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1.
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
2.
Yonsei Med J ; 61(9): 780-788, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32882762

RESUMO

PURPOSE: This research was designed to investigate how miR-542-5p regulates the progression of hyperglycemia and hyperlipoidemia. MATERIALS AND METHODS: An in vivo model with diabetic db/db mice and an in vitro model with forskolin/dexamethasone (FSK/DEX)-induced primary hepatocytes and HepG2 cells were employed in the study. Bioinformatics analysis was conducted to identify the expression of candidate miRNAs in the liver tissues of diabetic and control mice. H&E staining revealed liver morphology in diabetic and control mice. Pyruvate tolerance tests, insulin tolerance tests, and intraperitoneal glucose tolerance test were utilized to assess insulin resistance. ELISA was conducted to evaluate blood glucose and insulin levels. Red oil O staining showed lipid deposition in liver tissues. Luciferase reporter assay was used to depict binding between miR-542-5p and forkhead box O1 (FOXO1). RESULTS: MiR-542-5p expression was under-expressed in the livers of db/db mice. Further in vitro experiments revealed that FSK/DEX, which mimics the effects of glucagon and glucocorticoids, induced cellular glucose production in HepG2 cells and in primary hepatocytes cells. Notably, these changes were reversed by miR-542-5p. We found that transcription factor FOXO1 is a target of miR-542-5p. Further in vivo study indicated that miR-542-5p overexpression decreases FOXO1 expression, thereby reversing increases in blood glucose, blood lipids, and glucose-related enzymes in diabetic db/db mice. In contrast, anti-miR-542-5p exerted an adverse influence on blood glucose and blood lipid metabolism, and its stimulatory effects were significantly inhibited by sh-FOXO1 in normal control mice. CONCLUSION: Collectively, our results indicated that miR-542-5p inhibits hyperglycemia and hyperlipoidemia by targeting FOXO1.


Assuntos
Diabetes Mellitus Tipo 2/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperlipidemias/tratamento farmacológico , MicroRNAs/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Hiperglicemia/metabolismo , Hiperlipidemias/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , MicroRNAs/metabolismo , MicroRNAs/farmacologia
3.
Aquat Toxicol ; 227: 105586, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32882451

RESUMO

Estrogenic effects triggered by androgens have been previously shown in a few studies. Aromatization and direct binding to estrogen receptors (ERs) are the most proposed mechanisms. For example, previously, a modulation of vitellogenin A (VtgA) by testosterone (T), an aromatizable androgen, was reported in brown trout primary hepatocytes. The effect was reversed by an ER antagonist. In this study, using the same model the disruption caused by T and by the non-aromatizable androgen - dihydrotestosterone (DHT), was assessed in selected estrogenic targets. Hepatocytes were exposed (96 h) to six concentrations of each androgen. The estrogenic targets were VtgA, ERα, ERß1 and two zona pellucida genes, ZP2.5 and ZP3a.2. The aromatase CYP19a1 gene and the androgen receptor (AR) were also included. Modulation of estrogenic targets was studied by quantitative real-time PCR and immunohistochemistry, using an HScore system. VtgA and ERα were up-regulated by DHT (1, 10, 100 µM) and T (10, 100 µM). In contrast, ERß1 was down-regulated by DHT (10, 100 µM), and T (100 µM). ZP2.5 mRNA levels were increased by DHT and T (1, 10, 100 µM), while ZP3a.2 was up-regulated by DHT (100 µM) and T (10, 100 µM). Positive correlations were found between VtgA and ERα mRNA levels and ZPs and ERα, after exposure to both androgens. The mRNA levels of CYP19a1 were not changed, while AR expression tended to increase after micromolar DHT exposures. HScores for Vtg and ZPs corroborated the molecular findings. Both androgens triggered estrogen signaling through direct binding to ERs, most probably ERα.


Assuntos
Androgênios/toxicidade , Di-Hidrotestosterona/toxicidade , Estrogênios/metabolismo , Hepatócitos/efeitos dos fármacos , Testosterona/toxicidade , Truta/metabolismo , Poluentes Químicos da Água/toxicidade , Androgênios/metabolismo , Animais , Células Cultivadas , Di-Hidrotestosterona/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios/genética , Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Cultura Primária de Células , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Testosterona/metabolismo , Vitelogeninas/genética , Vitelogeninas/metabolismo , Poluentes Químicos da Água/metabolismo
4.
Int J Biol Sci ; 16(13): 2464-2476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760213

RESUMO

In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.


