Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 35
Filtrar
1.
J Appl Toxicol ; 44(8): 1198-1213, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38639436

RESUMO

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 µM) and demonstrated the effects of BBR (10, 50, and 100 µM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV.


Assuntos
Atorvastatina , Berberina , Hemorragia Cerebral , Peixe-Zebra , Animais , Atorvastatina/farmacologia , Hemorragia Cerebral/induzido quimicamente , Berberina/farmacologia , Animais Geneticamente Modificados , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos
2.
Toxicol Appl Pharmacol ; 469: 116529, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37100089

RESUMO

The most commonly reported side effect of azithromycin is gastrointestinal (GI) disorders, and the main acid degradation product is 3'-Decladinosyl azithromycin (impurity J). We aimed to compare the GI toxicity of azithromycin and impurity J on zebrafish larvae and investigate the mechanism causing the differential GI toxicity. Results of our study showed that the GI toxicity induced by impurity J was higher than that of azithromycin in zebrafish larvae, and the effects of impurity J on transcription in the digestive system of zebrafish larvae were significantly stronger than those of azithromycin. Additionally, impurity J exerts stronger cytotoxic effects on GES-1 cells than azithromycin. Simultaneously, impurity J significantly increased ghsrb levels in the zebrafish intestinal tract and ghsr levels in human GES-1 cells compared to azithromycin, and ghsr overexpression significantly reduced cell viability, indicating that GI toxicity induced by azithromycin and impurity J may be correlated with ghsr overexpression induced by the two compounds. Meanwhile, molecular docking analysis showed that the highest -CDOCKER interaction energy scores with the zebrafish GHSRb or human GHSR protein might reflect the effect of azithromycin and impurity J on the expression of zebrafish ghsrb or human ghsr. Thus, our results suggest that impurity J has higher GI toxicity than azithromycin due to its greater ability to elevate ghsrb expression in zebrafish intestinal tract.


Assuntos
Azitromicina , Peixe-Zebra , Animais , Humanos , Azitromicina/toxicidade , Larva , Simulação de Acoplamento Molecular , Intestinos
3.
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
4.
Yao Xue Xue Bao ; 51(4): 580-7, 2016 04.
Artigo em Zh | 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
5.
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
6.
Yao Xue Xue Bao ; 49(6): 843-8, 2014 Jun.
Artigo em Zh | 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
7.
Autophagy ; 20(10): 2238-2254, 2024 10.
Artigo em Inglês | MEDLINE | ID: mdl-38842055

RESUMO

ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12-ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug.Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein.


Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Proteínas Virais , Humanos , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Virais/metabolismo , Interferons/metabolismo , Interferon lambda , Células HEK293 , Regiões Promotoras Genéticas/genética , Autofagossomos/metabolismo , Interleucinas/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Ligação Proteica , Proteínas de Transporte Vesicular
8.
Yi Chuan ; 34(9): 1165-73, 2012 Sep.
Artigo em Zh | 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 , Teratogênicos/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
9.
Phytomedicine ; 101: 154130, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35526324

