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1.
Autophagy ; : 1-13, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996071

RESUMO

IFNL2 is a potent antiviral interferon, but the regulation of its gene expression is not fully clear. Here, we report the regulation of ATG10S for IFNL2 transcription. Through sequential deletion of the IFNL2 promoter sequence, we found LP1-1, a fragment of the promoter responding to ATG10S activity. Subcellular localization and DNA immunoprecipitation assays showed ATG10S translocating into the nucleus and binding to LP1-1. Online prediction for transcription factor binding sites showed an IRF1 targeting locus in LP1-1. Luciferase assays, RT-PCR, and western blot analysis revealed a core motif (CAAGAC) existing in LP1-1, which determined ATG10S and IRF1 activity; individual nucleotide substitution showed that the functional nucleotides of ATG10S targeting were C1, A3, and C6, and the ones associated with IRF1 were A3 and G4 within the core motif. Co-immunoprecipitation assays revealed ATG10S combination with KPNA1/importin α, KPNB1/importin ß, and IRF1. The knockdown of endogenous IRF1 increased ATG10S activity on IFNL2 transcription. These results indicate that ATG10S as a transcription factor competes with IRF1 for the same binding site to promote IFNL2 gene transcription.Abbreviations: ATG10: autophagy related 10; ATG10S: the shorter isoform of autophagy related 10; BD: binding domain; CM: core motif; co-IP: co-immunoprecipitation; GFP: green fluorescent protein; HCV: hepatitis C virus; IF: immunofluorescence; IFN: interferon; IRF: interferon regulatory factor; LP: lambda promoter; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RLU: relative light unit; SQSTM1: sequestosome 1.

2.
J Pharmacol Toxicol Methods ; 101: 106662, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31837439

RESUMO

The quantification and visualization of fluorescent staining at the whole organ level remain a challenge. Deconvolution image systems allow multi-dimensional imaging and stereo-measurement via rapid 3D reconstruction. To demonstrate this technique, we investigated doxorubicin-induced cardiotoxicity in zebrafish. Fluorogenic probe and immunofluorescence were employed to identify cardiac reactive oxygen species generation and myocardial apoptosis, respectively. We revealed the spatial distribution of fluorescent staining across the whole heart by this approach. In addition, the fluorescence intensities and fluorescence-dyed volumes in the zebrafish heart were quantified automatically. Importantly, doxorubicin treatment induced more ROS generation in the ventricle as compared to the atrium, while the levels of activated caspase-3 were much higher in the atrioventricular junction. These results would have been difficult to observe using traditional 2D images. Therefore, our deconvolution imaging strategy allows the 3D quantification and visualization of fluorescent staining at the whole organ level, and will thus support in vivo studies.

3.
Front Mol Neurosci ; 11: 378, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534049

RESUMO

Epilepsy is a neuronal dysfunction syndrome characterized by transient and diffusely abnormal discharges of neurons in the brain. Previous studies have shown that mutations in the syntaxin 1b (stx1b) gene cause a familial, fever-associated epilepsy syndrome. It is unclear as to whether the stx1b gene also correlates with other stimulations such as flashing and/or mediates the effects of antiepileptic drugs. In this study, we found that the expression of stx1b was present mainly in the brain and was negatively correlated with seizures in a pentylenetetrazole (PTZ)-induced seizure zebrafish model. The transcription of stx1b was inhibited by PTZ but rescued by valproate, a broad-spectrum epilepsy treatment drug. In the PTZ-seizure zebrafish model, stx1b knockdown aggravated larvae hyperexcitatory swimming and prompted abnormal trajectory movements, particularly under lighting stimulation; at the same time, the expression levels of the neuronal activity marker gene c-fos increased significantly in the brain. In contrast, stx1b overexpression attenuated seizures and decreased c-fos expression levels following PTZ-induced seizures in larvae. Thus, we speculated that a deficiency of stx1b gene expression may be related with the onset occurrence of clinical seizures, particularly photosensitive seizures. In addition, we found that berberine (BBR) reduced larvae hyperexcitatory locomotion and abnormal movement trajectory in a concentration-dependent manner, slowed down excessive photosensitive seizure-like swimming, and assisted in the recovery of the expression levels of STX1B. Under the downregulation of STX1B, BBR's roles were limited: specifically, it only slightly regulated the levels of the two genes stx1b and c-fos and the hyperexcitatory motion of zebrafish in dark conditions and had no effect on the overexcited swimming behavior seen in conjunction with lighting stimulation. These findings further demonstrate that STX1B protein levels are negatively correlated with a seizure and can decrease the sensitivity of the photosensitive response in a PTZ-induced seizure zebrafish larvae; furthermore, STX1B may partially mediate the anticonvulsant effect of BBR. Additional investigation regarding the relationship between STX1B, BBR, and seizures could provide new cues for the development of novel anticonvulsant drugs.

