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1.
Nat Commun ; 12(1): 98, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397935

RESUMO

Glucose metabolism and innate immunity evolved side-by-side. It is unclear if and how the two systems interact with each other during hepatitis B virus (HBV) infections and, if so, which mechanisms are involved. Here, we report that HBV activates glycolysis to impede retinoic acid-inducible gene I (RIG-I)-induced interferon production. We demonstrate that HBV sequesters MAVS from RIG-I by forming a ternary complex including hexokinase (HK). Using a series of pharmacological and genetic approaches, we provide in vitro and in vivo evidence indicating that HBV suppresses RLR signaling via lactate dehydrogenase-A-dependent lactate production. Lactate directly binds MAVS preventing its aggregation and mitochondrial localization during HBV infection. Therefore, we show that HK2 and glycolysis-derived lactate have important functions in the immune escape of HBV and that energy metabolism regulates innate immunity during HBV infection.


Assuntos
Vírus da Hepatite B/fisiologia , Imunidade Inata , Metaboloma , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anaerobiose , Animais , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Glucose/metabolismo , Glicólise , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Evasão da Resposta Imune , Interferons/metabolismo , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transdução de Sinais , Vírion/metabolismo
2.
Nat Commun ; 12(1): 66, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397952

RESUMO

IL11 is important for fibrosis in non-alcoholic steatohepatitis (NASH) but its role beyond the stroma in liver disease is unclear. Here, we investigate the role of IL11 in hepatocyte lipotoxicity. Hepatocytes highly express IL11RA and secrete IL11 in response to lipid loading. Autocrine IL11 activity causes hepatocyte death through NOX4-derived ROS, activation of ERK, JNK and caspase-3, impaired mitochondrial function and reduced fatty acid oxidation. Paracrine IL11 activity stimulates hepatic stellate cells and causes fibrosis. In mouse models of NASH, hepatocyte-specific deletion of Il11ra1 protects against liver steatosis, fibrosis and inflammation while reducing serum glucose, cholesterol and triglyceride levels and limiting obesity. In mice deleted for Il11ra1, restoration of IL11 cis-signaling in hepatocytes reconstitutes steatosis and inflammation but not fibrosis. We found no evidence for the existence of IL6 or IL11 trans-signaling in hepatocytes or NASH. These data show that IL11 modulates hepatocyte metabolism and suggests a mechanism for NAFLD to NASH transition.


Assuntos
Hepatócitos/metabolismo , Interleucina-11/metabolismo , Lipídeos/toxicidade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais , Adulto , Animais , Comunicação Autócrina/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Comportamento Alimentar , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Interleucina-6/metabolismo , Camundongos Knockout , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos
3.
Nat Metab ; 2(11): 1350-1367, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33168981

RESUMO

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.


Assuntos
Epitélio/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Macrófagos/patologia , Proteínas Proto-Oncogênicas c-rel/genética , Animais , Polaridade Celular/genética , Marcação de Genes , Hepatócitos/patologia , Hidroxiprolina/metabolismo , Cirrose Hepática/prevenção & controle , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/genética , Comunicação Parácrina/genética , Fosfofrutoquinase-2/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo
4.
PLoS One ; 15(10): e0240562, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33035276

RESUMO

Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.


Assuntos
Alanina Transaminase/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Furosemida/efeitos adversos , Glutamato Desidrogenase/sangue , Mitocôndrias Hepáticas , Mitofagia/efeitos dos fármacos , Acetaminofen/efeitos adversos , Animais , Biomarcadores/sangue , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo
5.
PLoS One ; 15(10): e0240478, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33044988

