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1.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205146

RESUMO

The objective of the present study was to investigate the proximate composition, antiradical properties and hepatoprotective activity of three species of shellfish, Corbicula japonica, Spisula sachalinensis, and Anadara broughtonii, from the coastal areas of Far East Russia. Biologically active peptides such as taurine (3.74 g/100 g protein) and ornithine (2.12 g/100 g protein) have been found in the tissues of A. broughtonii. C. japonica contains a high amount of ornithine (5.57 g/100 g protein) and taurine (0.85 g/100 g protein). The maximum DPPH and ABTS radical scavenging activity (36.0 µg ascorbic acid/g protein and 0.68 µmol/Trolox equiv/g protein, respectively) was determined for the tissue of C. japonica. The protein and peptide molecular weight distribution of the shellfish tissue water extracts was investigated using HPLC. It was found that the amount of low molecular weight proteins and peptides were significantly and positively correlated with radical scavenging activity (Pearson's correlation coefficient = 0.96), while the amount of high molecular weight proteins negatively correlated with radical scavenging activity (Pearson's correlation coefficient = -0.86). Hepatoprotective activity, measured by the survival rate of HepG2 hepatocytes after cotreatment with t-BHP, was detected for C. japonica. The highest protection (95.3 ± 2.4%) was achieved by the cold water extract of C. japonica at the concentration of 200 mg/mL. Moreover, oral administration of hot water extract of C. japonica to rats before the treatment with CCl4 exhibited a markedly protective effect by lowering serum levels of ALT and AST, inhibiting the changes in biochemical parameters of functional state of rat liver, including MDA, SOD, GSH and GST.


Assuntos
Antioxidantes/farmacologia , Arcidae/química , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Corbicula/química , Hepatócitos/citologia , Frutos do Mar/análise , Spisula/química , terc-Butil Hidroperóxido/efeitos adversos , Administração Oral , Animais , Antioxidantes/química , Tetracloreto de Carbono/efeitos adversos , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Peso Molecular , Ornitina/isolamento & purificação , Ratos , Federação Russa , Frutos do Mar/classificação , Taurina/isolamento & purificação
2.
Cells ; 10(5)2021 05 04.
Artigo em Inglês | MEDLINE | ID: covidwho-1223957

RESUMO

Liver injury in COVID-19 patients has progressively emerged, even in those without a history of liver disease, yet the mechanism of liver pathogenicity is still controversial. COVID-19 is frequently associated with increased serum ferritin levels, and hyperferritinemia was shown to correlate with illness severity. The liver is the major site for iron storage, and conditions of iron overload have been established to have a pathogenic role in development of liver diseases. We presented here six patients who developed severe COVID-19, with biochemical evidence of liver failure. Three cases were survived patients, who underwent liver biopsy; the other three were deceased patients, who were autopsied. None of the patients suffered underlying liver pathologies. Histopathological and ultrastructural analyses were performed. The most striking finding we demonstrated in all patients was iron accumulation into hepatocytes, associated with degenerative changes. Abundant ferritin particles were found enclosed in siderosomes, and large aggregates of hemosiderin were found, often in close contact with damaged mitochondria. Iron-caused oxidative stress may be responsible for mitochondria metabolic dysfunction. In agreement with this, association between mitochondria and lipid droplets was also found. Overall, our data suggest that hepatic iron overload could be the pathogenic trigger of liver injury associated to COVID-19.


Assuntos
COVID-19/diagnóstico , Sobrecarga de Ferro/etiologia , Falência Hepática/etiologia , Fígado/patologia , Índice de Gravidade de Doença , Adulto , Idoso , Antivirais , Biópsia , COVID-19/complicações , COVID-19/mortalidade , COVID-19/terapia , Feminino , Ferritinas/análise , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Ferro/análise , Ferro/metabolismo , Sobrecarga de Ferro/mortalidade , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/terapia , Fígado/citologia , Fígado/metabolismo , Falência Hepática/mortalidade , Falência Hepática/patologia , Falência Hepática/terapia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Respiração com Pressão Positiva , SARS-CoV-2/isolamento & purificação
3.
Molecules ; 26(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063700