4.
Front Immunol ; 9: 2176, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319633

RESUMO

Autophagy-related 10 (ATG10) is essential for autophagy since it promotes ATG5-ATG12 complex formation. Our previous study found that there are two isoforms of the ATG10 protein, ATG10 (a longer one) and ATG10S, which have identical sequences except an absence of a 36-amino acid fragment (peptide B) in ATG10S, yet exhibit distinct effects on HCV genome replication. Here, we report the existence of two amino acids, cysteine at residue 44 and 135 (Cys44 and Cys135, respectively), in ATG10 being related to differential effects of ATG10 on HCV replication and autophagy flux. Through a series of ATG10 mutation experiments and protein modeling prediction, we found that Cys44 was involved in the dual role of the two isoforms of ATG10 protein on HCV replication and autophagy flux, and that Cys135 plays similar roles as Cys44, but the disulfide bond of Cys44-Cys135 was not verified in the ATG10 protein. Further analyses by full HCV virion infection confirmed the roles of -SH of Cys44 and Cys135 on HCV replication. ATG10 with deleted or mutated Cys44 and/or Cys135 could activate expression of innate immunity-related genes, including il28a, irf-3, irf-7, and promote complete autophagy by driving autophagosomes to interact with lysosomes via IL28A-mediation. Subcellular localization assay and chromatin immunoprecipitation assay showed that ATG10 with the sulfydryl deletion or substitution of Cys44 and Cys135 could translocate into the nucleus and bind to promoter of IL28A gene; the results indicated that ATG10 with Cys44 and/or Cys135 absence might act as transcriptional factors to trigger the expression of anti-HCV immunological genes, too. In conclusion, our findings provide important information for understanding the differential roles on HCV replication and autophagy flux between ATG10 and ATG10S, and how the structure-function relationship of ATG10 transformed by a single -SH group loss on Cys44 and Cys135 in ATG10 protein, which may be a new target against HCV replication.


Assuntos
Proteínas Relacionadas à Autofagia/imunologia , Autofagia/imunologia , Hepacivirus/fisiologia , Proteínas de Transporte Vesicular/imunologia , Replicação Viral/imunologia , Substituição de Aminoácidos , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Cisteína/genética , Cisteína/imunologia , Células Hep G2 , Humanos , Mutação de Sentido Incorreto , Proteínas de Transporte Vesicular/genética , Replicação Viral/genética
5.
Artigo em Inglês | MEDLINE | ID: mdl-30026731

RESUMO

Dietary composition has important impact on nonalcoholic fatty liver disease (NAFLD). The purpose of this study was to explore the relationship between NAFLD and major dietary components using zebrafish larvae fed different diets. Zebrafish larvae fed with high cholesterol (HC), high fructose (HF) and extra feeding (EF) diets for 10 days displayed varying degrees steatosis. The incidence and degree of steatosis were the most severe in the EF group. A HC diet severely promoted lipid deposits in the caudal vein. The triglyceride and glucose contents of zebrafish significantly increased in the HF and EF groups compared with the control group. Moreover, the mRNA expression of oxidative stress gene gpx1a, endoplasmic reticulum stress genes ddit3 and grp78, inflammatory genes tnfa, glucose metabolism genes irs2, glut1 and glut2, and lipid metabolism genes cidec, chrebp, ppara and cpt1a were significantly increased in the HF group. The HC diet was associated with upregulation of grp78, and downregulation of irs2, glut1 and glut2. The mRNA expression of lipogenesis and glucose metabolism associated genes were decreased in the EF group. In addition, the autophagy associated genes atg3, atg5, atg7, and atg12, and protein expression of ATG3 and LC3BII were reduced and P62 were elevated in the HC group. We also performed comparative transcriptome analysis of the four groups. A total of 2,492 differentially expressed genes were identified, and 24 statistically significant pathways were enriched in the diet treatment groups. This study extends our understanding of the relationships between diet ingredients and host factors that contribute to the pathogenesis of NAFLD, which may provide new ideas for the treatment of NAFLD.