Assuntos
Biologia Computacional , Infecções por Coronavirus/imunologia , Neoplasias/imunologia , Pneumonia Viral/imunologia , Adulto , Betacoronavirus , Infecções por Coronavirus/complicações , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genótipo , Células Caliciformes/metabolismo , Hepatócitos/metabolismo , Humanos , Sistema Imunitário , Túbulos Renais/embriologia , Masculino , Neoplasias/complicações , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Prognóstico , RNA-Seq , Transdução de Sinais
5.
Int J Nanomedicine ; 15: 4859-4876, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764923

RESUMO

Introduction: CoenzymeQ10 (CoQ10) is a well-known antioxidant and anti-inflammatory agent with cardioprotective properties. However, clinical trials based on its oral administration have failed to provide significant effect on cardiac functionality. The main limitation of CoQ10 is based on its very low oral bioavailability and instability that limit dramatically its effects as a cardioprotective agent. Herein, we loaded CoQ10 in high bioavailable nano-emulsions (NEs) coated with chitosan or chitosan and hyaluronic acid in order to improve its performance. Methods: We tested cardioprotective and hepatoprotective effects of CoQ10-loaded nano-carriers against Doxorubicin and Trastuzumab toxicities in cardiomyocytes and liver cells through analysis of cell viability, lipid peroxidation, expression of leukotrienes, p65/NF-kB and pro-inflammatory cytokines involved in anticancer-induced cardio and hepatotoxicity. Results: Nano-carriers showed high stability and loading ability and increased cell viability both in hepatocytes and cardiomyocytes during anticancer treatments. We observed that these effects are mediated by the inhibition of lipid peroxidation and reduction of the inflammation. CoQ10-loaded nano-emulsions showed also strong anti-inflammatory effects reducing leukotriene B4 and p65/NF-κB expression and Interleukin 1ß and 6 production during anticancer treatments. Discussion: Anthracyclines and Human epidermal growth factor receptor (HER2) inhibitors have shown significant anticancer effects in clinical practice but their use is characterized by cardiotoxicity and hepatotoxicity. Nano-carriers loaded with CoQ10 showed cardio and hepatoprotective properties mediated by reduction of oxidative damages and pro-inflammatory mediators. These results set the stage for preclinical studies of cardio and hepatoprotection in HER2+ breast cancer-bearing mice treated with Doxorubicin and Trastuzumab.


Assuntos
Antraciclinas/efeitos adversos , Fígado/citologia , Miócitos Cardíacos/efeitos dos fármacos , Nanoestruturas/química , Trastuzumab/efeitos adversos , Ubiquinona/análogos & derivados , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Cápsulas , Cardiotônicos/química , Cardiotônicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Feminino , Hepatócitos/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ubiquinona/química , Ubiquinona/farmacologia
6.
Arterioscler Thromb Vasc Biol ; 40(9): 2095-2107, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757647

RESUMO

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.


Assuntos
Doenças da Aorta/prevenção & controle , Apolipoproteína C-III/deficiência , Aterosclerose/prevenção & controle , Doença da Artéria Coronariana/prevenção & controle , Triglicerídeos/sangue , Animais , Animais Geneticamente Modificados , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteína C-III/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Biomarcadores/sangue , HDL-Colesterol/sangue , VLDL-Colesterol/sangue , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Modelos Animais de Doenças , Feminino , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Lipoproteínas IDL/sangue , Fígado/metabolismo , Masculino , Oxirredução , Placa Aterosclerótica , Coelhos
7.
Int J Biol Sci ; 16(13): 2464-2476, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-695855

RESUMO

In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.


Assuntos
Biologia Computacional , Infecções por Coronavirus/imunologia , Neoplasias/imunologia , Pneumonia Viral/imunologia , Adulto , Betacoronavirus , Infecções por Coronavirus/complicações , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genótipo , Células Caliciformes/metabolismo , Hepatócitos/metabolismo , Humanos , Sistema Imunitário , Túbulos Renais/embriologia , Masculino , Neoplasias/complicações , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Prognóstico , RNA-Seq , Transdução de Sinais
8.
Mol Pharmacol ; 98(3): 267-279, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817462

RESUMO

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.


Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , Proteólise
9.
Cardiovasc Ther ; 2020: 9397109, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32821285

RESUMO

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients.