RESUMO

Momordica charantia is a popular vegetable associated with effective complementary and alternative diabetes management in some parts of the world. However, the molecular mechanism is less commonly investigated. In this study, we investigated the association between a major cucurbitane triterpenoid isolated from M. charantia, 3ß,7ß,25-trihydroxycucurbita-5,23(E)-dien-19-al (THCB) and peroxisome proliferator activated receptor gamma (PPARγ) activation and its related activities using cell culture and molecular biology techniques. In this study, we report on both M. charantia fruit crude extract and THCB in driving the luciferase activity of Peroxisome Proliferator Response Element, associated with PPARγ activation. Other than that, THCB also induced adipocyte differentiation at far less intensity as compared to the full agonist rosiglitazone. In conjunction, THCB treatment on adipocytes also resulted in upregulation of PPAR gamma target genes expression; AP2, adiponectin, LPL and CD34 at a lower magnitude compared to rosiglitazone's induction. THCB also induced glucose uptake into muscle cells and the mechanism is via Glut4 translocation to the cell membrane. In conclusion, THCB acts as one of the many components in M. charantia to induce hypoglycaemic effect by acting as PPARγ ligand and inducing glucose uptake activity in the muscles by means of Glut4 translocation.


Assuntos
Momordica/química , PPAR gama/metabolismo , Triterpenos/química , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Glucose/metabolismo , Hepatócitos/citologia , Hipoglicemia/tratamento farmacológico , Insulina/química , Ligantes , Camundongos , Células Musculares/citologia , Domínios Proteicos , Rosiglitazona/farmacologia , Triterpenos/farmacologia
4.
Cells ; 10(5)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064487

RESUMO

Liver injury in COVID-19 patients has progressively emerged, even in those without a history of liver disease, yet the mechanism of liver pathogenicity is still controversial. COVID-19 is frequently associated with increased serum ferritin levels, and hyperferritinemia was shown to correlate with illness severity. The liver is the major site for iron storage, and conditions of iron overload have been established to have a pathogenic role in development of liver diseases. We presented here six patients who developed severe COVID-19, with biochemical evidence of liver failure. Three cases were survived patients, who underwent liver biopsy; the other three were deceased patients, who were autopsied. None of the patients suffered underlying liver pathologies. Histopathological and ultrastructural analyses were performed. The most striking finding we demonstrated in all patients was iron accumulation into hepatocytes, associated with degenerative changes. Abundant ferritin particles were found enclosed in siderosomes, and large aggregates of hemosiderin were found, often in close contact with damaged mitochondria. Iron-caused oxidative stress may be responsible for mitochondria metabolic dysfunction. In agreement with this, association between mitochondria and lipid droplets was also found. Overall, our data suggest that hepatic iron overload could be the pathogenic trigger of liver injury associated to COVID-19.


Assuntos
COVID-19/diagnóstico , Sobrecarga de Ferro/etiologia , Falência Hepática/etiologia , Fígado/patologia , Índice de Gravidade de Doença , Adulto , Idoso , Antivirais , Biópsia , COVID-19/complicações , COVID-19/mortalidade , COVID-19/terapia , Feminino , Ferritinas/análise , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Ferro/análise , Ferro/metabolismo , Sobrecarga de Ferro/mortalidade , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/terapia , Fígado/citologia , Fígado/metabolismo , Falência Hepática/mortalidade , Falência Hepática/patologia , Falência Hepática/terapia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Respiração com Pressão Positiva , SARS-CoV-2/isolamento & purificação
5.
Biosensors (Basel) ; 11(5)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069382

RESUMO

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.


Assuntos
Reatores Biológicos , Hepatócitos/citologia , Sobrevivência Celular , Células Cultivadas , Desenvolvimento de Medicamentos/métodos , Preparações Farmacêuticas
6.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071368

RESUMO

Alpha-1-antitrypsin (AAT) and fibrinogen are secretory acute phase reactant proteins. Circulating AAT and fibrinogen are synthesized exclusively in the liver. Mutations in the encoding genes result in conformational abnormalities of the two molecules that aggregate within the rough endoplasmic reticulum (RER) instead of being regularly exported. That results in AAT-deficiency (AATD) and in hereditary hypofibrinogenemia with hepatic storage (HHHS). The association of plasma deficiency and liver storage identifies a new group of pathologies: endoplasmic reticulum storage disease (ERSD).


Assuntos
Afibrinogenemia/metabolismo , Retículo Endoplasmático/metabolismo , Fígado/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Afibrinogenemia/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Macrófagos do Fígado/metabolismo , Macrófagos do Fígado/ultraestrutura , Fígado/citologia , Microscopia Eletrônica de Transmissão , Mutação , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/genética
7.
FASEB J ; 35(6): e21648, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33993565

RESUMO

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.


Assuntos
Encéfalo/metabolismo , Hepatócitos/metabolismo , Serotonina/biossíntese , Triptofano Hidroxilase/metabolismo , Animais , Encéfalo/citologia , Hepatócitos/citologia , Camundongos
8.
J Med Chem ; 64(10): 6838-6855, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33950677

RESUMO

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.


