografia , Humanos , Degeneração Macular/diagnóstico , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Nevo/diagnóstico , Radioisótopos de Fósforo , Fotografação/instrumentação , Fotografação/métodos , UltrassonografiaRESUMO
Increases in monocyte/macrophage production of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), parallel the evolution of liver injury in rats and humans with alcoholic liver disease. However, the possibility that TNF-alpha expression may be induced in other cell populations before serious liver disease develops has not been evaluated. To clarify this issue, mRNAs and/or protein levels of TNF-alpha and cytokines [interleukin (IL)-6, IL-10, transforming growth factor-beta (TGF)-beta, IL-12, and interferon-gamma] that regulate its biological activity were measured in sera, liver, and adipose tissues of rats that had developed hepatic steatosis after consuming ethanol-containing diets for 6 weeks. Cytokine expression in the ethanol-fed groups was compared with that of pair-fed controls rats that had received isocaloric amounts of a similar, ethanol-free diet for the same time period. Animals were studied both before and after a surgical stress (partial hepatectomy) that is known to provoke cytokine production. Chronic ethanol consumption led to increased serum concentrations of TNF and related cytokines, at least in part, by inducing the overproduction of these factors in the liver and peripheral adipose tissues. Despite the pair-feeding protocol that ensured similar calorie consumption in both groups, adipose tissues in ethanol-fed rats also expressed more leptin, a TNF-alpha-inducible mRNA that encodes an appetite-suppressing hormone. Thus, white adipose tissue can be an important source of cytokines in nonobese animals and may be a target for ethanol's actions. These data implicate TNF-alpha as a potential mediator of the nutritional-metabolic aberrations that often accompany chronic alcohol intake, even in the absence of advanced liver disease.
Assuntos
Tecido Adiposo/imunologia , Alcoolismo/imunologia , Citocinas/sangue , Hepatopatias Alcoólicas/imunologia , Fígado/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Alcoolismo/patologia , Animais , Etanol/toxicidade , Fígado Gorduroso Alcoólico/imunologia , Fígado Gorduroso Alcoólico/patologia , Leptina , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias Alcoólicas/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND & AIMS: Tumor necrosis factor (TNF) alpha mediates both liver injury and regeneration. Kupffer cells are thought to produce TNF because gadolinium chloride (GdCl), a drug that depletes Kupffer cells, prevents TNF-mediated injury. However, GdCl increases liver TNF and regeneration after partial hepatectomy (PH), suggesting that other cells produce TNF during regeneration. The aim of this study was to identify the source(s) of TNF after PH in normal and Kupffer cell-depleted rats. METHODS: Livers were harvested at 0, 1, or 48 hours after PH from saline- or GdCl-treated rats. TNF expression was evaluated by in situ reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: In saline-treated rats, neither TNF transcripts nor protein was detected before PH, but both increased after PH. One hour after PH, 64% +/- 8% portal areas had TNF-positive bile ducts or veins and 61% +/- 1% central veins were TNF positive; by 48 hours, 57% +/- 1% portal areas, 40% +/- 1% central veins, and a few sinsusoidal cells expressed TNF. In GdCl-treated rats, TNF was expressed in 22% +/- 6% portal areas before PH; in 76% +/- 3% portal areas and 75% central veins at 1 hour; and in 88% +/- 2% portal areas and 80% +/- 9% central veins at 48 hours after PH. CONCLUSIONS: In the regenerating livers of both normal and Kupffer cell-depleted rats, bile ducts and veins are the predominant sources of TNF-alpha.