6.
Artigo em Inglês | MEDLINE | ID: mdl-29670865

RESUMO

Autophagy is a host mechanism for cellular homeostatic control. Intracellular stresses are symptoms of, and responses to, dysregulation of the physiological environment of the cell. Alternative gene transcription splicing is a mechanism potentially used by a host to respond to physiological or pathological challenges. Here, we aimed to confirm opposite effects of two isoforms of the human autophagy-related protein ATG10 on an HCV subgenomic replicon in zebrafish. A liver-specific HCV subreplicon model was established and exhibited several changes in gene expression typically induced by HCV infection, including overexpression of several HCV-dependent genes (argsyn, leugpcr, rasgbd, and scaf-2), as well as overexpression of several ER stress related genes (atf4, chop, atf6, and bip). Autophagy flux was blocked in the HCV model. Our results indicated that the replication of the HCV subreplicon was suppressed via a decrease in autophagosome formation caused by the autophagy inhibitor 3MA, but enhanced via dysfunction in the lysosomal degradation caused by another autophagy inhibitor CQ. Human ATG10, a canonical isoform in autophagy, facilitated the amplification of the HCV-subgenomic replicon via promoting autophagosome formation. ATG10S, a non-canonical short isoform of the ATG10 protein, promoted autophagy flux, leading to lysosomal degradation of the HCV-subgenomic replicon. Human ATG10S may therefore inhibit HCV replication, and may be an appropriate target for future antiviral drug screening.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Genoma Viral/genética , Hepacivirus/genética , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral/genética , Animais , Proteínas Relacionadas à Autofagia/genética , Hepacivirus/fisiologia , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Transporte Vesicular/genética , Peixe-Zebra
7.
Eur J Med Chem ; 143: 1053-1065, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29232582

RESUMO

Aloperine (1), a Chinese natural product with a unique endocyclic scaffold, was first identified to be a potent hepatitis C virus (HCV) inhibitor in our laboratory. Thirty-four new aloperine derivatives were designed, synthesized and evaluated for their anti-HCV activities taking 1 as the lead. Among them, compound 7f exhibited the potential potency with EC50 values in a micromolar range against both wild-type and direct-acting antiviral agents (DAAs)-resistant variants, and synergistically inhibited HCV replication with approved DAAs. Furthermore, it also owned a good oral pharmacokinetic and safety profile, suggesting a highly druglike nature. The primary mechanism showed that 7f might target host components, distinctly different from the DAAs currently used in clinic. Therefore, we consider aloperine derivatives to be a novel class of anti-HCV agents, and compound 7f has been selected as a promising antiviral candidate for further investigation.


Assuntos
Antivirais/farmacologia , Desenho de Drogas , Hepacivirus/efeitos dos fármacos , Piperidinas/farmacologia , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperidinas/administração & dosagem , Piperidinas/química , Relação Estrutura-Atividade , Fatores de Tempo , Replicação Viral/efeitos dos fármacos
8.
Sci Rep ; 7(1): 11250, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28900156