RESUMO

Kahweol is a diterpene found in coffee beans and unfiltered coffee drinks. Several studies have demonstrated that kahweol induces the nuclear factor erythroid-2 related factor 2/ hemeoxygenase-1 (Nrf2/HO-1) pathway; however, the mechanisms involved are currently unknown. Kelch-like ECH-associated protein 1 (Keap1) is a major regulator of Nrf2 expression and is degraded mostly by autophagy. The p62 protein enhances binding to Keap1 and contributes to the activation of Nrf2. Here, we examined the role of Keap1 regulation in the effect of kahweol on the Nrf2/HO-1 pathway in hepatocytes. In AML12 cells and primary mouse hepatocytes, kahweol increased the levels of Nrf2 and HO-1 protein without increasing expression of the Nrf2 mRNA. In addition, kahweol reduced Keap1 protein levels significantly without decreasing Keap1 mRNA levels. Although regulation of the Keap1-Nrf2-pathway by p62-dependent autophagy is well known, we confirmed here that the reduction of Keap1 protein levels by kahweol does not involve p62-dependent autophagy degradation or ubiquitination. In conclusion, kahweol increases the expression of Nrf2 in hepatocytes by inhibiting translation of the Keap1 mRNA.


Assuntos
Antioxidantes/farmacologia , Proteína 7 Relacionada à Autofagia/fisiologia , Diterpenos/farmacologia , Heme Oxigenase-1/metabolismo , Hepatócitos/patologia , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose , Autofagia , Sobrevivência Celular , Células Cultivadas , Heme Oxigenase-1/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
6.
Anticancer Res ; 40(11): 6075-6081, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109545

RESUMO

BACKGROUND: One of the most prevalent causes of cancer fatalities is hepatocellular carcinoma (HCC), which has been linked to metabolic syndrome. Circulating levels of the saturated fatty acid palmitate are elevated in metabolic syndrome and lead to cellular stress. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay, flow cytometry, and migration assays, we characterized the response of rat hepatoma cells to palmitate treatment. RESULTS: We detected a 60% increase in secretion of C-X-C motif ligand 1 (CXCL1) which was dose-dependent and coincided with apoptosis. We measured expression of C-X-C motif chemokine receptor 2 (CXCR2) and observed a 4.5-fold increase on apoptotic hepatoma cells. Furthermore, we assayed migration of hepatoma cells and saw a 2-fold increase in the number of migrating cells towards CXCL1. CONCLUSION: These findings suggest that HCC cells secrete CXCL1 in response to metabolic syndrome signals and may promote the progression of cancer through apoptosis recovery or metastasis.


Assuntos
Comunicação Autócrina , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Quimiocina CXCL1/metabolismo , Progressão da Doença , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Animais , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Glucose/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Camundongos , Ácido Palmítico/toxicidade , Ratos , Receptores de Interleucina-8B/metabolismo
7.
Aquat Toxicol ; 228: 105622, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32947073

RESUMO

Hexavalent chromium, a heavy metal toxicant, abundantly found in the environment showed hepatotoxic potential in zebrafish liver and instigated the Nrf2-Keap1-ARE pathway as a cellular stress response as reported in our previous studies. In the present study we have evaluated the ameliorating effect of shinorine, a mycosporine like amino acid (MAAs) and a mammalian Keap1 antagonist against chromium induced stress in zebrafish hepatocytes. Shinorine was found to be effective in increasing the cell viability of chromium treated hepatocytes through curtailing the cellular ROS content. Trigonelline, an Nrf2 inhibitor was found to reduce the viability of hepatocyte cultures co-exposed to shinorine and chromium. In other words, trigonelline being an Nrf2 blocker neutralised the alleviating effect of shinorine. This indicated that shinorine mediated cyto-protection in Cr [VI]-intoxicated cells is Nrf2 dependent. Further, qRT-PCR analysis revealed comparatively higher expression of nfe2l2 and nqo1 in shinorine + chromium treated hepatocytes than cells exposed to chromium alone indicating a better functioning of Nrf2-Keap1-Nqo1 axis. To further confirm if shinorine can lead to disruption of Nrf2-Keap1 interaction in zebrafish hepatocytes and render cytoprotection to chromium exposure, our in silico analysis through molecular docking revealed that shinorine could bind to the active amino acid residues of the DGR domain, responsible for Nrf2-Keap1 interaction of all the three Keap1s evaluated. This is the first report about shinorine that ameliorates chromium induced toxicity through acting as an Nrf2-Keap1 interaction disruptor. We additionally carried out in-silico pharmacokinetic and ADMET studies to evaluate druglikeness of shinorine whose promising results indicated its potential to be developed as an ideal therapeutic candidate against toxicant induced pathological conditions.