Assuntos
Ductos Biliares/metabolismo , Fígado/metabolismo , Veia Porta/metabolismo , Regeneração/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Genetically obese fatty/fatty rats and obese/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS). Among obese animals, females are more sensitive to endotoxin liver injury than males. LPS induction of tumor necrosis factor alpha (TNF alpha), the proven affecter of endotoxin liver injury, is no greater in the livers, white adipose tissues, or sera of obese animals than in those of lean controls. Indeed, the lowest serum concentrations of TNF occur in female obese rodents, which exhibit the most endotoxin-induced liver injury. Several cytokines that modulate the biological activity of TNF are regulated abnormally in the livers of obese animals. After exposure to LPS, mRNA of interferon gamma, which sensitizes hepatocytes to TNF toxicity, is overexpressed, and mRNA levels of interleukin 10, a TNF inhibitor, are decreased. The phagocytic activity of liver macrophages and the hepatic expression of a gene encoding a macrophage-specific receptor are also decreased in obesity. This new animal model of obesity-associated liver disease demonstrates that hepatic macrophage dysfunction occurs in obesity and suggests that this might promote steatohepatitis by sensitizing hepatocytes to endotoxin.
Assuntos
Fígado Gorduroso/fisiopatologia , Hepatite Animal/fisiopatologia , Fígado/patologia , Obesidade/fisiopatologia , Animais , Escherichia coli , Fígado Gorduroso/patologia , Feminino , Hepatite Animal/patologia , Células de Kupffer/patologia , Células de Kupffer/fisiologia , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Obesos , Obesidade/genética , Obesidade/patologia , Reação em Cadeia da Polimerase , Ratos , Ratos Zucker , Caracteres Sexuais , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Ethanol ingestion may interrupt the proregenerative signal transduction that is initiated by injury-related cytokines such as tumor necrosis factor (TNF)-alpha and TNF-alpha- inducible cytokines including interleukin (IL)-6. To test this theory, liver regeneration, TNF-alpha and IL-6 expression, and cytokine-regulated prereplicative events were compared in ethanol-fed rats and isocalorically fed controls after 70% partial hepatectomy (PH). Ethanol feeding inhibits hepatocyte replication and recovery of liver mass after PH but generally promotes induction of both cytokines in the liver and extrahepatic tissues (i.e., white adipose tissue). Cytokine-regulated events that occur early in the prereplicative period are influenced differentially. TNF-alpha-dependent increases in hepatic nuclear factor-kappaB (NF-kappaB) p50 and p65 expression and DNA binding activity are prevented, whereas IL-6-dependent inductions of hepatic Stat-3 phosphorylation and DNA binding activity occur normally. In contrast, events (e.g., induction of cyclin D1, cdk-1, cyclin D3, and p53 mRNA) that occur at the end of the prereplicative period are uniformly inhibited. These findings indicate that chronic ethanol ingestion arrests the regenerative process during the prereplicative period and demonstrate that increased TNF-alpha, IL-6 and Stat-3 are not sufficient to assure hepatocyte proliferation after PH.
Assuntos
Alcoolismo/fisiopatologia , Citocinas/genética , Citocinas/fisiologia , Regulação da Expressão Gênica , Regeneração Hepática/fisiologia , Fígado/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Tecido Adiposo/imunologia , Animais , Citocinas/biossíntese , Hepatectomia , Interleucina-6/biossíntese , Interleucina-6/genética , Fígado/imunologia , Fígado/fisiologia , NF-kappa B/biossíntese , NF-kappa B/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The impaired regenerative capacity of fatty livers might promote the progression of nonalcoholic fatty liver disease (NAFLD). To identify mechanisms involved, regenerative responses were compared in normal mice and ob/ob mice (a model for NAFLD) after partial hepatectomy (PH). We hypothesized that the usual PH activation of oxidant-sensitive, growth-regulatory kinase cascades would be abnormal in fatty hepatocytes, which have adapted to chronic oxidant stress, and expected that this might interfere with the induction of proliferative- and stress-related genes. The normal coordinated induction of Jun N-terminal kinases (Jnks) and extracellular regulated kinases (Erks) does not occur after PH in ob/ob mice, which cannot activate Jnks but can superinduce Erks. Jnk inhibition is associated with enhanced activation of Akt, which inhibits phosphoenolpyruvate carboxykinase (PEPCK) induction, causing severe hypoglycemia and increased lethality in the ob/ob group. Activation of nuclear factor kappaB (NF-kappaB) is also inhibited, but liver damage is increased only modestly, perhaps because Akt-regulated survival factors are protective. Despite enhanced Erk activity, induction of cyclin D-1, an NF-kappaB target gene, is abolished and this, together with hyperphosphorylated signal transducer and activator of transcription-3 (Stat-3) and reduced adenosine triphosphate (ATP) levels, arrests fatty hepatocytes in G(1). Thus, in mice with NAFLD that have adapted hepatocyte signaling mechanisms to survive chronic oxidative stress, the cellular response to an acute regenerative stimulus is altered. This contributes to NAFLD pathophysiology by inhibiting proliferation, increasing injury, and limiting function in fatty livers.