RESUMO

Autophagy and immune response are two defense systems that human-body uses against viral infection. Previous studies documented that some viral mechanisms circumvented host immunity mechanisms and hijacked autophagy for its replication and survival. Here, we focus on interactions between autophagy mechanism and innate-immune-response in HCV-subgenomic replicon cells to find a mechanism linking the two pathways. We report distinct effects of two autophagy-related protein ATG10s on HCV-subgenomic replication. ATG10, a canonical long isoform in autophagy process, can facilitate HCV-subgenomic replicon amplification by promoting autophagosome formation and by combining with and detaining autophagosomes in cellular periphery, causing impaired autophagy flux. ATG10S, a non-canonical short isoform of ATG10 proteins, can activate expression of IL28A/B and immunity genes related to viral ds-RNA including ddx-58, tlr-3, tlr-7, irf-3 and irf-7, and promote autophagolysosome formation by directly combining and driving autophagosomes to perinuclear region where lysosomes gather, leading to lysosomal degradation of HCV-subgenomic replicon in HepG2 cells. ATG10S also can suppress infectious HCV virion replication in Huh7.5 cells. Another finding is that IL28A protein directly conjugates ATG10S and helps autophagosome docking to lysosomes. ATG10S might be a new host factor against HCV replication, and as a target for screening chemicals with new anti-virus mechanisms.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Hepacivirus/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata , Isoformas de Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral , Linhagem Celular , Hepacivirus/fisiologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos
9.
J Biol Chem ; 292(44): 18062-18074, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28928221

RESUMO

Parkinson's disease (PD) is one of the most epidemic neurodegenerative diseases and is characterized by movement disorders arising from loss of midbrain dopaminergic (DA) neurons. Recently, the relationship between PD and autophagy has received considerable attention, but information about the mechanisms involved is lacking. Here, we report that autophagy-related gene 5 (ATG5) is potentially important in protecting dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in zebrafish. Using analyses of zebrafish swimming behavior, in situ hybridization, immunofluorescence, and expressions of genes and proteins related to PD and autophagy, we found that the ATG5 expression level was decreased and autophagy flux was blocked in this model. The ATG5 down-regulation led to the upgrade of PD-associated proteins, such as ß-synuclein, Parkin, and PINK1, aggravation of MPTP-induced PD-mimicking pathological locomotor behavior, DA neuron loss labeled by tyrosine hydroxylase (TH) or dopamine transporter (DAT), and blocked autophagy flux in the zebrafish model. ATG5 overexpression alleviated or reversed these PD pathological features, rescued DA neuron cells as indicated by elevated TH/DAT levels, and restored autophagy flux. The role of ATG5 in protecting DA neurons was confirmed by expression of the human atg5 gene in the zebrafish model. Our findings reveal that ATG5 has a role in neuroprotection, and up-regulation of ATG5 may serve as a goal in the development of drugs for PD prevention and management.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica , Terapia Genética , Transtornos Parkinsonianos/prevenção & controle , Proteínas de Peixe-Zebra/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , DNA Recombinante/uso terapêutico , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/patologia , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Larva , Microinjeções , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/uso terapêutico , Neuroproteção/efeitos dos fármacos , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
10.
Int J Biol Sci ; 13(8): 985-995, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28924380

RESUMO

Type 2 diabetes mellitus is characterized by insulin resistance. However, the complete molecular mechanism remains unclear. In this study, zebrafish were fed a long-term high-fat diet to induce type 2 diabetes, which resulted in a higher body weight, body mass index, more lipid vacuoles in liver, increased insulin transcription level in liver, brain and muscle, and high fasting blood glucose in the high-fat diet zebrafish. Oppositely, the transcription levels of insulin substrate-2 and glucose transporter 2 were significantly decreased, indicating insulin signaling pathway and glucose transport impaired in the insulin-targeting tissues. Transcription of the autophagy-related genes, ATG3, ATG4B, ATG5, ATG7, ATG12, and FOXO3, were decreased but autophagy inhibitor gene m-TOR increased, and autophagy-flux was inhibited in liver of the high-fat diet zebrafish. Main of these changes were confirmed in palmitic acid-treated HepG2 cells. Further, in co-immunoprecipitation and subcellular co-localization experiments, the conjunction of preproinsulin with cargo-recognition protein p62 increased, but conjuncts of autophagosome with p62-cargo, lysosomes with p62-cargo, and autolysosomes decreased apparently. Interestingly, lysosomes, autolysosomes and conjuncts of p62-insulin localized at the periphery of palmitic acid-treated cells, the margination of lysosomes may mediate deactivation of proteases activity. These findings suggest that intracellular high-lipid may trigger defective autophagy, defective downstream signaling of insulin and accumulated intracellular preproinsulin, leading to dysregulation of cell homeostasis mechanism, which may be one of reasons involved in insulin-resistance in type 2 diabetes.


