RESUMO
Metabolic syndrome is a multifactorial disease with high prevalence worldwide. It is related to cardiovascular disease, diabetes, and obesity. Approximately 80% of patients with metabolic syndrome have some degree of fatty liver disease. An adenosine derivative (IFC-305) has been shown to exert protective effects in models of liver damage as well as on elements involved in central metabolism; therefore, here, we evaluated the effect of IFC-305 in an experimental model of metabolic syndrome in rats induced by a high-fat diet and 10% sucrose in drinking water for 18 weeks. We also determined changes in fatty acid uptake in the Huh-7 cell line. In the experimental model, increases in body mass, serum triglycerides and proinflammatory cytokines were induced in rats, and the adenosine derivative significantly prevented these changes. Interestingly, IFC-305 prevented alterations in glucose and insulin tolerance, enabling the regulation of glucose levels in the same way as in the control group. Histologically, the alterations, including mitochondrial morphological changes, observed in response to the high-fat diet were prevented by administration of the adenosine derivative. This compound exerted protective effects against metabolic syndrome, likely due to its action in metabolic regulation, such as in the regulation of glucose blood levels and hepatocyte fatty acid uptake.
Assuntos
Síndrome Metabólica , Humanos , Ratos , Animais , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/prevenção & controle , Síndrome Metabólica/induzido quimicamente , Sacarose/metabolismo , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Adenosina/metabolismo , Glucose/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismoRESUMO
The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/veterinária , Núcleo Celular/metabolismo , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Dietilnitrosamina/toxicidade , Progressão da Doença , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/veterinária , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/genética , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.
Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular/genética , Metilação de DNA/genética , Fator 4 Nuclear de Hepatócito/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Desmetilação do DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Hepatócitos/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismoRESUMO
BACKGROUND: There is a correlation between the endocannabinoid system and hepatic fibrosis based on the activation of CB1 and CB2 receptors; where CB1 has profibrogenic effects. Gene therapy with a plasmid carrying a shRNA for CB1 delivered by hydrodynamic injection has the advantage of hepatic tropism, avoiding possible undesirable effects of CB1 pharmacological inhibition. OBJECTIVE: To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of a plasmid with the sequence of a shRNA for CB1 and its antifibrogenic effects. METHODS: Three shRNA (21pb) were designed for blocking CB1 mRNA at positions 877, 1232 and 1501 (pshCB1-A, B, C). Sequences were cloned in the pENTR™/U6. Safety was evaluated monitoring CB1 expression in brain tissue. The silencing effect was determined in rat HSC primary culture and CCl4 cirrhosis model. Hydrodynamic injection in cirrhotic liver was through iliac vein and with a dose of 3mg/kg plasmid. Serum levels of liver enzymes, mRNA levels of TGF-ß1, Col IA1 and α-SMA and the percentage of fibrotic tissue were analyzed. RESULTS: Hydrodynamic injection allows efficient CB1 silencing in cirrhotic livers and pshCB1-B (position 1232) demonstrated the main CB1-silencing. Using this plasmid, mRNA level of fibrogenic molecules and fibrotic tissue considerably decrease in cirrhotic animals. Brain expression of CB1 remained unaltered. CONCLUSION: Hydrodynamics allows a hepatotropic and secure transfection in cirrhotic animals. The sequence of the shCB1-B carried in a plasmid or any other vector has the potential to be used as therapeutic strategy for liver fibrosis.
Assuntos
Inativação Gênica , Hidrodinâmica , Cirrose Hepática/patologia , RNA Interferente Pequeno/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Actinas/genética , Actinas/metabolismo , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Masculino , Plasmídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
S-adenosylmethionine (SAM), biosynthesis from methionine and ATP, is markedly decreased in hepatocellularular carcinoma (HCC) for a diminution in ATP levels, and the down regulation of the liver specific MAT1a enzyme. Its metabolic activity is very important in the transmethylation reactions, the methionine cycle, the biosynthesis of glutathione (GSH) and the polyamine pathway, which are markedly affected in the HCC. The chemo-preventive effect of IFC305 in HCC induced by DEN, and the increase of ATP and SAM in CCl4-induced cirrhosis have been previously demonstrated. The aim of this work was to test whether this chemo-preventive effect is mediated by the induction of SAM biosynthesis and its metabolic flow. SAM hepatic levels and the methionine cycle were recovered with IFC305 treatment, restoring transmethylation and transsulfuration activities. IFC305 treatment, increased MAT1a levels and decrease MAT2a levels through modulation of their post-transcriptional regulation. This occurred through the binding of the AUF1 (binding factor 1 AU-rich sites) and HuR (human antigen R) ribonucleoproteins to Mat1a and Mat2a messenger RNAs, which maintained their nuclear localization. Finally, the compound inhibited the polyamine pathway favoring the recuperation of the normal methionine and one carbon cycle recuperating the metabolic flow of methionine, which probably facilitated its HCC chemo-preventive effect.
