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1.
Dis Model Mech ; 16(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35481478

RESUMO

Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.


Assuntos
Cardiomiopatias , Peixe-Zebra , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Cardiomiopatias/genética , Técnicas de Inativação de Genes , Genes Modificadores , RNA Guia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Braz. j. biol ; 83: e245202, 2023. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1285622

RESUMO

Abstract Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.


Resumo Embora a própolis tenha sido relatada por ter atividade anti-inflamatória, seus efeitos no sistema complemento, uma parte do sistema imunológico inato, não foram muito estudados. Esta pesquisa foi conduzida para descobrir os efeitos da própolis da Indonésia nos níveis de expressão de C3, C1r/s, Bf, MBL e C6 em larvas de peixe-zebra induzidas por lipopolissacarídeo (LPS). Foram realizadas contagens de macrófagos que migram para o saco vitelino e histologia do fígado. As larvas foram divididas em quatro grupos: CON (cultivadas apenas em meio E3), LPS (cultivadas em meio contendo 0,5 μg/L de LPS), LPSIBU (cultivadas em meio contendo LPS e, em seguida, tratadas com 100 μg/L de ibuprofeno por 24 horas) e LPSPRO (cultivado em meio contendo LPS, e então imerso em própolis 14,000 μg/L por 24 horas). Os resultados mostraram que a expressão do gene do complemento em larvas dos grupos LPSIBU e LPSPRO foi geralmente menor que em larvas do grupo LPS. O número de migrações de macrófagos para a gema no grupo LPSPRO também foi menor que no grupo LPS. A estrutura histológica do fígado em todos os grupos foi considerada normal. Este estudo mostra que a própolis indonésia tem potencial para ser utilizada como alternativa na substituição dos AINEs (anti-inflamatórios não esteroides).


Assuntos
Animais , Própole/farmacologia , Peixe-Zebra/genética , Regulação para Baixo , Lipopolissacarídeos/farmacologia , Indonésia , Larva/genética
3.
Cell Tissue Res ; 387(2): 275-285, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34820705

RESUMO

Isosteviol has been indicated as a cardiomyocyte protector. However, the underlying mechanism remains unclear. Thus, we sought to confirm the protective effect of isosteviol after myocardial infarction in a model of permanent coronary artery occlusion and investigate the potential proangiogenic activity in vitro and in vivo. A 4-week permanent coronary artery occlusion rat model was generated, and the protective effect of isosteviol was evaluated by echocardiographic imaging and hemodynamics assays. The coronary capillary density was tested by immunochemistry and micro-computed tomography (µCT) imaging. The effect of isosteviol on endothelial cells was determined in human umbilical vein endothelial cells (HUVECs) in vitro and Tg (kdrl: EGFP) zebrafish in vivo. We also examined the expression of related transcription factors by real-time polymerase chain reaction (RT-qPCR). Isosteviol increased ejection fraction (EF), fractional shortening (FS), cardiac systolic index (CI), maximum rate of increase of left ventricular pressure (Max dp/dt), and left ventricular systolic pressure (LVSP) by 32%, 40%, 25%, 26%, and 10%, respectively, in permanent coronary artery occlusion rats. Interestingly, it also promoted coronary capillary density by 2.5-fold. In addition, isosteviol promoted the proliferation and branching of HUVECs in vitro. It also rescued intersegmental vessel (ISV) development and improved endothelial cell proliferation by approximately fivefold (4-6) in zebrafish embryos in vivo. Isosteviol also upregulated the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in zebrafish by fourfold and 3.5-fold, respectively. Our findings suggest that isosteviol is a proangiogenic agent and that this activity is related to its protective effects against myocardial ischemia. After using the permanent coronary artery occlusion model, we demonstrated that isosteviol promotes angiogenesis directly and increases capillary density in myocardial ischemia rats. Isosteviol promotes angiogenesis in zebrafish in vivo and increases vascular endothelial cell proliferation in HUVECs and zebrafish. The angiogenesis activity of isosteviol may be correlated with VEGFA and HIF-1α signaling.