Assuntos
Cromo/toxicidade , Cicloexilaminas/farmacologia , Glicina/análogos & derivados , Hepatócitos/efeitos dos fármacos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cianobactérias/metabolismo , Cicloexilaminas/isolamento & purificação , Glicina/isolamento & purificação , Glicina/farmacologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Simulação de Acoplamento Molecular , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais
8.
PLoS One ; 15(9): e0238717, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915852

RESUMO

INTRODUCTION: Association between elevated cytokeratin 18 (CK-18) levels and hepatocyte death has made circulating CK-18 a candidate biomarker to differentiate non-alcoholic fatty liver from non-alcoholic steatohepatitis (NASH). Yet studies produced variable diagnostic performance. We aimed to provide summary estimates with increased precision for the accuracy of CK-18 (M30, M65) in detecting NASH and fibrosis among non-alcoholic fatty liver disease (NAFLD) adults. METHODS: We searched five databases to retrieve studies evaluating CK-18 against a liver biopsy in NAFLD adults. Reference screening, data extraction and quality assessment (QUADAS-2) were independently conducted by two authors. Meta-analyses were performed for five groups based on the CK-18 antigens and target conditions, using one of two methods: linear mixed-effects multiple thresholds model or bivariate logit-normal random-effects model. RESULTS: We included 41 studies, with data on 5,815 participants. A wide range of disease prevalence was observed. No study reported a pre-defined cut-off. Thirty of 41 studies provided sufficient data for inclusion in any of the meta-analyses. Summary AUC [95% CI] were: 0.75 [0.69-0.82] (M30) and 0.82 [0.69-0.91] (M65) for NASH; 0.73 [0.57-0.85] (M30) for fibrotic NASH; 0.68 (M30) for significant (F2-4) fibrosis; and 0.75 (M30) for advanced (F3-4) fibrosis. Thirteen studies used CK-18 as a component of a multimarker model. CONCLUSIONS: For M30 we found lower diagnostic accuracy to detect NASH compared to previous meta-analyses, indicating a limited ability to act as a stand-alone test, with better performance for M65. Additional external validation studies are needed to obtain credible estimates of the diagnostic accuracy of multimarker models.


Assuntos
Queratina-18/genética , Cirrose Hepática/diagnóstico , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Fragmentos de Peptídeos/genética , Adulto , Biomarcadores/metabolismo , Biópsia , Morte Celular/genética , Feminino , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia
9.
Ecotoxicol Environ Saf ; 203: 110928, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888618

RESUMO

Hexavalent chromium [Cr(VI)] is seriously harmful to ecosystems and living organisms due to its strong toxicity. Role of dynamin-related protein 1 (Drp1) and Drp1-associated mitochondrial fragmentation in mitophagy and cytotoxicity after Cr(VI) exposure has not been clarified so far. We confirmed that Cr(VI) caused mitochondrial fission by up-regulating Drp1 expression and enhancing Drp1 mitochondrial translocation. By applying the intracellular Ca2+ antagonist BAPTA-AM and mitochondrial Ca2+ antagonist Ru360, we demonstrated that Cr(VI)-induced excessive mitochondrial fission was in a Ca2+-Drp1 dependent manner. The administration of Drp1 siRNA significantly suppressed the overactivation of mitophagy in Cr(VI)-induced hepatotoxicity. The specific Drp1 inhibitor mitochondrial division inhibitor-1 (Mdivi-1) blocked the overactive mitophagy and subsequently ameliorated hepatotoxicity caused by Cr(VI) in vivo. We reached the conclusion that Drp1-dependent mitochondrial fission contributes to Cr(VI)-induced mitophagy and hepatotoxicity, which may provide experimental basis for the study of chromium-associated toxicity, especially for the prevention of health damage in chromium-exposed population.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cromo/toxicidade , Dinaminas/metabolismo , Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ecossistema , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , RNA Interferente Pequeno/metabolismo
10.
Life Sci ; 261: 118354, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32866517