Assuntos
Anti-Inflamatórios/farmacologia , Proteína C-Reativa/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Citocinas/metabolismo , Endotoxemia/tratamento farmacológico , Epigênese Genética/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Quinazolinonas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Proteína C-Reativa/genética , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Células Cultivadas , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Citocinas/genética , Modelos Animais de Doenças , Endotoxemia/genética , Endotoxemia/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas , Componente Amiloide P Sérico/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/metabolismo
10.
PLoS Comput Biol ; 16(7): e1008050, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687525

RESUMO

Machine learning algorithms trained to predict the regulatory activity of nucleic acid sequences have revealed principles of gene regulation and guided genetic variation analysis. While the human genome has been extensively annotated and studied, model organisms have been less explored. Model organism genomes offer both additional training sequences and unique annotations describing tissue and cell states unavailable in humans. Here, we develop a strategy to train deep convolutional neural networks simultaneously on multiple genomes and apply it to learn sequence predictors for large compendia of human and mouse data. Training on both genomes improves gene expression prediction accuracy on held out and variant sequences. We further demonstrate a novel and powerful approach to apply mouse regulatory models to analyze human genetic variants associated with molecular phenotypes and disease. Together these techniques unleash thousands of non-human epigenetic and transcriptional profiles toward more effective investigation of how gene regulation affects human disease.


Assuntos
Regulação da Expressão Gênica , Variação Genética , Aprendizado de Máquina , Algoritmos , Animais , Biologia Computacional , Bases de Dados Genéticas , Epigenômica , Genoma Humano , Genômica , Hepatócitos/metabolismo , Humanos , Camundongos , Modelos Genéticos , Modelos Estatísticos , Mutação , Redes Neurais de Computação , Locos de Características Quantitativas , Análise de Sequência de DNA , Software , Especificidade da Espécie
11.
Nat Commun ; 11(1): 3360, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620763

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate ß-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.


Assuntos
Ácidos Graxos/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Fígado/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adulto , Idoso , Animais , Conjuntos de Dados como Assunto , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Nanopartículas/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Oxirredução/efeitos dos fármacos , Cultura Primária de Células , RNA Interferente Pequeno/administração & dosagem , RNA-Seq
12.
PLoS One ; 15(7): e0235745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645073

RESUMO

Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Modelos Biológicos , Cultura Primária de Células/métodos , Esferoides Celulares , Animais , Antibacterianos/toxicidade , Anti-Inflamatórios não Esteroides/toxicidade , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/metabolismo , Imunidade Inata , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Esferoides Celulares/citologia , Esferoides Celulares/imunologia , Esferoides Celulares/metabolismo
13.
Environ Toxicol ; 35(11): 1202-1211, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32621570

RESUMO

Polybrominated diphenyl ethers (PBDEs) are ubiquitous and prolific contaminant in both the abiotic and biotic environment because of the wide industrial applications of these chemicals. In the present study, the effects of 2,2',4,4'-tetrabrominateddiphenyl ether (BDE-47) and 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153) exposure on the induction of hepatic oxidative stress, DNA damage, and the expression of apoptosis-related genes in adult zebrafish were investigated. The activities of antioxidant enzymes, such as catalase and superoxide dimutase, significantly increased when adult zebrafish was exposed to various concentrations of BDE-47 and BDE-153 for 7 and 15 days. BDE-47 and BDE-153 elicited significant alterations in zebrafish 7-Ethoxyresorufin-O-deethylase activity at 3, 7, or 15 days of exposure. In addition, the significant increase in comet assay parameters of zebrafish hepatocytes in a concentration-dependent manner indicated BDE-47 and BDE-153 induced DNA damage, probably due to observed oxidative stress. Furthermore, a monotonically upregulation of p53 and Caspase3, which are apoptotic-regulated genes, and decreased expression ratio of the anti-apoptotic B-cell lymphoma/leukaemia-2 and Bcl2-associated X protein genes for all BDE-47 and BDE-153 treatments at 7 and 15 days indicated apoptosis induction in zebrafish liver. Our findings help elucidate the mechanisms of BDE-47- and BDE-153-induced toxicity in zebrafish hepatocytes.