Assuntos
Fígado Gorduroso/fisiopatologia , Regeneração Hepática , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Obesidade/complicações , Proteínas Serina-Treonina Quinases , Trifosfato de Adenosina/metabolismo , Animais , Fase G1 , Interleucina-6/biossíntese , Canais Iônicos , Glicogênio Hepático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Estresse Oxidativo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fator de Necrose Tumoral alfa/biossíntese , Proteína Desacopladora 2RESUMO
Cytokines, such as TNF alpha, modulate the behavior of many cells by regulating the expression of a wide array of genes. When a cytokine binds to its receptor on the cell surface, the receptor becomes activated and activates signal transduction cascades. These cascades typically involve a series of phosphorylation reactions that lead to sequential activation of various kinases. The targets of these kinases include DNA binding proteins that regulate the transcription of target genes. The activity of DNA binding proteins, such as c-Jun and NF-kappa B, titrates the transcriptional activity of cytokine-regulated genes. Both acute and chronic alcohol consumption of ethanol increase hepatic expression of TNF alpha. After acute ethanol consumption, this is associated with increased induction of several TNF-dependent regenerative events, including the activation of c-Jun and increased binding activity of NF-kappa B. However, chronic consumption of ethanol appears to impede TNF alpha signaling in the liver because it attenuates the increases in c-JUN activity and NF-kappa B binding, which normally follow partial hepatectomy. These results suggest that one mechanism by which ethanol influences liver cell behavior is by influencing local expression of TNF alpha and changing the activity of TNF-regulated transcription factors.
Assuntos
Consumo de Bebidas Alcoólicas , Citocinas/fisiologia , Etanol/farmacologia , Fatores de Transcrição/metabolismo , Animais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Regeneração Hepática/fisiologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Injury-related cytokines, such as tumor necrosis factor-alpha (TNF), may preserve liver-specific gene expression during the subsequent regenerative response by modulating the activity of transcription factors, including CCAAT/enhancer binding proteins (C/EBPs), which regulate differentiated gene expression in hepatocytes. To test this theory, rats were treated with neutralizing antibody to TNF or nonimmune immunoglobulin before partial hepatectomy (PH) and regenerative changes in the messenger RNAs (mRNAs), proteins, and DNA-binding activities of C/EBP isoforms and the expression of a C/EBP-regulated gene, phosphoenol pyruvate carboxykinase (PEPCK), were compared. Before PH, the expressions of C/EBP-alpha, C/EBP-beta, and C/EBP-gamma were similar in the two treatment groups. Dimers containing C/EBP-alpha and C/EBP-beta accounted for virtually all of the C/EBP DNA binding activity and mRNA for PEPCK, the rate limiting hepatocyte enzyme for gluconeogenesis, was barely detected. After PH, in control rats, mRNA and nuclear protein concentrations of C/EBP-beta and C/EBP-gamma increased approximately fivefold by 3 hours after PH. This was accompanied by increased DNA binding activity of these C/EBP isoforms and decreased DNA binding activity of C/EBP-alpha. mRNA levels of PEPCK, a gene that is strongly transactivated by non-alpha C/EBP isoforms, increased fivefold. Pretreatment with anti-TNF antibodies prevented regenerative induction of C/EBP beta and gamma expression and DNA-binding activity. The nature of dimers binding to C/EBP cis-acting elements