RESUMO

AIMS: Recent studies have shown that the hyperactive Notch pathway is involved in cirrhosis and hepatocellular carcinoma (HCC) development by regulating differentiation of hepatic oval cells (HOCs) into cancer cells. The aim of this study was to investigate whether matrine can alleviate liver injury and promote HOC differentiation into hepatocytes by suppression of Notch pathway. MAIN METHODS: We evaluated the expression of Notch-1, Jagged-1, and Hes-1 in HCC tissue by immunohistochemistry. Stem cell characteristics of HOCs were evaluated by CCK-8, cell cycle, and apoptosis. The expression of Notch pathway, HOC markers and albumin (ALB) was detected by immunohistochemistry, QRT-PCR and western blotting. The effects of matrine in protecting liver in vivo were investigated in a rat Solt-Farber precancerous model. KEY FINDINGS: We found an abnormal activated Notch pathway in HCC tissue, and the hyperactive Notch pathway was strongly associated with poor liver function in patients with cirrhosis with HCC. Using siNotch-1 to inhibit Notch pathway confirmed that Notch pathway could maintain stem cell characteristics of HOCs. Matrine inhibited stem cell characteristics of HOCs, the expression of Notch pathway and HOC markers but upregulated ALB. Matrine in combined with siNotch-1 RNA decreased the more potently inhibited HOC markers and Notch pathway. In rat Solt-Farber precancerous model, prophylactic application of matrine alleviated liver injury, downregulated Notch pathway and HOC markers, and upregulated ALB in a dose-dependent manner. SIGNIFICANCE: Matrine could promote the differentiation of HOCs into hepatocytes by inhibiting the Notch signalling pathway and alleviate liver injury.


Assuntos
Alcaloides/farmacologia , Diferenciação Celular , Hepatócitos/patologia , Fígado/lesões , Fígado/patologia , Quinolizinas/farmacologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Estimativa de Kaplan-Meier , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley
11.
Am J Physiol Endocrinol Metab ; 319(5): E863-E876, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32924526

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a growing epidemic linked to metabolic disease. The first stage of NAFLD is characterized by lipid accumulation in hepatocytes, but this can progress into nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). Western diets, high in fats, sugars, and cholesterol, are linked to NAFLD development. Murine models are often used to study NAFLD; however, there remains debate on which diet-induced model best mimics both human disease progression and pathogenesis. In this study, we performed a side-by-side comparison of two popular diet models of murine NAFLD/NASH and associated HCC, a high-fat diet supplemented with 30% fructose water (HFHF) and a Western diet high in cholesterol (WDHC), and these were compared with a common grain-based chow diet (GBD). Mice on both experimental diets developed liver steatosis, and WDHC-fed mice had greater levels of hepatic inflammation and fibrosis than HFHF-fed mice. In contrast, HFHF-fed mice were more obese and developed more severe metabolic syndrome, with less pronounced liver disease. Despite these differences, WDHC-fed and HFHF-fed mice had similar tumor burdens in a model of diet-potentiated liver cancer. Response to diet and resulting phenotypes were generally similar between sexes, albeit delayed in females. This study shows that modest differences in diet can significantly uncouple glucose homeostasis and liver damage. In conclusion, long-term feeding of either HFHF or WDHC is a reliable method to induce NASH and diet-potentiated liver cancer in mice of both sexes; however, the choice of diet involves a trade-off between severity of metabolic syndrome and liver damage.