RESUMO

BACKGROUND: Doxorubicin (DOX)-induced cardiotoxicity is related to abnormal autophagy and apoptosis in the heart. Berberine (BBR) is a well-known natural compound with potential cardioprotective and autophagic modulatory properties. HYPOTHESIS: We hypothesized that BBR ameliorates DOX-induced cardiotoxicity by balancing cardiomyocyte autophagy and apoptosis. STUDY DESIGN/METHODS: DOX was used to generate in vivo and in vitro cardiotoxic models. Larval and adult zebrafish and human AC16 cells were used to study (i) the effects of BBR on autophagy and apoptosis upon DOX challenge and (ii) the underlying mechanisms. RESULTS: BBR protected AC16 cells and zebrafish hearts from DOX-induced cytotoxicity and apoptosis. Bcl-xL knockdown in AC16 cells and zebrafish demonstrated that Bcl-xL is required for BBR's anti-apoptotic activity. DOX treatment promoted Beclin1 binding to Bcl-xL, disrupted mitophagy, and increased ROS accumulation in AC16 cells. In AC16 cells and zebrafish hearts, pretreatment with BBR enhanced mitophagy via dissociation of the Bcl-xL-Beclin1 complex and decreased ROS accumulation. Inhibition of autophagy attenuated this effect of BBR. Intriguingly, BBR increased Bcl-xL binding to Bnip3, sequestration, and mitophagy, indicating that Bcl-xL may play a beneficial role in BBR-induced mitophagy. Additionally, BBR significantly ameliorated DOX-induced cardiac dysfunction in zebrafish, whereas Bcl-xL knockdown abolished this effect. Notably, we discovered that BBR exerts biphasic dose-response effects in response to DOX; the cardioprotective properties were observed upon treatment with low-dose BBR (≤ 1 µM in cells, ≤ 10 µM in zebrafish), but not with relatively high-dose BBR. CONCLUSION: These findings indicate that the protective effects of low-dose BBR against DOX-induced cardiotoxicity are mediated by Bcl-xL.


Assuntos
Berberina , Cardiotoxicidade , Animais , Apoptose , Proteína Beclina-1/metabolismo , Berberina/farmacologia , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/metabolismo , Doxorrubicina/farmacologia , Mitofagia , Miócitos Cardíacos , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra/metabolismo
10.
Front Pharmacol ; 13: 860702, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35444552

RESUMO

Impurities in pharmaceuticals of potentially hazardous materials may cause drug safety problems. Macrolide antibiotic preparations include active pharmaceutical ingredients (APIs) and different types of impurities with similar structures, and the amount of these impurities is usually very low and difficult to be separated for toxicity evaluation. Our previous study indicated that hepatotoxicity induced by macrolides was correlated with c-fos overexpression. Here, we report an assessment of macrolide-related liver toxicity by ADMET prediction, molecular docking, structure-toxicity relationship, and experimental verification via detection of the c-fos gene expression in liver cells. The results showed that a rapid assessment model for the prediction of hepatotoxicity of macrolide antibiotics could be established by calculation of the -CDOCKER interaction energy score with the FosB/JunD bZIP domain and then confirmed by the detection of the c-fos gene expression in L02 cells. Telithromycin, a positive compound of liver toxicity, was used to verify the correctness of the model through comparative analysis of liver toxicity in zebrafish and cytotoxicity in L02 cells exposed to telithromycin and azithromycin. The prediction interval (48.1∼53.1) for quantitative hepatotoxicity in the model was calculated from the docking scores of seven macrolide antibiotics commonly used in clinics. We performed the prediction interval to virtual screening of azithromycin impurities with high hepatotoxicity and then experimentally confirmed by liver toxicity in zebrafish and c-fos gene expression. Simultaneously, we found the hepatotoxicity of azithromycin impurities may be related to the charge of nitrogen (N) atoms on the side chain group at the C5 position via structure-toxicity relationship of azithromycin impurities with different structures. This study provides a theoretical basis for improvement of the quality of macrolide antibiotics.

11.
Yao Xue Xue Bao ; 46(1): 50-7, 2011 Jan.
Artigo em Zh | MEDLINE | ID: mdl-21465809

RESUMO

The pharmaceutical ethynylestradiol (EE) is a potent endocrine modulator. Application enlargement of ethynylestradiol in clinics and abuse in livestock farming and fishing make it important to explore ethynylestradiol toxicological action on vertebrate embryonic development and to establish an in vivo method for EE toxicity detection efficiently and conveniently. In the present study, using a model animal zebrafish and 17alpha-ethynylestradiol as a representative compound, we have investigated EE2 teratogenicity, target tissues and target genes on zebrafish embryo. The results show that median teratogenesis concentration (TC50) of EE2 is 0.8 microg x mL(-1), and median lethal dose (LD50) is 3.3 microg x mL(-1). Targets of EE2 action were implicated in brain, eyes, heart, muscle, skeleton, pigment and viscera. Embryonic cardiac arrhythmia caused by EE2 is probably resulted from heart abnormal structure. The embryonic stage sensitive to EE2 mainly started at cleavage and last up to the organogenesis with time-accumulating effect. RT-PCR results indicate that EE2 treatment disturbed gene expression pattern at the early period of zebrafish embryonic development by suppressing transcription of gene boz that promotes brain development, upregulating genes for trunk and tail, such as ntl, spt, shh, and perturbing Nodal signal expression of TGFbeta superfamily, for example, cyc, sqt and oep. Using zebrafish, an efficient in vivo method for quick evaluation of EE toxicity on embryonic development has been developed.