Assuntos
Morfolinas/química , Nucleotídeos/química , RNA Interferente Pequeno/metabolismo , Acetilgalactosamina/química , Animais , Células Cultivadas , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos/síntese química , Nucleotídeos/metabolismo , Pré-Albumina/antagonistas & inibidores , Pré-Albumina/genética , Pré-Albumina/metabolismo , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química
9.
Nat Commun ; 12(1): 2858, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001902

RESUMO

Tissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues.


Assuntos
Rim/metabolismo , Fígado/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Miocárdio/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Contagem de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Rim/citologia , Fígado/citologia , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas Analíticas Microfluídicas/instrumentação , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Reprodutibilidade dos Testes
10.
J Biosci Bioeng ; 132(1): 71-80, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33895082

RESUMO

Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6 nM-16 µM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.


Assuntos
Membrana Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Glicolipídeos/química , Trealose/química , Trealose/farmacologia , Animais , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos
11.
J Med Chem ; 64(8): 5037-5048, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33848153

RESUMO

Propionic acidemia (PA) and methylmalonic acidemia (MMA) are rare autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, caused by a deficiency in the enzymes P-CoA carboxylase and methylmalonyl-CoA (M-CoA) mutase, respectively. PA and MMA are classified as intoxication-type inborn errors of metabolism because the intramitochondrial accumulation of P-CoA, M-CoA, and other metabolites results in secondary inhibition of multiple pathways of intermediary metabolism, leading to organ dysfunction and failure. Herein, we describe the structure-activity relationships of a series of short-chain carboxylic acids which reduce disease-related metabolites in PA and MMA primary hepatocyte disease models. These studies culminated in the identification of 2,2-dimethylbutanoic acid (10, HST5040) as a clinical candidate for the treatment of PA and MMA. Additionally, we describe the in vitro and in vivo absorption, distribution, metabolism, and excretion profile of HST5040, data from preclinical studies, and the synthesis of the sodium salt of HST5040 for clinical trials.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Butiratos/uso terapêutico , Acidemia Propiônica/tratamento farmacológico , Acil Coenzima A/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Área Sob a Curva , Butiratos/química , Butiratos/metabolismo , Células Cultivadas , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Camundongos , Modelos Biológicos , Acidemia Propiônica/patologia , Curva ROC , Ratos , Relação Estrutura-Atividade
12.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33922101

RESUMO

3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-ß1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-ß1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).


Assuntos
Biomarcadores/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Macrófagos do Fígado/metabolismo , Cirrose Hepática/metabolismo , Análise de Célula Única/métodos , Fator de Crescimento Transformador beta1/farmacologia , Técnicas de Cultura de Células , Proliferação de Células , Regulação da Expressão Gênica , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Macrófagos do Fígado/citologia , Macrófagos do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Prognóstico
13.
Front Immunol ; 12: 555095, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746941

RESUMO

Hepatocytes compose up to 80% of the total liver and have been indicated as important players in the induction of immunologic tolerance in this organ. We show that hepatocytes possess the molecular machinery required for the cross-presentation of extracellular antigens. Using a derivative of the model antigen ovalbumin (OVA) covalently modified with a polymer containing multiple N-acetylgalactosamine residues (pGal-OVA) that enhance extracellular antigen uptake by mimicking the glycome of apoptotic debris, we show efficient hepatocyte-dependent induction of cross-tolerance of both adoptively transferred OT-I cells and endogenous OVA-specific CD8+ T lymphocytes, for example inducing tolerance to OVA-expressing skin transplants. Our study confirms that hepatocytes are capable of inducing peripheral tolerogenesis and provides proof of concept that they may be a valuable candidate for in vivo targeted tolerogenic treatments.


Assuntos
Acetilgalactosamina/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Hepatócitos/imunologia , Tolerância Imunológica/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transferência Adotiva/métodos , Animais , Apresentação do Antígeno/imunologia , Células Cultivadas , Hepatócitos/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Transplante de Pele/métodos , Solubilidade , Proteínas de Transporte Vesicular/imunologia , Proteínas de Transporte Vesicular/metabolismo
14.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-1109085

RESUMO

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Ocludina/genética , Ocludina/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/análogos & derivados , Cultura Primária de Células , Serina Endopeptidases/genética
15.
FASEB J ; 35(4): e21471, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33683737