Assuntos
Autofagia/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Western Blotting , Transportador de Glucose Tipo 2/metabolismo , Células Hep G2 , Humanos , Imunoprecipitação , Precursores de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra
11.
Front Pharmacol ; 8: 403, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28694779

RESUMO

Cefazolin sodium is an essential drug that is widely used in clinical therapy for certain infective diseases caused by bacteria. As drug impurities are considered to be one of the most important causes of drug safety issues, we studied embryotoxicity, cardiotoxicity, and neurotoxicity of nine cefazolin sodium impurities in zebrafish embryo and larvae for the objective control of impurity profiling. LC-MS/MS was employed to analyze the compound absorbance in vivo, and the structure-toxicity relationship was approached. Our results suggested that the structure of MMTD (2-mercapto-5-methyl-1, 3, 4-thiadiazole) is the main toxic functional group for embryo deformities; the 7-ACA (7-aminocephalosporanic acid) structure mainly affects motor nerve function; and both the MMTD and 7-ACA structures are responsible for cardiac effects. Impurity G (7-ACA) presented with the strongest toxicity; impurity A was most extensively absorbed to embryo and larvae; and impurity F (MMTD) exhibited the strongest apparent toxic effect; Therefore, impurities F and G should be monitored from the cefazolin sodium preparations.

12.
Yao Xue Xue Bao ; 51(4): 580-7, 2016 04.
Artigo em Chinês | MEDLINE | ID: mdl-29859527

RESUMO

Epilepsy is a kind of neurogenic diseases with high prevalence and characterized by seizure, brain paradoxical discharge and convulsion in spontaneous, transient, recurrent and uncontrolled manner. Development of new anti-epilepsy drugs requires a new reliable and high-performance animal models in screening of leading compounds. In this study, an epilepsy model in larval zebrafish was established using pentylenetetrazole (PTZ) compound. The results show that PTZ induced epilepsy-like seizure behavior such as irregular circular swimming, exciting locomotion, high swim velocity and convulsion in zebrafish. Expression patterns of two epilepsy-related gene c-fos and lgi1 were analyzed using RT-PCR and in situ hybridization; c-fos was enhanced and extended and lgi1 expression was reduced in PTZ concentration-dependent in the larval brain. When the model larvae exposed to anticonvulsant valproate(VPA), the epilepsy-like symptom decreased or disappeared, the marker genes c-fos and lgi1, as well as NeuN protein recovered to the normal levels. These responses to PTZ and to antiepileptic drug VPA are consistent with the observations in clinical studies and mouse models. Using this model, we evaluated anti-epilepsy activity of compounds Y53 and BMT, two homolog of berberine. The results show that the model larvae seizure triggered by lighting was partly remedied by Y53; and the larval exciting locomotion under the condition of no stimulation was suppressed by BMT. The findings indicate that the zebrafish larval epilepsy model is able to distinguish compounds with different activities in eleptiform seizure. We conclude that the zebrafish epilepsy model may be as a reliable and useful platform in screening of new anti-epilepsy candidates, which is suitable for basic research in epilepsy pathogenesis.


Assuntos
Modelos Animais de Doenças , Epilepsia/fisiopatologia , Convulsões/fisiopatologia , Peixe-Zebra , Animais , Anticonvulsivantes , Encéfalo/metabolismo , Epilepsia/induzido quimicamente , Larva , Proteínas do Tecido Nervoso/metabolismo , Pentilenotetrazol , Proteínas Proto-Oncogênicas c-fos/metabolismo , Convulsões/induzido quimicamente , Natação , Ácido Valproico , Proteínas de Peixe-Zebra/metabolismo
13.
PLoS One ; 10(5): e0124805, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25938774

RESUMO

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.