Assuntos
Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Etinilestradiol/toxicidade , Teratogênicos/toxicidade , Peixe-Zebra/embriologia , Anormalidades Induzidas por Medicamentos/etiologia , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/embriologia , Relação Dose-Resposta a Droga , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Etinilestradiol/administração & dosagem , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/anormalidades
12.
Yao Xue Xue Bao ; 46(8): 928-35, 2011 Aug.
Artigo em Zh | 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
13.
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.


Assuntos
Traumatismos Cardíacos/diagnóstico por imagem , Traumatismos Cardíacos/fisiopatologia , Imageamento Tridimensional/métodos , Animais , Cardiotoxicidade , Caspase 3/metabolismo , Doxorrubicina/toxicidade , Fluorescência , Testes de Função Cardíaca/efeitos dos fármacos , Traumatismos Cardíacos/induzido quimicamente , Relação Quantitativa Estrutura-Atividade , Espécies Reativas de Oxigênio/metabolismo , Análise Espacial , Peixe-Zebra
14.
Autophagy ; 16(12): 2167-2179, 2020 12.
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.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interleucinas/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Hep G2 , Humanos , Fator Regulador 1 de Interferon/química , Interleucinas/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ativação Transcricional/genética
15.
Cell Death Dis ; 11(3): 200, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32205851

RESUMO

Interferon lambda-2 (IL28A) has a wide antiviral effect with fewer side-effects. Autophagy is a host mechanism to maintain intracellular homeostasis and defends invasion of pathogenic microorganisms. HCV NS5A can disable host defense systems to support HCV replication. Thus, molecular mechanism of interaction among interferon lambda, autophagy, and HCV was concerned and explored in this study. We report that HCV NS5A activated an incomplete autophagy by promoting the autophagic ubiquitylation-like enzymes ATG3, ATG5, ATG7, ATG10, and autophagosome maker LC3B, but blocked autophagy flux; IL28A bound to NS5A at NS5A-ISDR region, and degraded HCV-NS5A by promoting autolysosome formations in HepG2 cells. A software prediction of IL28A protein conformation indicated a potential structure of IL28A homotetramer; the first α-helix of IL28A locates in the interfaces among the four IL28A chains to maintain IL28A homotetrameric conformation. Co-IP and cell immunofluorescence experiments with sequential deletion mutants demonstrate that IL28A preferred a homotetramer conformation to a monomer in the cells; the IL28A homotetramer is positively correlated with autolysosomal degradation of HCV NS5A and the other HCV proteins. Summarily, the first α-helix of IL28A protein is the key domain for maintaining IL28A homotetramer which is required for promoting formation of autolysosomes and degradation of HCV proteins in vitro.


Assuntos
Hepacivirus/metabolismo , Interleucinas/metabolismo , Lisossomos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células Hep G2 , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Humanos , Interleucinas/química , Interleucinas/genética , Modelos Moleculares , Transfecção , Proteínas não Estruturais Virais/genética
16.
Toxicology ; 441: 152501, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32454074