RESUMO

Acute liver failure constitutes a devastating condition that needs novel cell and molecular therapies. To elicit synergisms in cell types of therapeutic interest, we studied hepatocytes and liver sinusoidal endothelial in mice with acetaminophen-induced acute liver failure. The context of regenerative signals was examined by transplants in peritoneal cavity because it possesses considerable capacity and allows soluble signals to enter the systemic circulation. Whereas transplanted hepatocytes and liver sinusoidal endothelial cells engrafted in peritoneal cavity, only the former could rescue mice in liver failure by improving injury outcomes, activating hepatic DNA damage repair, and inducing liver regeneration. The cytokines secreted by donor hepatocytes or liver sinusoidal endothelial cells differed and in hepatocytes from mice undergoing acetaminophen toxicity major cytokines were even rendered deficient (eg, G-CSF, VEGF, and others). Significantly, recapitulating hepatotoxicity-related DNA damage response in cultured cells identified impairments in ATM and JAK/STAT3 intersections since replacing cytokines produced less from injured hepatocytes restored these pathways to avoid acetaminophen hepatotoxicity. Similarly, hepatocyte transplantation in acute liver failure restored ATM and JAK/STAT3 pathways to advance DNA damage/repair and liver regeneration. The unexpected identification of novel hepatic G-CSF receptor expression following injury allowed paradigmatic studies of G-CSF supplementation to confirm the centrality of this paracrine ATM and STAT3 intersection. Remarkably, DNA damage/repair and hepatic regeneration directed by G-CSF concerned rebalancing of regulatory gene networks overseeing inflammation, metabolism, and cell viability. We conclude that healthy donor hepatocytes offer templates for generating specialized cell types to replace metabolic functions and regenerative factors in liver failure.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Hepatócitos/citologia , Falência Hepática Aguda/terapia , Regeneração Hepática/efeitos dos fármacos , Acetaminofen/farmacologia , Animais , Sobrevivência Celular/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Humanos , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Regeneração Hepática/fisiologia , Fator de Transcrição STAT3/metabolismo
16.
FASEB J ; 35(5): e21469, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33788981

RESUMO

Mycotoxins are toxic secondary metabolites produced by food-contaminating fungi, which lead to global epigenetic changes and cause toxicity to both farm animals and humans. However, whether mycotoxins induce gene-specific epigenetic alterations associated with inducible downstream gene expression is unclear as are the underlying regulatory mechanisms. Here, we found that T-2 toxin and its deacetylated metabolites but not deoxynivalenol (DON) or other representative mycotoxins highly induced the expression of cytochrome P450 1A4 (CYP1A4) in both Leghorn male hepatoma (LMH) cells and chicken primary hepatocytes, and this effect was related to the regulation of both aryl hydrocarbon receptor (AhR) and DNA methylation. We used methylation-sensitive restriction enzyme digestion-qPCR (MSRE-qPCR) and chromatin immunoprecipitation (ChIP) assays and found that the binding of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) to highly methylated CpG island 3-2 at the enhancer of CYP1A4 was accompanied by the recruitment of the repressive histone modification marker H3K27me3, inducing a silent state. In turn, T-2 toxin stimulation enriched the binding of AhR to demethylated CpG island 3-2, which facilitated p300 and H3K9ac recruitment and ultimately generated an activated chromatin structure at the enhancer by increasing the active histone modification markers, including H3K4me3, H3K27ac, and H3K14ac. Interestingly, T-2 toxin-induced AhR activation also facilitated RNA polymerase II binding to CpG island 2, which may form a transcriptionally active chromatin structure at the promoter and ultimately transactivate CYP1A4. Our findings provide novel insights into the epigenetic regulation of T-2 toxin-induced gene expression.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas Aviárias/metabolismo , Carcinoma Hepatocelular/patologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Toxina T-2/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Proteínas Aviárias/genética , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Galinhas , Ilhas de CpG , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Transcrição Genética
17.
Biochem Biophys Res Commun ; 552: 150-156, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33744763

RESUMO

Hepatocyte apoptosis is a crucial factor affecting liver quality in brain-dead donors. The identification of key molecular proteins involved in brain-death (BD)-induced hepatocyte apoptosis may help determine an effective method for improving the quality of livers from brain-dead donors. In this study, we used in vivo and in vitro models to investigate the role of chitinase-3-like protein 1 (CHI3L1) in promoting liver cell apoptosis after BD. Chitin was used to inhibit CHI3L1 in a rat model of BD. Macrophage polarization of THP-1 cells and hypoxia/reoxygenation (H/R) of LO-2 cells were used to mimic BD-induced cell stress in liver. We found that CHI3L1 played a vital role in promoting liver cell apoptosis. Six hours after BD, CHI3L1 expression was significantly upregulated in liver macrophages and was associated with BD-induced M1 polarization of these cells. In liver cells cultured under H/R conditions, recombinant CHI3L1 activated the protease-activated receptor 2 (PAR2)/c-June N-terminal kinase (JNK) apoptotic pathway and aggravated apoptosis. Compared with the control group, chitin particles inhibited the expression of CHI3L1 in the liver of brain dead rats, thereby reducing activation of the hepatocyte surface receptor, PAR2, and its downstream JNK/caspase-3 signaling pathway, ultimately reducing hepatocyte apoptosis. In conclusion, our results indicate that CHI3L1 relies on a PAR2/JNK-mediated mechanism to promote BD-induced hepatocyte apoptosis.