Assuntos
Carcinoma Hepatocelular/metabolismo , Metabolismo dos Lipídeos/fisiologia , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Dieta Hiperlipídica , Dieta Ocidental , Modelos Animais de Doenças , Progressão da Doença , Feminino , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia
12.
Sci Rep ; 10(1): 15558, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968110

RESUMO

Advanced fibrosis and portal hypertension influence short-term mortality. Lipocalin 2 (LCN2) regulates infection response and increases in liver injury. We explored the role of intrahepatic LCN2 in human alcoholic hepatitis (AH) with advanced fibrosis and portal hypertension and in experimental mouse fibrosis. We found hepatic LCN2 expression and serum LCN2 level markedly increased and correlated with disease severity and portal hypertension in patients with AH. In control human livers, LCN2 expressed exclusively in mononuclear cells, while its expression was markedly induced in AH livers, not only in mononuclear cells but also notably in hepatocytes. Lcn2-/- mice were protected from liver fibrosis caused by either ethanol or CCl4 exposure. Microarray analysis revealed downregulation of matrisome, cell cycle and immune related gene sets in Lcn2-/- mice exposed to CCl4, along with decrease in Timp1 and Edn1 expression. Hepatic expression of COL1A1, TIMP1 and key EDN1 system components were elevated in AH patients and correlated with hepatic LCN2 expression. In vitro, recombinant LCN2 induced COL1A1 expression. Overexpression of LCN2 increased HIF1A that in turn mediated EDN1 upregulation. LCN2 contributes to liver fibrosis and portal hypertension in AH and could represent a new therapeutic target.


Assuntos
Colágeno Tipo I/genética , Hepatite Alcoólica/genética , Lipocalina-2/genética , Cirrose Hepática/genética , Animais , Tetracloreto de Carbono/toxicidade , Modelos Animais de Doenças , Etanol/toxicidade , Feminino , Regulação da Expressão Gênica/genética , Hepatite Alcoólica/sangue , Hepatite Alcoólica/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipertensão Portal/sangue , Hipertensão Portal/genética , Hipertensão Portal/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Análise em Microsséries/métodos , Inibidor Tecidual de Metaloproteinase-1/genética
13.
Life Sci ; 260: 118373, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32898530

RESUMO

AIMS: Endoplasmic reticulum stress (ERS) as an emerging factor is involved in insulin resistance (IR), which is the pathological basis of diabetes mellitus. Accumulation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase is associated with IR, but the underlying mechanisms have not been elucidated. This study was to reveal the important role of ADMA in IR and determine whether endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats and hepatocytes. MATERIALS AND METHODS: Diabetic rat model was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg). Phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) was detected to evaluate IR. The protein kinase PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, x-box binding protein-1 (XBP-1) splicing, glucose-regulated protein 78 (GRP78) and C/EBP homologues protein (CHOP) expressions were measured to assess ERS. KEY FINDINGS: Endogenous ADMA content was significantly increased and positively correlated with either IR as evidenced by increased IRS1 at serine and reduced Akt phosphorylation or ERS as indicated by upregulations of PERK and eIF2α phosphorylation, XBP-1 splicing, GRP78 and CHOP expressions in the liver of diabetic rats compared with control rats. Exogenous ADMA directly caused IR and ERS in dose- and time-dependent manners in primary mouse hepatocytes. Pretreatment with ERS inhibitor 4-phenylbutyrate or ADMA antagonist L-arginine not only improved ADMA-associated or -induced hepatic IR but also attenuated ADMA-associated or -induced ERS in diabetic rats or hepatocytes. SIGNIFICANCE: These findings indicate that endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats.


Assuntos
Arginina/análogos & derivados , Diabetes Mellitus Experimental/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Intolerância à Glucose/patologia , Resistência à Insulina , Insulina/metabolismo , Fígado/patologia , Animais , Apoptose , Arginina/toxicidade , Diabetes Mellitus Experimental/induzido quimicamente , Intolerância à Glucose/induzido quimicamente , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
14.
PLoS One ; 15(9): e0239540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966316

RESUMO

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.


