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1.
Zhongguo Zhong Yao Za Zhi ; 44(17): 3780-3785, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31602953

RESUMO

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.


Assuntos
Cálculos Biliares/química , Hepatócitos/citologia , Soro/química , Animais , Apoptose , Bovinos , Células Cultivadas , Fígado Gorduroso , Frutose , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Peroxidação de Lipídeos , Fígado , Medicina Tradicional Chinesa , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos
2.
Nature ; 574(7779): 538-542, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645727

RESUMO

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.


Assuntos
Células Clonais/citologia , Células Clonais/patologia , Fibrose/genética , Fibrose/patologia , Fígado/citologia , Fígado/metabolismo , Mutação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Filogenia , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/patologia
3.
Postepy Biochem ; 65(3): 193-201, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643166

RESUMO

Liver diseases that lead to its failure are one of the most frequent causes of death worldwide. Taking into account liver's complexity, there are no drug for acute or acute on chronic liver failure treatment. So far the only effective therapy is the liver transplantation. Unfortunately donor shortage is a main problem of this therapy. Due to this fact scientists have been looking for a new alternatives. The most promising are cell transplantation and bioartificial support systems. Without doubt hepatocytes are the best source of cells to use. But isolated human hepatocytes dedifferentiate very quickly and lose their functions ex vivo. Therefore, the new sources of cells, which could replace hepatocytes, are highly sought after. It is believed that, in order to help patients suffering from liver disease, the approach to solve this problem should be considered on different levels.


Assuntos
Doença Hepática Terminal/terapia , Hepatócitos/citologia , Hepatócitos/transplante , Fígado Artificial , Doença Hepática Terminal/patologia , Doença Hepática Terminal/fisiopatologia , Hepatócitos/patologia , Humanos
4.
Int J Nanomedicine ; 14: 6035-6060, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534335

RESUMO

Background: The clearance of nanomaterials (NMs) from the liver is essential for clinical safety, and their hepatic clearance is primarily determined by the co-disposition process of various types of hepatic cells. Studies of this process and the subsequent clearance routes are urgently needed for organic NMs, which are used as drug carriers more commonly than the inorganic ones. Materials and methods: In this study, the co-disposition of chitosan-based nanoparticles (CsNps) by macrophages and hepatocytes at both the cellular and animal levels as well as their subsequent biological elimination were investigated. RAW264.7 and Hepa1-6 cells were used as models of Kupffer cells and hepatocytes, respectively. Results: The cellular studies showed that CsNps released from RAW264.7 cells could enter Hepa1-6 cells through both clathrin- and caveolin-mediated endocytosis. The transport from Kupffer cells to hepatocytes was also studied in mice, and it was observed that most CsNps localized to the hepatocytes after intravenous injection. Following the distribution in hepatocytes, the hepatobiliary-fecal excretion route was shown to be the primary elimination route for CsNps, besides the kidney-urinary excretion route. The elimination of CsNps in mice was a lengthy process, with a half time of about 2 months. Conclusion: The demonstration in this study of the transport of CsNps from macrophages to hepatocytes and the subsequent hepatobiliary-fecal excretion provides basic information for the future development and clinical application of NMs.


Assuntos
Quitosana/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Nanopartículas/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Exocitose , Hepatócitos/efeitos dos fármacos , Cinética , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Nanopartículas/ultraestrutura , Fótons
5.
Adv Exp Med Biol ; 1155: 567-581, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468432

RESUMO

Here, we investigated the hepatoprotective effect of a hot water extract from Loliolus beka gray meat (LBMH) containing plentiful taurine in H2O2-induced oxidative stress in hepatocytes. LBMH potently scavenged the 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and exhibited the good reducing power and the oxygen radical absorbance capacity (ORAC) value. Also, LBMH improved the cell viability against H2O2-induced hepatic damage in cultured hepatocytes by reducing intracellular reactive oxygen species (ROS) production. In addition, LBMH inhibited apoptosis via a reduction in sub-G1 cell population, as well as inhibition of apoptotic body formation from H2O2-induced oxidative damage in hepatocytes. Moreover, LBMH regulated the expression levels of Bax, a pro-apoptotic molecule and Bcl-2, an anti-apoptotic molecule in H2O2-treated hepatocytes. Additionally, pre-treatment with LBMH increased the expression of heme oxygenase 1 (HO-1), which is a hepatoprotective enzyme, by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) in H2O2-treated hepatocytes. Taken together, LBMH may be useful as a food ingredient for treatment of liver disease by regulating the Nrf2/HO-1 signal pathway.