remained similar to that observed in liver before PH and increases in PEPCK mRNA were blunted. These results support the theory that TNF helps maintain liver-specific gene expression during liver regeneration by altering transcription factor complexes that regulate differentiated gene expression in hepatocytes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Regeneração Hepática , Fígado/fisiologia , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Fígado/citologia , Masculino , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Tumor necrosis factor alpha (TNF), initiates a cytokine cascade that promotes hepatocyte proliferation after 70% partial hepatectomy (PH) but the mechanisms regulating TNF production after PH are unknown. We previously reported that gadolinium chloride (GdCl), an agent that depletes the liver of phagocytically active Kupffer cells, enhances hepatic expression of TNF messenger RNA (mRNA) and promotes liver regeneration after subsequent PH. This suggests that GdCl interferes with Kupffer cell mechanisms that normally constrain TNF production after PH. To evaluate this, the pre- and post-PH expression of TNF, TNF-inducible cytokines (interleukin [IL]-1, IL-6) and cytokines (transforming growth factor [TGF] beta 1 and IL-10) that down-regulate TNF were compared in controls and GdCl-treated rats. In controls, TNF, IL-1, IL-6, and IL-10 increase within 3 hours after PH, whereas TGF-beta 1 is induced much later (> 24 hours after PH). GdCl causes sustained overexpression of TNF mRNA and transient overexpression of circulating TNF protein after PH; both TNF-inducible cytokines are also relatively overexpressed. Cytokines that down-regulate TNF are effected differentially by GdCl. Regenerative induction of IL-10 is abolished but TGF-beta 1 induction is unaltered. Because IL-10 is known to shorten the half-life of TNF mRNA, these results suggest that Kupffer cell production of IL-10 is an important mechanism that down-regulates TNF production during liver regeneration.
Assuntos
Interleucina-10/biossíntese , Células de Kupffer/fisiologia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Sequência de Bases , Citocinas/biossíntese , Citocinas/genética , Primers do DNA/genética , Gadolínio/farmacologia , Expressão Gênica/efeitos dos fármacos , Hepatectomia , Interleucina-10/genética , Células de Kupffer/efeitos dos fármacos , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-DawleyRESUMO
Hepatocytes were cultured in the presence of recombinant tumor necrosis factor (TNF) alpha or mutated TNF alpha peptides that specifically activate either p55 or p75 TNF receptors to determine if TNF alpha can activate cytokine-inducible CCAAT/enhancer binding protein (C/EBP) isoforms by post-transcriptional mechanisms that are initiated by TNF receptors. Within 5-10 min after treatment with any of these agents, nuclear concentrations of C/EBP beta and C/EBP delta double and remain 2-4-fold greater than control cultures for 30 min (p < 0.01). Consistent with these results, gel mobility shift assays demonstrate 3-fold increased nuclear C/EBP beta- and C/EBP delta-DNA binding activity in TNF alpha-treated cells, and immunocytochemistry confirms rapid redistribution of these C/EBP isoforms into the nucleus. In contrast, mRNA and whole cell protein concentrations of C/EBP beta and delta are not altered by TNF alpha exposure, and nuclear concentrations of another C/EBP isoform, C/EBP alpha, are decreased by 80%. This novel evidence that TNF alpha initiates post-transcriptional activation of cytokine-inducible C/EBP isoforms identifies a mechanism that enables hepatocytes to respond immediately to inflammatory stress.