Assuntos
Aflatoxina B1/toxicidade , Hepatócitos/efeitos dos fármacos , Ativação Metabólica , Aflatoxina B1/administração & dosagem , Aflatoxina B1/farmacocinética , Animais , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/patologia , Hepatócitos/transplante , Humanos , Técnicas In Vitro , Dose Letal Mediana , Transplante de Fígado , Masculino , Camundongos , Camundongos SCID , RNA Interferente Pequeno/genética , Quimeras de Transplante , Vacúolos/efeitos dos fármacos , Vacúolos/patologia
15.
J Toxicol Sci ; 45(8): 475-492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32741898

RESUMO

By analysis of the data from the Toxicogenomics Database (TG-GATEs), histidine decarboxylase gene (Hdc) was identified as largely and commonly upregulated by three fibrates, clofibrate, fenofibrate, and WY-14,643, which are known to induce hepatocellular hypertrophy and proliferation via stimulation of peroxisome proliferator-activated receptor α (PPARα) in rodents. As histamine has been reported to be involved in the proliferation of liver cells, the present study was conducted to focus on Hdc. Among other genes related to histidine and histamine, the expression of the gene of histamine ammonia lyase (Hal) was exclusively mobilized by the three fibrates. The expression of Hdc, which was usually very low in the liver, was increased with the repeated administration of fibrates, and concomitantly, the constitutive expression of Hal was suppressed. An interpretation is that the formation of urocanic acid from histidine under the normal condition switches to the formation of histamine. The mobilization of gene expression of Hdc and Hal by PPARα agonists could not be reproduced in primary cultured hepatocytes. The Hdc mRNA appeared to be translated to a protein which is processed differently from brain but similarly to gastric mucosa. Surprisingly, the fibrates caused hepatic hypertrophy but no induction of Hdc mRNA at all in mice. These results revealed that the changes in the histidine catabolism by PPARα agonists might be partially, but not directly, involved in the hepatocyte proliferation in rats, and there is a large genetic distance even between rat and mouse.


Assuntos
Clofibrato/efeitos adversos , Bases de Dados Genéticas , Fenofibrato/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Histidina Descarboxilase/metabolismo , Fígado/metabolismo , Fígado/patologia , PPAR alfa/agonistas , Pirimidinas/efeitos adversos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Hepatócitos/patologia , Histidina Descarboxilase/genética , Hipertrofia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
16.
Gene ; 758: 144946, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32649978

RESUMO

Hepatic injury is one of the most challenging diseases in clinical medicine. Hepatic injury is accompanied by hepatocyte apoptosis and leads to hepatic fibrosis and cirrhosis, which may cause liver cancer and increased mortality. Therefore, it is essential to investigate the regulation mechanism and therapeutic strategies for hepatic injury. In the study, the effects of Thymosin ß4 (Tß4) on Long intergenic noncoding RNA-p21 (lincRNA-p21)-mediated liver injury were investigated. Results showed that lincRNA-p21 overexpression promoted hepatocytes apoptosis, which was blocked by Tß4. Besides, Tß4 reversed the levels of cleaved caspase-3 and caspase-9 induced by lincRNA-p21. LincRNA-p21 overexpression also caused the pathological injury and fibrosis in hepatic tissues and increased the levels of fibrosis-related proteins (Collagen I, α-SMA and TIMP-1), and induced hydroxyproline and ALT production. However, Tß4 reversed the effects of overexpression of lincRNA-p21 on hepatic injury and fibrosis. In vitro experiments, after lincRNA-p21 was overexpressed in hepatic stellate cells (HSCs), the proliferation ability and the levels of HSCs markers α-SMA and Desmin were increased. However, Tß4 reversed the effects of lincRNA-p21 on HSCs. Furthermore, the PI3K-AKT-NF-κB pathway was activated by lincRNA-p21, which was then reversed by the Tß4 administration. After the mice treated by insulin-like growth factor-1 (IGF-1) (the activator of PI3K-AKT), the inhibitory effect of Tß4 on activated the PI3K-AKT-NF-κB pathway was abrogated. Besides, IGF-1 abolished the protective effects of Tß4 on hepatic apoptosis and fibrosis induced by lincRNA-p21. Therefore, Tß4 reversed. lincRNA-p21-mediated liver injury through inhibiting PI3K-AKT-NF-κB pathway. Tß4 may be a promising drug for fibrosis therapy.