Assuntos
Absorção Fisiológica/efeitos dos fármacos , Antibacterianos/toxicidade , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Peixe-Zebra/metabolismo , Animais , Córion , Embrião não Mamífero/efeitos dos fármacos , Dose Letal Mediana , Reprodutibilidade dos Testes , Peixe-Zebra/embriologia
14.
Int J Mol Med ; 35(3): 791-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25572289

RESUMO

Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs.


Assuntos
Genoma Viral , Hepacivirus/fisiologia , Replicação Viral , Animais , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Hepacivirus/efeitos dos fármacos , Humanos , Ribavirina/farmacologia , Replicação Viral/efeitos dos fármacos , Peixe-Zebra
15.
J Appl Toxicol ; 35(3): 241-52, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24853142

RESUMO

Drug-induced cardiotoxicity is a leading factor for drug withdrawals, and limits drug efficacy and clinical use. Therefore, new alternative animal models and methods for drug safety evaluation have been given great attention. Anthracyclines (ANTs) are widely prescribed anticancer agents that have a cumulative dose relationship with cardiotoxicity. We performed experiments to study the toxicity of ANTs in early developing zebrafish embryos, especially their effects on the heart. LC50 values for daunorubicin, pirarubicin, doxorubicin (DOX), epirubicin and DOX-liposome at 72 h post-fertilization were 122.7 µM, 111.9 µM, 31.2 µM, 108.3 µM and 55.8 µM, respectively. At the same time, zebrafish embryos were exposed to ANTs in three exposure stages and induced incomplete looping of the heart tube, pericardia edema and bradycardia in a dose-dependent manner, eventually leading to death. DOX caused the greatest heart defects in the treatment stages and its liposome reduced the effects on the heart, while daunorubicin produced the least toxicity. Genes and proteins related to heart development were also identified to be sensitive to ANT exposure and downregulated by ANTs. It revealed ANTs could disturb the heart formation and development. ANTs induced cardiotoxicity in zebrafish has similar effects in mammalian models, indicating that zebrafish may have a potential value for assessment of drug-induced developmental cardiotoxicity.


Assuntos
Antraciclinas/toxicidade , Antineoplásicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Cardiopatias Congênitas/induzido quimicamente , Peixe-Zebra , Animais , Cardiotoxicidade , Relação Dose-Resposta a Droga , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Peixe-Zebra/embriologia
16.
Yao Xue Xue Bao ; 49(6): 843-8, 2014 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-25212030

RESUMO

To investigate vincristine-induced dopaminergic neurons toxicity and mechanism, and explore the molecular target to reduce the toxicity, zebrafish was chosen as a model animal, based on RT-PCR, Western blotting, whole mount in situ immunofluorescence and other technical means. The results showed that the transcription levels of tyrosine hydroxylase gene and dopamine transporter protein gene were inhibited. Furthermore, the number of dopaminergic neurons was decreased by vincristine. Autophagy was suppressed and beclin1 gene expression was inhibited in a dose-dependent manner by vincristine in larval zebrafish. Up-regulated beclin1 partly reduced vincristine-induced neurotoxicity, and down-regulated beclin1 increased toxicity. Beclin1 plays an important role in vincristine-induced dopaminergic neurons toxicity.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Vincristina/efeitos adversos , Proteínas de Peixe-Zebra/metabolismo , Animais , Autofagia , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Larva/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra
17.
PLoS One ; 8(3): e56985, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469178

RESUMO

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-ß, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.


Assuntos
Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , RNA Mensageiro/antagonistas & inibidores , Proteínas do Core Viral/genética , Peixe-Zebra/virologia , Animais , Antivirais/farmacologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Genes Reporter , Proteínas de Fluorescência Verde , Hepacivirus/crescimento & desenvolvimento , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Larva/efeitos dos fármacos , Larva/virologia , Fígado/efeitos dos fármacos , Fígado/virologia , Proteínas Mutantes Quiméricas/antagonistas & inibidores , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Ribavirina/farmacologia , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/metabolismo , Vitamina B 12/farmacologia
18.
PLoS One ; 8(3): e58675, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23516533

RESUMO

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD50>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations.