Assuntos
Éteres Difenil Halogenados/toxicidade , Bifenil Polibromatos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3 , Ensaio Cometa , Citocromo P-450 CYP1A1/metabolismo , Dano ao DNA , Éteres Difenil Halogenados/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/metabolismo
14.
Metabolism ; 110: 154306, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32621820

RESUMO

BACKGROUND: Iron is finely regulated due to its vital roles in organisms and the peroxidase reactivity if excess. Solute Carrier Family 46 Member 1 (SLC46A1), also named PCFT or HCP1, is the main importer of heme­iron in the intestine, but has a high abundance in the liver. Since the liver has a central role in iron homeostasis, whether SLC46A1 regulates hepatic iron metabolism is of interest to be identified. METHODS: The recombinant adeno-associated virus vectors were used to hepatic-specifically inhibit SLC46A1 expression to observe its effects on hepatic iron metabolism. Then the abilities of SLC46A1 in importing heme and folate, and consequent alterations of iron content in hepatocytes were determined. Furthermore, effects of iron on SLC46A1 expression were investigated both in vitro and in vivo. RESULTS: The hepatocyte-specific inhibition of SLC46A1 decreases iron content in the liver and increases iron content in serum. Expressions of iron-related molecules, transferrin receptor 1, hepcidin and ferroportin, are correspondingly altered. Interestingly, free heme concentration in serum is increased, indicating a decreased import of heme by the liver. In hepatocytes, SLC46A1 is capable of importing hemin, increasing intracellular iron content. The import of hemin by SLC46A1 is unaffected by its other substrate, folate. Instead, hemin treatment decreases SLC46A1 expression, reducing the import of folate. In addition, SLC46A1 itself shows to be iron-responsive both in vivo and in vitro, making it available for regulating iron metabolism. CONCLUSION: The results elucidate that SLC46A1 regulates iron metabolism in the liver through a folate-independent manner of importing heme. The iron-responsive characters of SLC46A1 give us a new clue to link heme or iron overload with folate deficiency diseases.


Assuntos
Heme/metabolismo , Ferro/metabolismo , Fígado/metabolismo , Transportador de Folato Acoplado a Próton/fisiologia , Animais , Células Cultivadas , Hemina/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transportador de Folato Acoplado a Próton/antagonistas & inibidores
15.
Arterioscler Thromb Vasc Biol ; 40(9): 2108-2113, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640904

RESUMO

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.


Assuntos
Angiotensina II/metabolismo , Angiotensinogênio/metabolismo , Aterosclerose/metabolismo , Pressão Sanguínea , Hepatócitos/metabolismo , Hipertensão/metabolismo , Renina/metabolismo , Substituição de Aminoácidos , Angiotensinogênio/deficiência , Angiotensinogênio/genética , Animais , Aterosclerose/genética , Aterosclerose/patologia , Modelos Animais de Doenças , Feminino , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Camundongos Knockout , Placa Aterosclerótica , Receptores de LDL/genética , Receptores de LDL/metabolismo , Especificidade da Espécie
16.
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
17.
Anticancer Res ; 40(8): 4491-4504, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727779

RESUMO

BACKGROUND: Peroxiredoxin II (PRDX2) performs unique roles in cells. It can reduce peroxides through cysteine residues, and helps prevent the effects of oxidative stress on cells. It is closely related to the occurrence and development of various diseases, especially alcoholic liver injury and even liver cancer. The metabolism of alcohol in hepatocytes leads to the increase in the levels of reactive oxygen species (ROS), oxidative stress, injury, and apoptosis. Therefore, this study focused on the investigating the protection conferred by PRDX2 against alcohol-induced apoptosis of hepatocytes. MATERIALS AND METHODS: PRDX2 inhibition of alcohol-induced apoptosis in L02 hepatocytes was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescence microscopy, flow cytometry, western blotting and hematoxylin and eosin staining. RESULTS: The results showed that the levels of reactive oxygen species, protein kinase B, ß-catenin, B-cell lymphoma-2 (BCL2), BCL-XL, BCL2-associated X, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in PRDX2-silenced cells were increased significantly after the treatment of cells with ethanol. Similar results were obtained in an in vivo Prdx2-knockout mouse model of alcoholic liver injury. Therefore, PRDX2 may regulate the phosphorylation of the AKT signal protein by eliminating reactive oxygen species from cells, and it inhibits the downstream mitochondria-dependent apoptosis pathway, and, thereby, the apoptosis of cells. CONCLUSION: Thus, PRDX2 may be a potential molecular target for the prevention and treatment of alcoholic liver injury.