Assuntos
Adenosina/análogos & derivados , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Metionina Adenosiltransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , S-Adenosilmetionina/metabolismo , Adenosina/farmacologia , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Metionina Adenosiltransferase/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cirrhosis is a liver pathology originated by hepatocytes, Kupffer and hepatic stellate cells interactions and transformations. This pathology is associated with inflammation and fibrosis, originated by molecular signals secreted by immunological and parenchymal cells, such as cytokines and chemokines, like IL-1ß, IL-6, TNF-α or MCP-1, driven by Kupffer cells signals. As part of inflammation resolution, the same activated Kupffer cells contribute to anti-inflammatory effects with IL-10 and MMP-9 secretion. In a Wistar rat model, cirrhosis induced with CCl4 is characterized by increased inflammatory cytokines, IL-6, IL-1ß, MCP-1, and TNF-α, in plasma and liver tissue. The IFC-305 compound, an adenosine derivative salt, reverses the cirrhosis in this model, suggesting that immune mechanisms related to inflammation should be explored. The IFC-305 reduced inflammatory cytokines, supporting the anti-inflammatory effects induced by the elevation of IL-10, as well as the reduction of M1 inflammatory macrophages (CD11b/c+/CD163+) and the increase of M2 anti-inflammatory macrophages (HIS36+/CD11b+), measured by flow cytometry. Furthermore, the IFC-305 enhances the metabolic activity of arginase and moderates the inducible nitric oxide synthetase, evaluated through biochemical and immunohistochemical methods. These results contribute to understand the function of the IFC-305, which modulates the immune response in the Wistar rat model of CCl4-induced cirrhosis and support the hepatic protective action through an anti-inflammatory effect, mainly mediated by Kupffer cells.
Assuntos
Adenosina/análogos & derivados , Anti-Inflamatórios/uso terapêutico , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Adenosina/uso terapêutico , Animais , Arginase/metabolismo , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Tetracloreto de Carbono , Diferenciação Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose/induzido quimicamente , Fibrose/imunologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Masculino , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Wistar , Equilíbrio Th1-Th2RESUMO
Regulatory T cells (Tregs) are considered key players in the prevention of allograft rejection in transplanted patients. Belatacept (BLT) is an effective alternative to calcineurin inhibitors that appears to preserve graft survival and function; however, the impact of this drug in the homeostasis of Tregs in transplanted patients remains controversial. Here, we analyzed the phenotype, function, and the epigenetic status of the Treg-specific demethylated region (TSDR) in FOXP3 of circulating Tregs from long-term kidney transplant patients under BLT or Cyclosporine A treatment. We found a significant reduction in the proportion of CD4+CD25hiCD127lo/-FOXP3+ T cells in all patients compared to healthy individual (controls). Interestingly, only BLT-treated patients displayed an enrichment of the CD45RA+ "naïve" Tregs, while the expression of Helios, a marker used to identify stable FOXP3+ thymic Tregs remained unaffected. Functional analysis demonstrated that Tregs from transplanted patients displayed a significant reduction in their suppressive capacity compared to Tregs from controls, which is associated with decreased levels of FOXP3 and CD25. Analysis of the methylation status of the FOXP3 gene showed that BLT treatment results in methylation of CpG islands within the TSDR, which could be associated with the impaired Treg suppression function. Our data indicate that analysis of circulating Tregs cannot be used as a marker for assessing tolerance toward the allograft in long-term kidney transplant patients. Trial registration number IM103008.