Assuntos
Compartimento Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Proteínas Estimuladoras de Ligação a CCAAT , Fígado/citologia , Dados de Sequência Molecular , Ligação Proteica , Receptores do Fator de Necrose Tumoral/agonistas , Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Although obesity-related fatty livers are vulnerable to damage from endotoxin, the mechanisms involved remain obscure. The purpose of this study was to determine if immunologic priming might be involved by determining if fatty livers resemble normal livers that have been sensitized to endotoxin damage by Propionibacterium acnes infection. The latter induces interleukin (IL)-12 and -18, causing a selective reduction of CD4+NK T cells, diminished IL-4 production, deficient production of T-helper type 2 (Th-2) cytokines (e.g., IL-10), and excessive production of Th-1 cytokines (e.g., interferon gamma [IFN-gamma]). Liver and spleen lymphocyte populations and hepatic cytokine production were compared in genetically obese, ob/ob mice (a model for obesity-related fatty liver) and lean mice. Obese mice have a selective reduction of hepatic CD4+NK T cells. Serum IL-18 is also increased basally, and the hepatic mRNA levels of IL-18 and -12 are greater after endotoxin challenge. Thus, up-regulation of IL-18 and IL-12 in fatty livers may reduce hepatic CD4+NK T cells. In addition, mononuclear cells from fatty livers have decreased expression of the adhesion molecule, leukocyte factor antigen-1 (LFA-1), which is necessary for the hepatic accumulation of CD4+NK T cells. Consistent with reduced numbers of hepatic CD4+NK T cells, mononuclear cells from fatty livers produce less IL-4. Furthermore, after endotoxin treatment, hepatic induction of IL-10 is inhibited, while that of IFN-gamma is enhanced. Thus, fatty livers have inherent immunologic alterations that may predispose them to damage from endotoxin and other insults that induce a proinflammatory cytokine response.
Assuntos
Fígado Gorduroso/complicações , Subpopulações de Linfócitos/imunologia , Obesidade/complicações , Animais , Separação Celular , Fígado Gorduroso/imunologia , Imunização Passiva , Interleucina-12/análise , Interleucina-18/análise , Interleucina-4/análise , Lipopolissacarídeos , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/imunologiaRESUMO
The growth-stimulatory actions of tumor necrosis factor alpha (TNF-alpha) after partial hepatectomy (PH) are difficult to reconcile with its well-established role in the genesis of liver injury. The lethal actions of TNF are thought to involve the induction of oxidant production by mitochondria. It is not known if TNF initiates mitochondrial oxidant production after PH. Furthermore, if this potentially toxic response follows PH, it is not clear how hepatocytes defend themselves sufficiently so that replication, rather than death, occurs. These studies test the hypothesis that TNF does increase mitochondrial oxidant production after PH but that these oxidants primarily promote the induction of antioxidant defenses in regenerating hepatocytes. Consistent with this concept, H2O2 production by liver mitochondria increases from 5 minutes to 3 hours after PH, beginning before the transient inductions of hepatic NF kB activity (which peaks at 30 minutes post-PH) and uncoupling protein-2 (UCP-2) (which begins around 30 minutes and peaks from 6-24 hours post-PH). Pretreatment with neutralizing anti-TNF antibodies, which inhibits hepatocyte DNA synthesis after PH, also reduces post-PH hepatic mitochondrial oxidant production by 80% and inhibits NF kappaB activation and UCP-2 induction by 50% and 80%, respectively. In contrast, pretreatment with D609, an agent that inhibits phosphatidylcholine-specific phospholipase C, neither inhibits regenerative induction of mitochondrial oxidant production, UCP-2 expression, nor hepatocyte DNA synthesis, although it inhibits NF kappaB activation by 50%. Given published evidence that NF kappaB is antiapoptotic and that UCP-2 may decrease mitochondrial oxidant production in some cells, these results suggest that TNF-dependent increases in oxidant production by liver mitochondria promote the induction of antioxidant defenses in the regenerating liver.