Assuntos
Etanol/efeitos adversos , Hepatócitos/citologia , Peroxirredoxinas/genética , Transdução de Sinais , Apoptose , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo
18.
Am J Physiol Cell Physiol ; 319(3): C552-C560, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32697600

RESUMO

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-ß1 signaling. With TGF-ß1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.


Assuntos
Hepatócitos/metabolismo , Microfluídica , Animais , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura/métodos , Microfluídica/métodos , Fenótipo , Esferoides Celulares/metabolismo
19.
PLoS One ; 15(6): e0233767, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32531779

RESUMO

Functional and anatomical connection between the liver and the spleen is most clearly manifested in various pathological conditions of the liver (cirrhosis, hepatitis). The mechanisms of the interaction between the two organs are still poorly understood, as there have been practically no studies on the influence exerted by the spleen on the normal liver. Mature male Sprague-Dawley rats of 250-260 g body weight, 3 months old, were splenectomized. The highest numbers of Ki67+ hepatocytes in the liver of splenectomized rats were observed at 24 h after the surgery, simultaneously with the highest index of Ki67-positive hepatocytes. After surgical removal of the spleen, expression of certain genes in the liver tissues increased. A number of genes were upregulated in the liver at a single time point of 24 h, including Ccne1, Egf, Tnfa, Il6, Hgf, Met, Tgfb1r2 and Nos2. The expression of Ccnd1, Tgfb1, Tgfb1r1 and Il10 in the liver was upregulated over the course of 3 days after splenectomy. Monitoring of the liver macrophage populations in splenectomized animals revealed a statistically significant increase in the proportion of CD68-positive cells in the liver (as compared with sham-operated controls) detectable at 24 h and 48 h after the surgery. The difference in the liver content of CD68-positive cells between splenectomized and sham-operated animals evened out by day 3 after the surgery. No alterations in the liver content of CD163-positive cells were observed in the experiments. A decrease in the proportion of CD206-positive liver macrophages was observed at 48 h after splenectomy. The splenectomy-induced hepatocyte proliferation is described by us for the first time. Mechanistically, the effect is apparently induced by the removal of spleen as a major source of Tgfb1 (hepatocyte growth inhibitor) and subsequently supported by activation of proliferation factor-encoding genes in the liver.


Assuntos
Proliferação de Células , Hepatócitos/metabolismo , Esplenectomia/efeitos adversos , Animais , Hepatócitos/fisiologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Masculino , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
20.
Life Sci ; 257: 117889, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32502541

RESUMO

PURPOSE: Gestational diabetes mellitus (GDM) has many adverse effects on offspring, such as abnormal glycolipid metabolism, obesity, insulin resistance, mental retardation, schizophrenia and so on. METHODS: We established a GDM rat model by injecting 1% streptozotocin associated with a high-fat diet one week before pregnancy, and offspring rats were sacrificed at 8 weeks of age to obtain liver tissue for study. We used hematoxylin-eosin (HE) staining to observe liver morphological changes, Tunel staining for hepatocyte apoptosis, transmission electron microscope for liver ultrastructure, and western blot for protein expression in liver tissue. RESULTS: Compared with normal offspring rats, hepatocytes of GDM offspring rats showed obvious edema, liver organ index increased, and hepatocyte apoptosis and autophagosome in the liver were significantly increased; Bax, cleaved-caspase3/caspase3, LCII, Beclin 1, P-IKBα/IKBα and P-p65/p6 protein expression in the liver were significantly increased; Bcl2, p62 and PPARγ protein expression in the liver were significantly decreased. Tau prevented the GDM-related effects in the offspring: Tau decreased hepatocyte edema (or even disappears), liver organ index, hepatocyte apoptosis and the number of autophagosomes in the liver. In addition, Tau also decreased Bax, cleaved-caspase3/caspase3, LCII, Beclin 1, P-IKBα/IKBα and P-p65/p6 protein expression, and increased Bcl2, p62 and PPARγ protein expression in the liver of GDM offspring rats. CONCLUSION: Taurine should be considered as a potential gestational nutritional supplement to prevent liver damage in GDM offspring.


Assuntos
Diabetes Gestacional/tratamento farmacológico , Taurina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Diabetes Mellitus Experimental , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Fígado/efeitos dos fármacos , Masculino , Obesidade/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Ratos , Ratos Sprague-Dawley , Taurina/metabolismo
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