Assuntos
Apoptose/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Fígado/lesões , RNA Longo não Codificante/genética , Timosina/farmacologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Hepatócitos/patologia , Proteínas I-kappa B/antagonistas & inibidores , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-1/metabolismo
17.
Sci Rep ; 10(1): 10578, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601309

RESUMO

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active ß-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.


Assuntos
Hepatócitos/citologia , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Xenoenxertos , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Esferoides Celulares , alfa-Fetoproteínas/metabolismo
18.
Toxicol Lett ; 333: 49-61, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32726682

RESUMO

Hepatotoxicity induced by Mylabris has been reported in both clinical and animal experiments. Cantharidin (CTD), the main active compound of Mylabris was responsible for the hepatotoxicity, which aroused widespread concern. However, the mechanism of CTD hepatotoxicity remained unclear. In this study, LO2 cells were exposed to two doses of CTD (6.25 and 25 µM) for 12 h, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. The metabolites in LO2 cells were profiled by LC-MS. Partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis were used for screening potential biomarkers. The MetPA software was used for clustering and pathway analysis. Network pharmacology was used to predict the genes acted with potential biomarkers. Compared with the control group, the levels of ALT, AST, and LDH was significantly increased after CTD treatment. A total of 46 potential biomarkers for hepatotoxicity induced by CTD were identified. And downregulated potential biomarkers reflected the inhibitory effects of CTD toxicity on metabolism of LO2. Moreover, CTD-induced liver toxicity of LO2 cells is mainly related to three pathways: cysteine and methionine metabolism; glutathione metabolism; and glycine, serine, and threonine metabolism. Furtherly, the mRNA expression of CES2, DNMT1, NOS1, NOS3, S1PR2, and CES1 screened by network pharmacology were regulated by CTD. These studies provide valuable mechanistic insights into CTD-associated hepatotoxicity that will aid in the development of therapeutic prevention and treatment options for this liver disease.


Assuntos
Cantaridina/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica/métodos , Estresse Oxidativo/efeitos dos fármacos , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cromatografia Líquida/métodos , Relação Dose-Resposta a Droga , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Espectrometria de Massas/métodos , Metabolômica/instrumentação
19.
Proc Natl Acad Sci U S A ; 117(29): 17177-17186, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631996

RESUMO

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.


Assuntos
Ciclo Celular/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout
20.
Biochem Pharmacol ; 180: 114173, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32717228

RESUMO

A large variety of model systems are used in hepatobiliary research. In this review, we aim to provide an overview of established and emerging models for specific research questions. We specifically discuss the value and limitations of these models for research on metabolic associated fatty liver disease (MAFLD), (previously named non-alcoholic fatty liver diseases/non-alcoholic steatohepatitis (NAFLD/NASH)) and cholestasis-related diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). The entire range of models is discussed varying from immortalized cell lines, mature or pluripotent stem cell-based models including organoids/spheroids, to animal models and human ex vivo models such as normothermic machine perfusion of livers and living liver slices. Finally, the pros and cons of each model are discussed as well as the need in the scientific community for continuous innovation in model development to better mimic the human (patho)physiology.


Assuntos
Bile/metabolismo , Modelos Animais de Doenças , Hepatócitos/metabolismo , Cirrose Hepática Biliar/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Linhagem Celular Transformada , Hepatócitos/patologia , Humanos , Fígado/patologia , Cirrose Hepática Biliar/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Técnicas de Cultura de Órgãos
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