RESUMO
Introduction. Cirrhosis is a chronic degenerative illness characterized by changes in normal liver architecture, failure of hepatic function, and impairment of proliferative activity. The aim of this study is to know how IFC-305 compound induces proliferation of the liver during reversion of cirrhosis. Methods. Once cirrhosis has been installed by CCl(4) treatment for 10 weeks in male Wistar rats, they were divided into four groups: two received saline and two received the compound; all were euthanized at 5 and 10 weeks of treatment. Liver homogenate, mitochondria, and nucleus were used to measure cyclins, CDKs, and cell cycle regulatory proteins PCNA, pRb, p53, E2F, p21, p27, HGF, liver ATP, and mitochondrial function. Results. Diminution and small changes were observed in the studied proteins in the cirrhotic animals without treatment. The IFC-305-treated rats showed a clear increase in most of the proteins studied mainly in PCNA and CDK6, and a marked increased in ATP and mitochondrial function. Discussion/Conclusion. IFC-305 induces a recovery of the cell cycle inhibition promoting recovery of DNA damage through the action of PCNA and p53. The increase in energy and preservation of mitochondrial function contribute to recovering the proliferative function.
RESUMO
We have shown that adenosine administration is capable of reversing fibrosis in the carbon tetrachloride-induced rat cirrhotic liver, stimulating the diminished proliferative potential of the cirrhotic liver. To characterize adenosine actions on liver cellular proliferation, we used rats subjected to one-third partial hepatectomy (PH). In PH animals acutely administered with adenosine (25-200 mg/kg b.w.), parameters indicative of cell proliferation were determined. In addition, hepatocyte growth factor (HGF), epidermal growth factor, and transforming growth factor-alpha, cyclins, members of the E2F family, proto-oncogenes, and adenosine-receptors were determined through Western blot analyses. Adenosine (100 mg/kg body weight) induced an earlier increase in liver cell proliferation as evidenced by enhanced levels of proliferating cell nuclear antigen, nuclear Ki-67 antigen, and those for cyclins (D1, E, A, and B1), as well as by an increased mitotic index. These effects were also accompanied for a long-lasting increase of serum and liver levels of HGF and liver expression of c-Met and HGF liver activator. Adenosine effects on cell proliferation could be mediated by an early increase in E2F-1 and by that of c-Myc, despite the fact that phosphorylation of the Rb protein and expression of E2F-3 were decreased. Moreover, the liver amount of specific receptors for adenosine was not significantly changed by PH and/or adenosine treatment. In conclusion, these data suggest that adenosine actions can accelerate and increase proliferation in a "primed" liver, mainly through enhancing c-Myc, E2F family, cell-cycle cyclins, and HGF expression. Therefore, these pharmacological adenosine effects suggest a modulating role for the nucleoside on mitogenic events once the liver has been triggered to proliferate.
Assuntos
Adenosina/administração & dosagem , Ciclo Celular/fisiologia , Hepatectomia , Regeneração Hepática/fisiologia , Adenosina/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hepatectomia/métodos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Fígado/cirurgia , Regeneração Hepática/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
We have proposed that controlled peroxidative modifications of membranes could be playing a role in the early steps of liver regeneration. Hence, lipid peroxidation (LP) was modified in vivo by treatment with vitamin E in rats subjected to partial hepatectomy (PH), and its influence on liver regeneration was evaluated. Our results, using several methods to monitor LP, indicate that vitamin E administration promoted a decreased LP rate in liver subcellular membranes. Vitamin E drastically diminished cytosolic LP, shifting earlier increased LP in plasma membranes, and promoted a higher increase of nuclear LP in animals subjected to PH. Pretreatment with vitamin E induced a striking reduction of liver mass recovery and nuclear bromodeoxyuridine labeling (clearly shown at 24 hours after surgery), as well as promoted a decreased expression of cyclin D1 and of the proliferating cell nuclear antigen after PH. These effects seem to lead to a decreased mitotic index at 48 hours after PH. Vitamin E pretreatment also diminished PH-induced hypoglycemia but elevated serum bilirubin level, which was not observed in PH animals without vitamin treatment. In conclusion, an enhanced but controlled LP seems to play a critical role during the early phases of liver regeneration. Decreasing magnitude or time course of the PH-promoted enhanced LP (at early post-PH stages) by in vivo treatment with vitamin E could promote an early termination of preparative cell events, which lead to the replicative phase, during PH-promoted liver proliferation. The latter could have a significant implication in the antitumorigenic effect ascribed to the treatment with vitamin E.