Assuntos
Apoptose/genética , Morte Encefálica/fisiopatologia , Caspase 3/genética , Proteína 1 Semelhante à Quitinase-3/genética , Hepatócitos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Receptor PAR-2/genética , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Quitina/farmacologia , Proteína 1 Semelhante à Quitinase-3/metabolismo , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Interferência de RNA , Ratos Sprague-Dawley , Receptor PAR-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células THP-1
18.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33641565

RESUMO

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peróxido de Hidrogênio/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Ocludina/genética , Ocludina/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/análogos & derivados , Cultura Primária de Células , Serina Endopeptidases/genética
19.
Biomed Res Int ; 2021: 8621464, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33542926

RESUMO

In addition to serving as the building blocks for protein synthesis, amino acids can be used as an energy source, through catabolism. The transamination, oxidative deamination, and decarboxylation processes that occur during amino acid catabolism are catalyzed by specific enzymes, including aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC); however, the overall molecular mechanisms through which amino acid catabolism occurs remain largely unknown. To examine the role of mechanistic target of rapamycin complex 1 (mTORC1) on amino acid catabolism, mTORC1 was inactivated by rapamycin or shRNA targeting Raptor, versus activated by overexpressing Rheb or amino acids in human hepatocytes. The expression of amino acid catabolic genes and related transcription factor was investigated by RT/real-time PCR and western blot analysis. A few types of amino acid metabolite were examined by ELISA and HPLC analysis. The data showed that inactivated mTORC1 resulted in inhibition of NF-κB and the expression of AST, GDH, GAD, and ODC, whereas activated mTORC1 enhanced NF-κB activation and the expression levels of the catabolism-associated genes. Further, inhibition of NF-κB reduced the expression levels of AST, GDH, GAD, and ODC. mTORC1 upregulated NF-κB activation and the expression of AST and ODC in response to glutamate and ornithine treatments, whereas rapamycin inhibited the utilization of glutamate and ornithine in hepatocytes. Taken together, these results indicated that the mTORC1/NF-κB axis modulates the rate of amino acid catabolism by regulating the expression of key catabolic enzymes in hepatocytes.


Assuntos
Aminoácidos/metabolismo , Hepatócitos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Antibióticos Antineoplásicos/farmacologia , Células Cultivadas , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Ornitina Descarboxilase/metabolismo , Transdução de Sinais , Sirolimo/farmacologia
20.
PLoS One ; 16(2): e0244070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33556073

RESUMO

A major bottleneck in the study of human liver physiology is the provision of stable liver tissue in sufficient quantity. As a result, current approaches to modelling human drug efficacy and toxicity rely heavily on immortalized human and animal cell lines. These models are informative but do possess significant drawbacks. To address the issues presented by those models, researchers have turned to pluripotent stem cells (PSCs). PSCs can be generated from defined genetic backgrounds, are scalable, and capable of differentiation to all the cell types found in the human body, representing an attractive source of somatic cells for in vitro and in vivo endeavours. Although unlimited numbers of somatic cell types can be generated in vitro, their maturation still remains problematic. In order to develop high fidelity PSC-derived liver tissue, it is necessary to better understand the cell microenvironment in vitro including key elements of liver physiology. In vivo a major driver of zonated liver function is the oxygen gradient that exists from periportal to pericentral regions. In this paper, we demonstrate how cell culture conditions for PSC-derived liver sphere systems can be optimised to recapitulate physiologically relevant oxygen gradients by using mathematical modelling. The mathematical model incorporates some often-understated features and mechanisms of traditional spheroid systems such as cell-specific oxygen uptake, media volume, spheroid size, and well dimensions that can lead to a spatially heterogeneous distribution of oxygen. This mathematical modelling approach allows for the calibration and identification of culture conditions required to generate physiologically realistic function within the microtissue through recapitulation of the in vivo microenvironment.


Assuntos
Hepatócitos/metabolismo , Fígado/metabolismo , Oxigênio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Hepatócitos/citologia , Humanos , Fígado/citologia , Modelos Teóricos , Células-Tronco Pluripotentes/citologia
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