Assuntos
Antioxidantes , Extratos Celulares/farmacologia , Decapodiformes/química , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo , Taurina/farmacologia , Animais , Células Cultivadas , Heme Oxigenase-1/metabolismo , Hepatócitos/citologia , Humanos , Peróxido de Hidrogênio , Carne , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
6.
Nihon Yakurigaku Zasshi ; 154(2): 72-77, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31406046

RESUMO

In drug disposition, the liver and small intestine are very important as tissues involving in drug metabolism, absorption, and excretion. Thus, in drug development studies, it is necessary to evaluate the pharmacokinetics in these tissues accurately including the contributions of drug-metabolizing enzymes and drug transporters. Currently, all kinds of evaluation systems have been used for the pharmacokinetic prediction; however, there are some issues in these systems. Therefore, the researches for the development of human induced pluripotent stem (iPS) cell-derived hepatocytes and enterocytes, as novel systems besides existing ones, are being advanced. Because human iPS cells have abilities of pluripotency and almost infinite proliferation, it is thought to be possible to stably provide the high-quality cells that have similar characteristics to human normal tissue cells by using human iPS cells. In this review, we describe current status of differentiation studies of human iPS cell-derived hepatocytes and enterocytes and the functional characteristics of these cells centered on pharmacokinetic functions.


Assuntos
Enterócitos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Diferenciação Celular , Avaliação Pré-Clínica de Medicamentos , Enterócitos/citologia , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Intestino Delgado , Fígado , Farmacocinética
7.
Adv Exp Med Biol ; 1155: 597-610, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468434

RESUMO

Octopus ocellatus meat (OM) is well known as a plentiful protein source. In this study, we evaluated the hepatoprotective effect of an aqueous extract of OM (OMA) against H2O2-triggered oxidative stress in human hepatocytes. First of all, taurine rich OMA showed a good ORAC value and reducing power and it was similar with that of ascorbic acid, which is known as a strong antioxidant. Also, OMA significantly improved H2O2-decreased cell viability by reducing the generation of intracellular reactive oxygen species (ROS) in hepatocytes. Interestingly, the stimulation of H2O2-induced the formations of apoptotic bodies and sub-G1 DNA content, whereas they were inhibited by the treatment with OMA. Furthermore, OMA regulated the protein expression levels of apoptotic molecules, such as Bax and Bcl-2. Taken together, this study suggests that OMA, which contains an abundant amount of taurine, protects hepatocytes from H2O2-triggered oxidative stress and might be a functional food material with hepatoprotective effects.


Assuntos
Extratos Celulares/farmacologia , Hepatócitos/efeitos dos fármacos , Octopodiformes/química , Estresse Oxidativo , Animais , Apoptose , Células Cultivadas , Hepatócitos/citologia , Humanos , Peróxido de Hidrogênio/efeitos adversos , Carne , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo
8.
Adv Exp Med Biol ; 1155: 691-703, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468440

RESUMO

In this study, we investigated the hepatoprotective activity of the water extract derived from Octopus vulgaris meat (OM). First of all, a water extract prepared from OM (OMW) showed the high extraction yield (48.22%) and the highest taurine content (39.84%) in free amino acids. OMW exhibited the high value of reducing power, ABTS and hydrogen peroxide radical scavenging activities in dose-dependent manner. The taurine-rich OMW also led to the reduced intracellular reactive oxygen species (ROS) generation with the increased cell survival in H2O2-treated Chang liver cells. In addition, OMW decreased the apoptotic phenomenon, including the formations of apoptotic bodies and sub-G1 DNA contents by regulating the protein expressions of apoptosis-related molecules such as Bcl-2 and Bax. From these results, this study indicated the taurine-rich OMW protected hepatocytes against oxidative stress. These findings suggest that OWM may be a novel potential antioxidant resource.