RESUMO

Macrolide antibiotics (macrolides) are among the most commonly prescribed antibiotics worldwide and are used for a wide range of infections, but macrolides also expose people to the risk of adverse events include hepatotoxicity. Here, we report the liver toxicity of macrolides with different structures in zebrafish. The absorption, distribution, metabolism, excretion and toxicology (ADMET) parameters of macrolide compounds were predicted and contrasted by utilizing in silico analysis. Fluorescence imaging and Oil Red O stain assays showed all the tested macrolide drugs induced liver degeneration, changed liver size and liver steatosis in larval zebrafish. Through RNA-seq analysis, we found seven co-regulated differentially expressed genes (co-DEGs) associated with metabolism, apoptosis and immune system biological processes, and two co-regulated significant pathways including amino sugar and nucleotide sugar metabolism and apoptosis signaling pathway. We found that only fosab of seven co-DEGs was in the two co-regulated significant pathways. fosab encoded proto-oncogene c-Fos, which was closely associated with liver diseases. The whole-mount in situ hybridization showed high transcription of c-Fos induced by macrolide compounds mainly in the liver region of zebrafish larvae. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) leakage assays revealed that macrolides exerts significant cytotoxic effects on L02 cells. qRT-PCR and western blot analysis demonstrated macrolides also promoted human c-Fos expression in L02 cells. The c-Fos overexpression significantly reduced cell viability by using CCK-8 assay. These data indicate that hepatotoxicity induced by macrolides may be correlated with c-Fos expression activated by these compounds. This study may provide a biomarker for the further investigations on the mechanism of hepatotoxicity induced by macrolide drugs with different structures, and extend our understanding for improving rational clinical application of macrolides.


Assuntos
Antibacterianos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Macrolídeos/toxicidade , Animais , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Doença Hepática Induzida por Substâncias e Drogas/patologia , Simulação por Computador , Fígado Gorduroso/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Larva , Fígado/diagnóstico por imagem , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Proteínas Luminescentes/metabolismo , Imagem Óptica , Proto-Oncogene Mas , Reação em Cadeia da Polimerase em Tempo Real , Relação Estrutura-Atividade , Peixe-Zebra , Proteína Vermelha Fluorescente
17.
Front Pharmacol ; 10: 1504, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31969822

RESUMO

Background: The prevalence of non-alcohol fatty liver disease (NAFLD) is increasing in children and adolescents who are mostly resulted from overfeeding. Previous studies demonstrate that berberine (BBR), a compound derived from plant, has beneficial effects on NAFLD in adults but poorly understood in the pediatric population. This study employed a larval zebrafish model to mimic the therapeutic effects of BBR in the pediatric population and the mechanisms underlying its hepatoprotection. Methods: High-cholesterol diet (HCD)-fed zebrafish exposed to BBR at doses of 0, 1, 5, and 25 µM. After the larvae were treated with BBR for 10 days, its effect on hepatic steatosis was evaluated. We introduced Raman imaging and three-dimensional (3D) molecular imaging to detect changes in the biochemical composition and reactive oxygen species (ROS) levels of zebrafish liver. Gene expression microarray was performed to identify differentially expressed genes (DEGs) followed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and functional category analysis. Results: BBR (5 and 25 µM) administration prevented HCD-induced liver lipid accumulation in larval zebrafish. The result was further confirmed by the pathological observation. Raman mapping indicated that the biochemical composition in the liver of BBR-treated group shifted to the control. The quantitative analysis of 3D imaging showed that the ROS level was significantly decreased in the liver of BBR-treated larvae. In the livers of the BBR group, we found 468 DEGs, including 172 genes with upregulated expression and 296 genes with downregulated expression. Besides, GO enrichment, KEGG pathway, and functional category analysis showed that various processes related to glucolipid metabolism, immune response, DNA damage and repair, and iron were significantly enriched with DEGs. The expression levels of the crucial genes from the functional analysis were also confirmed by quantitative PCR (qPCR). Conclusion: BBR can significantly improve hepatic steatosis in HCD-fed zebrafish larvae. Its mechanisms might be associated with the regulation of lipid metabolism, oxidative stress, and iron homeostasis. Raman imaging in larval zebrafish might become a useful tool for drug evaluation. Mainly, the gene expression profiles provide molecular information for BBR on the prevention and treatment of pediatric NAFLD.

18.
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
19.
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.

20.
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.

SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa