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1.
Biomed Res Int ; 2020: 5204348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33344639

RESUMO

Zhibai Dihuang Wan (ZDW) is an eight-herbal formula of traditional Chinese medicine. Clinically, it regulated immune activity and was used to treat diabetes and renal disease. In this study, we aimed to explore the nephroprotective effect of ZDW in an aristolochic acid- (AA-) intoxicated zebrafish model. We used a green fluorescent kidney transgenic zebrafish to evaluate the nephroprotective effects of ZDW by recording subtle changes in the kidney. Our results demonstrated that ZDW treatment can attenuate AA-induced kidney malformations (60% for AA-treated, 47% for pretreatment with ZDW, and 17% for cotreatment ZDW with AA, n = 50). Furthermore, we found that the expression levels of tnfα and mpo were decreased either in pretreatment or cotreatment groups. In conclusion, our findings revealed that AA-induced nephrotoxicities can be attenuated by ZDW. Therefore, we believe that zebrafish represent an efficient model for screening AA-protective Chinese medicine.

2.
Toxics ; 8(4)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233497

RESUMO

(1) Background: Amikacin is an aminoglycoside antibiotic used for treating gram-negative bacterial infections in cancer patients. In this study, our aims are to investigate the migratory inhibition effects of amikacin in human MDA-MB-231 cells. (2) Methods: We used a wound-healing assay, trans-well analysis, Western blotting, immunostaining and siRNA knockdown approaches to investigate how amikacin influenced MDA-MB-231 cell migration and invasion. (3) Results: Wound healing showed that the MDA-MB-231 cell migration rates decreased to 44.4% in the presence of amikacin. Trans-well analysis showed that amikacin treatment led to invasion inhibition. Western blotting demonstrated that amikacin induced thioredoxin-interacting protein (TXNIP) up-regulation. TXNIP was knocked down using siRNA in MDA-MB-231 cell. Using immunostaining analysis, we found that inhibition of TXNIP expression led to MDA-MB-231 pseudopodia extension; however, amikacin treatment attenuated the cell extension formation. (4) Conclusions: We observed inhibition of migration and invasion in MDA-MB-231 cells treated with amikacin. This suggests inhibition might be mediated by up-regulation of TXNIP.

3.
Genes (Basel) ; 11(9)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899868

RESUMO

The Klotho gene functions as an anti-aging gene. A previous klotho-knockout mice study indicated that neither male nor female gametocytes could accomplish the first meiotic division. It suggested that Klotho might regulate cell division. In this study, we determined the roles of Klotho in cytokinesis in cultural human cells (HEK293 and HeLa) and in zebrafish embryos. Immunoprecipitation, mass spectrometry analysis, and a zebrafish model were used in this study. The results showed that Klotho is located in the midbody, which correlated with cytokinesis related kinases, Aurora kinase B and citron kinases, in the late stage of cytokinesis. There was a spatial correlation between the abscission site and the location of Klotho in the cytokinesis bridge. A three-dimensional structural reconstruction study demonstrated there was a spatial correlation among Klotho, Aurora kinase B, and citron kinases in the midbody. In addition, Klotho depletion inactivated Aurora kinases; it was also indicated that Klotho depletion caused aberrant cell cycle and delayed cytokinesis in a cell model. The study with zebrafish embryos suggested that klotho knockdown caused early embryo development abnormality due to dysregulated cytokinesis. In conclusion, Klotho might have a critical role in cytokinesis regulation by interacting with the cytokinesis related kinases.

4.
Int J Biol Macromol ; 126: 159-169, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30586584

RESUMO

To fulfill the properties of membrane for guided bone tissue regeneration, chitosan (CS) and calcium phosphates were blended to produce porous hybrid membranes by lyophilization. We synthesized three different calcium phosphates: calcium deficient hydroxyapatite (CDHA), biphasic calcium phosphate (BCP) and ß­tricalcium phosphate (TCP) by a reverse emulsion method followed by calcination, and compared their efficacy on bone regeneration. The CDHA/CS, BCP/CS, and TCP/CS membranes had an interconnected pore structure with porosity of 91-95% and pore size of 102-147 µm. These hybrid membranes could promote the permeability and adhesiveness to bone cells as demonstrated by in-vitro cell culture of primary osteoblast. Particularly, the CDHA/CS and BCP/CS could further increase the cell attachment and differentiation, whereas the BCP/CS and TCP/CS could enhance cell proliferation. Finally, these hybrid membranes were assessed for guided bone regeneration in the critical-size calvarial bone defects created in SD rats. Histological and histomorphometric analyses revealed that the BCP/CS membrane had the most effective bone regeneration compared to the other two hybrid membranes. At three-week post-surgery, the BCP/CS membrane could enhance new bone generation up to 57% of the original bone defect area. The BCP/CS membrane thus has the potential to be applied for guided bone regeneration.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Quitosana/farmacologia , Regeneração Tecidual Guiada , Membranas Artificiais , Animais , Fosfatos de Cálcio/química , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Liofilização , Peso Molecular , Muramidase/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Porosidade , Ratos Sprague-Dawley , Crânio/efeitos dos fármacos , Crânio/fisiologia , Resistência à Tração , alfa-Amilases/metabolismo
5.
J Clin Med ; 7(12)2018 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-30544995

RESUMO

Lower extremities varicose veins (VV) are among the most easily recognized venous abnormalities. The genetic mechanism of VV is largely unknown. In this study, we sought to explore the global expressional change of VV and identify novel genes that might play a role in VV. We used next-generation ribonucleic acid (RNA) sequence (RNA seq) technology to study the global messenger RNA expressional change in the venous samples of five diseased and five control patients. We identified several differentially expressed genes, which were further confirmed by conventional reverse transcription polymerase chain reaction (RT-PCR). Using these significant genes we performed in silico pathway analyses and found distinct transcriptional networks, such as angiogenesis, cell adhesion, vascular injury, and carbohydrate metabolisms that might be involved in the mechanism of VV. Among these significant genes, we also found hyaluronan synthases 2 gene (HAS2) played a pivotal role and governed all these pathways. We further confirmed that HAS2 expression was decreased in the venous samples of patients with VV. Finally, we used a zebrafish model with fluorescence emitting vasculature and red blood cells to see the morphological changes of the venous system and blood flow. We found that HAS2 knockdown in zebrafish resulted in dilated venous structural with static venous flow. HAS2 may modulate the transcriptional networks of angiogenesis, cell adhesion, vascular injury, and carbohydrate metabolisms in venous tissues and downregulation of HAS2 may underlie the mechanism of VV.

6.
Environ Toxicol ; 33(12): 1321-1328, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30259639

RESUMO

4-methylimidazole (4-MI) is an imidazole-derived organic chemical compound that can be used as a raw material in the manufacture of diverse chemicals and has been identified as an ingredient of caramel color in soybean sauce, beers, and other soft drinks. The aim of the present study was to investigate the teratogenic effects of 4-MI during zebrafish embryogenesis. Zebrafish embryos were treated with different dosages of 4-MI (0-120 mM) for different exposure durations (12-60 hours). The percentages of embryos with malformed phenotypes increased as the exposure dosages and duration time of 4-MI increased. We also used immunofluorescence and transmission microscopy to evaluate the subtle changes in the myofibril alignment and ultrastructure of muscle organization. Our data showed that 4-MI treatment disturbs muscle fiber alignment. Electron microscopy data indicated that Z-lines were undetectable in the 4-MI-treated embryos. Although the thick and thin filaments were visible, they were all disorganized. In addition, zebrafish embryos treated by 4-MI exhibited aberrant expression of 2 muscle-specific genes, myod and myogenin. Taken together, we concluded that early exposure to 4-MI affects zebrafish myogenesis, especially in myofibril alignment.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Imidazóis/toxicidade , Desenvolvimento Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Miofibrilas/fisiologia , Proteínas de Peixe-Zebra/metabolismo
7.
Antiviral Res ; 158: 199-205, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30138642

RESUMO

Porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) discovered in the 1970s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. It can cause extreme dehydration and death in neonatal piglets. In Asia, modified live attenuated vaccines have been used to control PEDV infection in recent years. However, a new strain of PEDV that belongs to genogroup 2a appeared in the USA in 2013 and then quickly spread to Canada and Mexico as well as Asian and European countries. Due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. The emergence of this new strain increases the importance of understanding PEDV as well as strategies for inhibiting it. Coronaviral proteases, including main proteases and papain-like proteases, are ideal antiviral targets because of their essential roles in viral maturation. Here we provide a first description of the expression, purification and structural characteristics of recombinant PEDV papain-like protease 2, moreover present our finding that 6-thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on SARS- and MERS-CoV papain-like proteases, is a noncompetitive inhibitor of PEDV papain-like protease 2.


Assuntos
Antivirais/farmacologia , Papaína/antagonistas & inibidores , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Tioguanina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus , Cinética , Simulação de Acoplamento Molecular , Papaína/química , Papaína/genética , Papaína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/genética , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes
8.
Sci Rep ; 8(1): 8085, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29795395

RESUMO

We aimed to analyze the associations of single nucleotide polymorphisms (SNP) in the 5' regulatory region of the human organic anion transporter 1 (OAT1) gene with chronic kidney disease (CKD). A case-control study including age- and sex-matched groups of normal subjects and patients with CKD (n = 162 each) was designed. Direct sequencing of the 5' regulatory region (+88 to -1196 region) showed that patients with CKD had a higher frequency of the -475 SNP (T > T/G) than normal subjects (14/162 vs. 2/162). The luciferase activity assay results indicated that the -475G SNP had a higher promoter efficiency than the -475T SNP. Chromatin immunoprecipitation (ChIP) and LC/MS/MS analyses showed that the -475G SNP up-regulated 26 proteins and down-regulated 74 proteins. The Southwestern blot assay results revealed that the -475G SNP decreased the binding of Hepatoma-derived growth factor (HDGF), a transcription repressor, compared to the -475T SNP. Overexpression of HDGF significantly down-regulated OAT1 in renal tubular cells. Moreover, a zebrafish animal model showed that HDGF-knockdown zebrafish embryos had higher rates of kidney malformation than wild-type controls [18/78 (23.1%) vs. 1/30 (3.3%)]. In conclusion, our results suggest that an OAT1 SNP might be clinically associated with CKD. Renal tubular cells with the -475 SNP had increased OAT1 expression, which resulted in increased transportation of organic anion toxins into cells. Cellular accumulation of organic anion toxins caused cytotoxicity and resulted in CKD.


Assuntos
Região 5'-Flanqueadora/genética , Proteína 1 Transportadora de Ânions Orgânicos/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Insuficiência Renal Crônica/genética , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/epidemiologia , Fatores de Risco
9.
Antiviral Res ; 150: 155-163, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29289665

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PLpros) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PLpro but as a competitive (or mixed) inhibitor of SARS-CoV PLpro. The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PLpro by disulfiram, while synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs.


Assuntos
Antivirais/farmacologia , Dissulfiram/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Peptídeo Hidrolases/metabolismo , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/enzimologia , Dissulfiram/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Modelos Moleculares , Conformação Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ligação Proteica , Vírus da SARS/genética
10.
Int J Mol Sci ; 18(8)2017 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-28783116

RESUMO

Topiramate is commonly used for treating epilepsy in both children and adults. Recent clinical data suggests that administration of topiramate to women during pregnancy increases the risk of oral clefts in their offspring. To better understand the potential effects of topiramate, we dosed adult female zebrafish with topiramate, and investigated the altered morphologies in adult females and their offspring. It showed that topiramate-treated female fish had reduced oocyte maturation, and the survival rates of their offspring were seriously decreased during embryogenesis. In addition, around 23% of offspring displayed cartilage malformation in the craniofacial area, such as loss of ceratobranchial cartilages as well as impaired ceratohyal, Meckel's cartilage and ethmoid plate development. Moreover, mineralization of ceratohyal, Meckel's cartilage, and vertebrae were downregulated during bone development. Taken together, we concluded that topiramate impaired oogenesis in the maternal reproductive system, and then caused offspring cartilage malformation or bone dysplasia.


Assuntos
Frutose/análogos & derivados , Teratogênese/efeitos dos fármacos , Teratogênios/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Anormalidades Craniofaciais/induzido quimicamente , Feminino , Frutose/farmacologia , Frutose/toxicidade , Modelos Animais , Oócitos/efeitos dos fármacos , Oócitos/patologia , Oogênese/efeitos dos fármacos , Teratogênios/toxicidade , Topiramato , Peixe-Zebra
11.
Int J Mol Sci ; 17(6)2016 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-27314333

RESUMO

R-spondin 1 (Rspo1) plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1) domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER) quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.


Assuntos
Heparina/metabolismo , Processamento de Proteína Pós-Traducional , Via Secretória , Trombospondinas/metabolismo , Sítios de Ligação , Retículo Endoplasmático/metabolismo , Glicosilação , Células HEK293 , Humanos , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Trombospondinas/química
12.
Bioorg Med Chem Lett ; 26(3): 774-777, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26764188

RESUMO

Direct coupling of a hydrophobic drug and a hydrophilic natural product via an ester bond produced an amphiphilic adduct that formed liposomes. Liposomes of resveratrol-norcantharidin adduct are capable of forming a tadpole-like nanoparticle and exhibited high toxicity in zebrafish embryos to give the better transportation and the effective concentration into cells. Using fluorescent chromophore showed the liposome in the stomach and intestinal villi rather than in the skin and muscle. This result may provide an insight into the mechanism of action of traditional Chinese medicines, which often contain a significant amount of flavonoids and polyphenol analogs.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Nanopartículas/química , Estilbenos/química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/toxicidade , Portadores de Fármacos/química , Embrião não Mamífero/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Medicina Tradicional Chinesa , Tamanho da Partícula , Resveratrol , Estilbenos/toxicidade , Peixe-Zebra/crescimento & desenvolvimento
13.
J Toxicol Pathol ; 28(3): 141-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26441476

RESUMO

The aim of this study was to evaluate the teratogenic effects of three common Chinese medical prescriptions, Si Jun Zi Tang (SJZT), Liu Jun Zi Tang (LJZT) and Shenling Baizhu San (SLBS), during zebrafish pronephros development. We used the transgenic zebrafish line Tg(wt1b:EGFP) to assess the teratogenic effects using 12 different protocols, which comprised combinations of 4 doses (0, 25, 250, 1,250 ng/mL) and 3 exposure methods [methods I, 12-36 hours post fertilization (hpf), II, 24-48 hpf, and III, 24-36 hpf]. As a result, few defects in the kidneys were observed in the embryos exposed to 25 ng/mL of each medical prescription. The percentage of kidney malformation phenotypes increased as the exposure concentrations increased (25 ng/mL, 0-10%; 250 ng/mL, 0-60%; 1,250 ng/mL, 80-100%). Immunohistochemistry for α6F, which is a basolateral and renal tubular differentiation marker, revealed no obvious defective phenotypes in either SJZT- or LJZT-treated embryos, indicating that these Chinese medical prescriptions had minimal adverse effects on the pronephric duct. However, SLBS-treated embryos displayed a defective phenotype in the pronephric duct. According to these findings, we suggest (1) that the Chinese medical prescriptions induced kidney malformation phenotypes that are dose dependent and (2) that the embryonic zebrafish kidney was more sensitive to SLBS than SJZT and LJZT.

14.
Molecules ; 20(7): 12512-24, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-26184137

RESUMO

The aim of this study was to investigate novel chalcones with potent angiogenic activities in vivo. Chalcone-based derivatives were evaluated using a transgenic zebrafish line with fluorescent vessels to real-time monitor the effect on angiogenesis. Results showed that the chalcone analogues did not possess anti-angiogenic effect on zebrafish vasculatures; instead, some of them displayed potent pro-angiogenic effects on the formation of the sub-intestinal vein. Similar pro-angiogenic effects can also be seen on wild type zebrafish embryos. Moreover, the expression of vegfa, the major regulator for angiogenesis, was also upregulated in their treatment. Taken together, we have synthesized and identified a series of novel chalcone-based derivatives as potent in vivo pro-angiogenic compounds. These novel compounds hold potential for therapeutic angiogenesis.


Assuntos
Indutores da Angiogênese/farmacologia , Chalconas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/agonistas , Proteínas de Peixe-Zebra/agonistas , Indutores da Angiogênese/síntese química , Animais , Animais Geneticamente Modificados , Chalconas/síntese química , Embrião não Mamífero , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Estrutura Molecular , Morfogênese/efeitos dos fármacos , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
In Vitro Cell Dev Biol Anim ; 51(10): 1023-32, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26194803

RESUMO

von Hippel-Lindau (pVHL)-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability. In the present study, using zebrafish as a model, we showed that specific knockdown of endogenous vhl leads to pronephros malformation and renal failure. Knockdown of vhl resulted in abnormal kidney development, including curved and cystic pronephric tubule or/and cystic and atrophic glomerulus. Co-injecting capped vhl messenger RNA (mRNA) partially rescued pronephros morphant phenotype, confirming the specificity of the morpholino oligonucleotide (MO)-induced pronephric defects. In keeping with the pronephros phenotype, renal function was affected as well in vhl morphants. Dextran clearance abilities of vhl morphants were significantly reduced as compared with those of control embryos. Further analysis indicated that glomerular integrity is impaired in vhl morphants, while the organization of pronephric duct was minimally affected. Vhl morphants display global increased vegf signaling and angiogenesis. In addition, we found that vhl morphants displayed elevated expression of vegfa in podocytes and increased angiogenesis at pronephric glomerulus and the nearby vessels. Treatment of vegf inducer to embryos also caused pronephros phenotype resembling vhl morphants, further supporting that increased vegfa signaling contribute to the pronephros morphant phenotype. Our study establishes the zebrafish as an alternative vertebrate model system for studying Vhl function during kidney development.


Assuntos
Glomérulos Renais/embriologia , Organogênese/genética , Pronefro/embriologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Dextranos/metabolismo , Técnicas de Silenciamento de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Modelos Animais , Morfolinos/genética , Neovascularização Fisiológica/genética , Podócitos/metabolismo , Policitemia/genética , Pronefro/anormalidades , RNA Mensageiro/genética , Transdução de Sinais/genética , Ubiquitinação , Peixe-Zebra/genética , Proteína da Zônula de Oclusão-1/metabolismo
16.
Toxicol Sci ; 147(1): 246-54, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26136230

RESUMO

Aristolochic acid (AA) nephropathy is complicated with early onset and severe anemia. The molecular pathological mechanism of AA-induced anemia remains unclear. The aim of this study was to evaluate the putative pathological roles of the erythropoietin receptor (EPOR) in AA-induced anemia in both AA nephropathy zebrafish and cultured human renal tubular cells (HK2). Immunofluorescence staining experiments revealed that AA colocalizes with the EPOR in zebrafish embryos as well as in the cytoplasm of HK2 cells. After exogenous EPO stimulation, the EPOR was detected in the plasma membrane of HK cells. However, cotreatment with AA and EPO inhibited EPOR signaling and its membrane localization upon EPO stimulation. The results of studies with a protein synthesis inhibitor and a lysosome inhibitor revealed that AA accelerates the lysosomal degradation of EPOR. The molecular docking results suggest that AA may interact with the N-terminus of EPOR. Together with the results of light absorption and in vitro competition assays, we concluded that AA treatment impairs EPOR membrane localization, accelerates its lysosomal degradation, and consequently downregulates EPOR signaling by direct targeting. The results of this study may further detail the pathological mechanism of severe anemia complicated with AA nephropathy.


Assuntos
Ácidos Aristolóquicos/toxicidade , Receptores da Eritropoetina/efeitos dos fármacos , Anemia/induzido quimicamente , Anemia/metabolismo , Anemia/patologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Lisossomos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra
17.
PLoS One ; 10(5): e0126750, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25978439

RESUMO

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.