Assuntos
Extratos Celulares/farmacologia , Hepatócitos/citologia , Octopodiformes/química , Estresse Oxidativo , Taurina/farmacologia , Animais , Antioxidantes/farmacologia , Linhagem Celular , Humanos , Peróxido de Hidrogênio , Carne , Espécies Reativas de Oxigênio , Água
9.
Nat Commun ; 10(1): 3051, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296864

RESUMO

Treatment of liver metastasis experiences slow progress owing to the severe side effects. In this study, we demonstrate a strategy capable of eliminating metastatic cancer cells in a selective manner. Nucleus-targeting W18O49 nanoparticles (WONPs) are conjugated to mitochondria-selective mesoporous silica nanoparticles (MSNs) containing photosensitizer (Ce6) through a Cathepsin B-cleavable peptide. In hepatocytes, upon the laser irradiation, the generated singlet oxygen species are consumed by WONPs, in turn leading to the loss of their photothermally heating capacity, thereby sparing hepatocyte from thermal damage induced by the laser illumination. By contrast, in cancer cells, the cleaved peptide linker allows WONPs and MSNs to respectively target nucleus and mitochondria, where the therapeutic powers could be unleashed, both photodynamically and photothermally. This ensures the energy production of cancer cells can be abolished. We further assess the underlying molecular mechanism at both gene and protein levels to better understand the therapeutic outcome.


Assuntos
Portadores de Fármacos/metabolismo , Neoplasias Hepáticas/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Catepsina B/metabolismo , Núcleo Celular/metabolismo , Portadores de Fármacos/química , Feminino , Células HCT116 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Injeções Intravenosas , Lasers , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Fosforilação Oxidativa , Óxidos/química , Fotoquimioterapia/instrumentação , Espécies Reativas de Oxigênio , Dióxido de Silício/química , Resultado do Tratamento , Tungstênio/química , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Chem Biol Interact ; 311: 108761, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348918

RESUMO

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters - MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and -RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.


Assuntos
Glutationa/química , Microcistinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzobromarona/química , Benzobromarona/metabolismo , Cromatografia Líquida de Alta Pressão , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Microcistinas/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em Tandem
11.
Nat Cell Biol ; 21(8): 1015-1026, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332348

RESUMO

Human liver cancers, including hepatocellular carcinomas and intra-hepatic cholangiocarcinomas, are often diagnosed late with poor prognosis. A better understanding of cancer initiation could provide potential preventive therapies and increase survival. Models for studying human liver cancer initiation are largely missing. Here, using directly reprogrammed human hepatocytes (hiHeps) and inactivation of p53 and RB, we established organoids possessing liver architecture and function. HiHep organoids were genetically engineered to model the initial alterations in human liver cancers. Bona fide hepatocellular carcinomas were developed by overexpressing c-Myc. Excessive mitochondrion-endoplasmic reticulum coupling induced by c-Myc facilitated hepatocellular carcinoma initiation and seemed to be a target of preventive treatment. Furthermore, through the analysis of human intra-hepatic cholangiocarcinoma-enriched mutations, we demonstrate that the RAS-induced lineage conversion from hepatocytes to intra-hepatic cholangiocarcinoma cells can be prevented by the combined inhibition of Notch and JAK-STAT. Together, hiHep organoids represent a system that can be genetically manipulated to model cancer initiation and identify potential preventive therapies.


Assuntos
Hepatócitos/citologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Organoides/citologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Hepáticas/genética , Camundongos , Proteína Supressora de Tumor p53/genética
12.
J Dairy Sci ; 102(8): 7522-7535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155243

RESUMO

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.


Assuntos
Bovinos/genética , Hepatócitos/metabolismo , Fígado/citologia , Receptores da Somatotropina/genética , Animais , Bovinos/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Feminino , Hormônio do Crescimento/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Gravidez , Cultura Primária de Células , Receptores da Somatotropina/metabolismo , Vimentina/genética , Vimentina/metabolismo
13.
Gen Physiol Biophys ; 38(4): 343-352, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31241045