Assuntos
Alcaloides/química , Alcaloides/farmacologia , Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSC70/metabolismo , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Quinolizinas/química , Quinolizinas/farmacologia , Replicação Viral/efeitos dos fármacos , Alcaloides/efeitos adversos , Alcaloides/farmacocinética , Animais , Antivirais/efeitos adversos , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Quinolizinas/efeitos adversos , Quinolizinas/farmacocinética , Segurança , Relação Estrutura-Atividade
19.
Yi Chuan ; 34(9): 1165-73, 2012 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-23017458

RESUMO

To further understand the neural toxicity and teratogenicity of antiepileptic drugs in clinic, we established a zebrafish model for antiepileptic toxicity using trimethadione as a probe drug. The results indicated that embryonic malformation occurred under trimethadione treatment in a concentration-dependent manner. The defects included growth retardation, small head, eyes and acoustic capsule, deficient semicircular canals and otolith, and abnormal cardiovascular system. The number of hair cells in neuromast ML2 was obviously reduced in the treated larvae. Whole mount in situ hybridization indicated that the gene expression patterns of brain marker genes, such as zic1 and xb51, and autophagic gene atg5 was changed significantly. The result of RT-PCR showed that the expressions of hearing genes val and hmx2 were also changed in the trimethadione-treated embryos. All these findings suggest that brain tissue and the neural sensors for body balance and hearing are the main targets of trimethadione toxicity, and that zebrafish is able to mimic mammal responses to the teratogenicity and the neural toxicity of trimethadione in the embryonic and larva development.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Teratogênios/toxicidade , Trimetadiona/toxicidade , Peixe-Zebra/embriologia , Anormalidades Múltiplas/induzido quimicamente , Animais , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos
20.
Yao Xue Xue Bao ; 46(8): 928-35, 2011 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-22007517

RESUMO

Aminoglycoside antibiotics, due to their strong antibacterial effects and broad antimicrobial spectra, have been very commonly used in clinical practice in the past half century. However, aminoglycoside antibiotics manifest severe ototoxicity and nephrotoxicity, and are one of top factors in hearing loss. In this study, three members of the aminoglycoside antibiotics family, gentamycin, neomycin and streptomycin, were chosen as the representatives to be investigated for their toxicity to the embryonic development and the larva hair cells in zebrafish, and also to their target genes associated with hearing-related genes. The results showed that: (1) the lethal effect of all three drugs demonstrated a significant dependence on concentration, and the severity order of the lethal effect was streptomycin > neomycin > gentamycin; (2) all the three drugs caused the larva trunk bending in resting state at 5 dpf (day past fertilization), probably due to their ototoxicity in the physical imbalance and postural abnormalities; (3) impairment and reducing of the hair cells were observed in all three cases of drug treatment; (4) four genes, eya1, val, otx2 and dlx6a, which play an important role in the development of hearing organs, showed differential and significant decrease of gene expression in a drug concentration-dependent manner. This study for the first time reports the relevance between the expression of hearing genes and the three ototoxic antibiotics and also proved the feasibility of establishing a simple, accurate, intuitive and fast model with zebrafish for the detection of drug ototoxicity.


Assuntos
Aminoglicosídeos/toxicidade , Antibacterianos/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Transtornos da Audição/induzido quimicamente , Animais , Regulação da Expressão Gênica , Gentamicinas/toxicidade , Células Ciliadas Auditivas/citologia , Transtornos da Audição/genética , Transtornos da Audição/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/efeitos dos fármacos , Sistema da Linha Lateral/efeitos dos fármacos , Fator de Transcrição MafB/metabolismo , Modelos Animais , Neomicina/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição Otx/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , Proteínas Tirosina Fosfatases/metabolismo , Estreptomicina/toxicidade , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
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