Assuntos
Hipóxia/patologia , Neovascularização Patológica/patologia , Retina/patologia , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Larva/metabolismo , Neovascularização Patológica/metabolismo , RNA Mensageiro/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra
18.
Toxins (Basel) ; 7(1): 97-109, 2015 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-25590276

RESUMO

The nephrotoxicity of aristolochic acid (AA) is well known, but information regarding the attenuation of AA-induced toxicity is limited. The aim of the present study was to study the nephroprotective effects of resveratrol (Resv) and ursolic acid (UA) in a zebrafish model. We used two transgenic lines, Tg(wt1b:EGFP) and Tg(gata1:DsRed), to evaluate the nephroprotective effects of Resv and UA by recording subtle changes in the kidney and red blood cell circulation. Our results demonstrated that both Resv and UA treatment can attenuate AA-induced kidney malformations and improve blood circulation. Glomerular filtration rate assays revealed that both Resv and UA treatment can restore renal function (100% for Mock; 56.1% ± 17.3% for AA-treated; 80.2% ± 11.3% for Resv+AA; and 83.1% ± 8.1% for UA+AA, n = 15). Furthermore, real-time RT-PCR experiments showed that pre-treatment with either Resv or UA suppresses expression of pro-inflammatory genes. In conclusion, our findings reveal that AA-induced nephrotoxicities can be attenuated by pre-treatment with either Resv or UA. Therefore, we believe that zebrafish represent an efficient model for screening AA-protective natural compounds.


Assuntos
Anti-Inflamatórios/uso terapêutico , Substâncias Protetoras/uso terapêutico , Insuficiência Renal/tratamento farmacológico , Estilbenos/uso terapêutico , Triterpenos/uso terapêutico , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/farmacologia , Ácidos Aristolóquicos , Embrião não Mamífero , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/metabolismo , Resveratrol , Estilbenos/farmacologia , Triterpenos/farmacologia , Peixe-Zebra
19.
J Appl Toxicol ; 35(3): 287-94, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25186829

RESUMO

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.


Assuntos
Compostos de Boro/toxicidade , Cafeína/toxicidade , Cálcio/metabolismo , Citosol/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Citosol/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica de Transmissão , Miofibrilas/ultraestrutura
20.
J Am Soc Nephrol ; 26(2): 281-90, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25012179

RESUMO

Indoxyl sulfate and p-cresol sulfate have been suggested to induce kidney tissue remodeling. This study aimed to clarify the molecular mechanisms underlying this tissue remodeling using cultured human proximal renal tubular cells and half-nephrectomized mice treated with indoxyl sulfate or p-cresol sulfate as study models. Molecular docking results suggested that indoxyl sulfate and p-cresol sulfate dock on a putative interdomain pocket of the extracellular EGF receptor. In vitro spectrophotometric analysis revealed that the presence of a synthetic EGF receptor peptide significantly decreased the spectrophotometric absorption of indoxyl sulfate and p-cresol sulfate. In cultured cells, indoxyl sulfate and p-cresol sulfate activated the EGF receptor and downstream signaling by enhancing receptor dimerization, and increased expression of matrix metalloproteinases 2 and 9 in an EGF receptor-dependent manner. Treatment of mice with indoxyl sulfate or p-cresol sulfate significantly activated the renal EGF receptor and increased the tubulointerstitial expression of matrix metalloproteinases 2 and 9. In conclusion, indoxyl sulfate and p-cresol sulfate may induce kidney tissue remodeling through direct binding and activation of the renal EGF receptor.


Assuntos
Cresóis/farmacologia , Receptores ErbB/efeitos dos fármacos , Indicã/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Toxinas Biológicas/farmacologia , Animais , Células Cultivadas , Cresóis/administração & dosagem , Humanos , Técnicas In Vitro , Indicã/administração & dosagem , Injeções Intraperitoneais , Rim/cirurgia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Modelos Animais , Nefrectomia , Transdução de Sinais/efeitos dos fármacos , Toxinas Biológicas/administração & dosagem
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