RESUMO

Non-alcoholic fatty liver disease and its complications are frequent causes of liver-related morbidity and mortality. Incretin glucagon-like peptide-1 (GLP-1) affects liver functions and metabolism. Although GLP-1 analogues are widely used in clinical practice, information regarding their potential toxic effect on hepatocytes in vitro is missing. Therefore, we evaluated the effect of GLP-1 analogue liraglutide on activity of caspases 3/7, cell viability and oxidative stress in primary cultures of hepatocytes. Primary cultures isolated from male Wistar rats fed a standard (ST1-group, 10% energy from fat) or a high-fat diet (HF-group, 71% fat) for 10 weeks were incubated with liraglutide (0.1-1000 nmol/l) for 24 h. Activities of caspases 3/7 and cellular dehydrogenases (WST-1), lactate dehydrogenase (LDH) leakage and oxidative stress (malondialdehyde concentration and DCFDA assay) were evaluated. HF-groups vs. ST1-groups showed higher caspases activity, LDH leakage and MDA production (p < 0.001) and lower cellular dehydrogenases activity (p < 0.01). Liraglutide induced a dose-dependent decrease of caspases activity in both groups, reduction of oxidative stress in HF-animals and exerted no negative effects on other parameters. In conclusion, GLP-1 analogue liraglutide decreased activity of caspases 3/7, reduced ROS production and didn't exhibit negative effects on cell viability and oxidative stress in primary cultures of hepatocytes isolated from lean and steatotic livers.


Assuntos
Separação Celular , Fígado Gorduroso/patologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Liraglutida/farmacologia , Fígado/citologia , Animais , Células Cultivadas , Masculino , Ratos , Ratos Wistar
14.
Emerg Microbes Infect ; 8(1): 879-894, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179847

RESUMO

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Hepatócitos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Animais , Membrana Celular/genética , Proliferação de Células , Feminino , Células Hep G2 , Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Simportadores/metabolismo
15.
J Microbiol ; 57(9): 812-820, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31201723

RESUMO

Sanghuangporus sanghuang is a well-known pharmacodynamic and economically important edible fungus associated with mulberry (Morus spp.). A distinctly new exopolysaccharide (EPS), designated SHP-2 was obtained from S. sanghuang P0988 broth, and its structure and anti-aging prosperity were characterized. SHP-2 was found to be composed of a back-bone of →4)-ß-Manp-(1→4)-α-Araf-(1→3,4)-α-Glcp(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→6)-α-Galp-(1→4)-ß-Manp-(1→ and five branches, including four α-D-Glcp-(1→ and one α-D-Manp-(1→SHP-2 was shown to increase antioxidant enzyme activities including catalase (CAT) and superoxide dismutase (SOD) activities, as well as trolox equivalent antioxidant (TEAC) capacity in serum of mice pre-treated with D-Gal, while reducing lipofuscin levels. SHP-2 exerted a favorable influence on immune organ coefficients and ameliorated the histopathological hepatic lesions and apoptosis in hepatocytes of D-galactose-aged mice almost in a dose-dependent manner. Using the same analytical methods, on comparison with previously studied EPS compounds (i.e. SHP-1), SHP-2 was found to have more complex structure, larger molecule weight, and different anti-aging properties. The results presented here suggest that not only does EPS bioactivity vary with respect to molecular structures and molecule weight, but that multiple structures with different activity can be expressed by a single fungal strain. These results may help understanding the anti-aging prosperity of these polysaccharides for use in health foods or dietary supplements.


Assuntos
Basidiomycota/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sequência de Carboidratos , Catalase/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Lipofuscina/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Peso Molecular , Capacidade de Absorbância de Radicais de Oxigênio , Superóxido Dismutase/metabolismo
16.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207892

RESUMO

AIM: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. METHODS: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. RESULTS: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. CONCLUSION: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.


Assuntos
Desdiferenciação Celular , Hepatócitos/citologia , Metabolismo dos Lipídeos , MicroRNAs/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Células Cultivadas , Citocromos/genética , Citocromos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Regulação para Cima
17.
Chem Biol Interact ; 307: 147-153, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31071334

RESUMO

The development of novel agrochemical compounds to reduce the use of pesticides with high ecological impact is urgently needed. A complex of Mg with two flavonoids hesperidin and phenanthroline [Mg(hesp)2(phen)], referred to as MgHP, results in high insecticidal activity against urban, agricultural and forest insect pests. In vitro cytotoxicity biomarkers were used to assess the mechanism of action MgHP on fish cells, as this insecticide can reach the aquatic environment and affect its biota. The cytotoxic effects of MgHP were evaluated at different concentrations (0, 0.1, 1, 10, 100 and 1000 ng mL-1) in a zebrafish hepatocyte cell line (ZF-L). Twenty-four hours of exposure to high concentrations (10 and 1000 ng mL-1) of MgHP affected cell confluence and morphology. Mitochondrial activity and lysosomal retention ability decreased as the MgHP concentration was increased. Cell membrane injury, apoptosis, and necrosis were not induced. These results suggested that toxicity to ZF-L cells was due loss of organellar activity caused by MgHP, which may also include activation of an alternative cell death mechanism. However, after 96 h of exposure, the toxic effects of MgHP may be mitigated, even at high concentrations, enabling cellular population recovery. These data provide important information on the mechanism of action of MgHP on hepatocyte fish cells and stimulate analyses to elucidate the cellular responses to MgHP.


Assuntos
Complexos de Coordenação/toxicidade , Inseticidas/toxicidade , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Inseticidas/química , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra
18.
BMC Vet Res ; 15(1): 134, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064364

RESUMO

BACKGROUND: Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. RESULTS: The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. CONCLUSIONS: GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.


Assuntos
Vírus da Hepatite do Pato/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Patos , Gynostemma/química , Hepatite Viral Animal/tratamento farmacológico , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fosforilação , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Replicação Viral/efeitos dos fármacos
19.
Food Chem ; 294: 302-308, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126467

RESUMO

Pereskia aculeata Miller, known worldwide as ora-pro-nobis, is a highly nutritive species of the Cactaceae family from the Brazilian Atlantic Forest. In this work, we report inedited information on the phenolic profile of P. aculeata leaves, besides a broad study of their antioxidant potential using a set of five different methods. A total of ten phenolic compounds were identified, such as two phenolic acids (caffeic acid derivatives) and eight flavonoids (quercetin, kaempferol and isorhamnetin glycoside derivatives). Caftaric acid was the extract's major phenolic constituent, accounting for more than 49% of the phenolic content, followed by quercetin-3-O-rutinoside (14.99%) and isorhamnetin-O-pentoside-O-rutinoside (9.56%). Overall, the ora-pro-nobis leaf extract showed relevant values of antioxidant capacity, with higher activities than the Trolox in the DPPH and ABTS trials. The antimicrobial activity exhibited by the extract against both Gram-positive and Gram-negative bacteria suggests the presence of a broad spectrum of phytochemicals with antibiotic activity.


Assuntos
Cactaceae/química , Compostos Fitoquímicos/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antioxidantes/química , Brasil , Cactaceae/metabolismo , Células Cultivadas , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Florestas , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fenóis/química , Fenóis/isolamento & purificação , Fenóis/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Folhas de Planta/química , Folhas de Planta/metabolismo
20.
Chem Biol Interact ; 308: 185-193, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31132328

RESUMO

Cytochrome P450 3A (CYP3A) activity is inhibited, and its expression is suppressed during many diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanism is controversial. Here, we report that PXR may not take part in the downregulation of CYP3A during NAFLD. Hepatic CYP3A11 (major subtype of mouse CYP3A) mRNA and protein expression was significantly decreased in both mice fed a high-fat diet (HFD) for 8 weeks and palmitate (PA)-treated mouse primary hepatocytes. Similarly, in HepG2 cells, PA treatment significantly suppressed the CYP3A4 (major subtype of human CYP3A) mRNA level and promoter transcription activity. However, Western blotting analysis found an induction of PXR nuclear translocation during NAFLD in both in vivo and in vitro models. Moreover, immunofluorescence determination also found nuclear translocation effect of PXR by PA stimulation in HepG2 cells. In addition, the siRNA knockdown of PXR did not affect the suppressive effects of PA on the CYP3A4 promoter transcription activity and mRNA levels in HepG2 cells. Similarly, PXR knockdown also did not affect the suppressive effects of PA on CYP3A11 mRNA and protein expression levels in mouse primary hepatoctyes. Taken together, the results showed that the suppressive effect of CYP3A transcription was independent of PXR regulation.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Dieta Hiperlipídica , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Pregnano X/metabolismo , Animais , Citocromo P-450 CYP3A/genética , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/veterinária , Palmitatos/farmacologia , Receptor de Pregnano X/antagonistas & inibidores , Receptor de Pregnano X/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Genética/efeitos dos fármacos
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