Assuntos
Infarto do Miocárdio , Fator A de Crescimento do Endotélio Vascular , Animais , Diterpenos do Tipo Caurano , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-X , Peixe-Zebra/metabolismo
4.
J Transl Med ; 20(1): 388, 2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36058942

RESUMO

BACKGROUND: Radiotherapy is the primary therapeutic option for glioblastoma. Some studies proved that radiotherapy increased the release of exosomes from cells. The mechanism by which these exosomes modify the phenotype of microglia in the tumor microenvironment to further determine the fate of irradiated glioblastoma cells remains to be elucidated. METHODS: We erected the co-culture system of glioblastoma cells and microglia. After radiation, we analyzing the immunophenotype of microglia and the proliferation of radiated glioblastoma cells. By whole transcriptome sequencing, we analyzed of circRNAs in exosomes from glioblastoma cells and microglia. We used some methods, which included RT-PCR, dual-luciferase reporter, et al., to identify how circ_0012381 from radiated glioblastoma cell-derived exosomes regulated the immunophenotype of microglia to further affect the proliferation of radiated glioblastoma cells. RESULTS: Radiated glioblastoma cell-derived exosomes markedly induced M2 microglia polarization. These M2-polarized microglia promoted the proliferation of irradiated glioblastoma cells. Circ_0012381 expression was increased in the irradiated glioblastoma cells, and circ_0012381 entered the microglia via exosomes. Circ_0012381 induced M2 microglia polarization by sponging with miR-340-5p to increase ARG1 expression. M2-polarized microglia suppressed phagocytosis and promoted the growth of the irradiated glioblastoma cells by CCL2/CCR2 axis. Compared with the effects of radiotherapy alone, the inhibition of exosomes significantly inhibited the growth of irradiated glioblastoma cells in a zebrafish model. CONCLUSIONS: Our data suggested that the inhibition of exosome secretion might represent a potential therapeutic strategy to increase the efficacy of radiotherapy in patients with glioblastoma.


Assuntos
Exossomos , Glioblastoma , MicroRNAs , Animais , Exossomos/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , MicroRNAs/genética , MicroRNAs/metabolismo , Microglia/metabolismo , Microambiente Tumoral , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
5.
Cell Transplant ; 31: 9636897221116085, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36062473

RESUMO

Nasopharyngeal carcinoma (NPC) is a unique malignant tumor of the head and neck. Despite higher survival rates by the combination of radiotherapy and chemotherapy, the recurrence or metastasis of NPC still occurs at about 10%. Therefore, there is urgent demand to develop more effective in vivo models for preclinical trials to investigate the mechanisms of NPC development and progression and to explore better treatment approaches. In this study, we transplanted human NPC CNE1 cells into zebrafish embryos to establish a xenograft model of NPC, where the proliferation and invasion behaviors of NPC cells were investigated in vivo. Combining in vitro and in vivo analyses, we found that activating transcription factor 7 (ATF7) was involved in the occurrence and development of NPC regulated by peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The zebrafish NPC xenograft model established here thereby provides an in vivo tool for exploring the occurrence and development of NPC, which may help to identify new tumor markers and develop new therapeutic strategies for the treatment of NPC.


Assuntos
Carcinoma , Neoplasias Nasofaríngeas , Animais , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Peixe-Zebra/metabolismo
6.
Zhonghua Yi Xue Za Zhi ; 102(33): 2619-2623, 2022 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-36058688

RESUMO

Objective: To construct zebrafish models for the screening of intracranial hemorrhage (ICH) associated genes. Methods: ICH zebrafish models were constructed through morpholino oligonucleotides (MOs) technique and microinjection technique, and multiple verification was performed from macro and micro perspectives. First, the normal wild-type AB strain zebrafish injected with control MO was used as the control group, and AB zebrafish embryos microinjected with MOs of genes related to development of neural crest-derived cells (NCDCs) were used as the study group, such as col8a1 MO, tfap2α MO, msx1a MO, msx2 MO, and dkk1a MO. Preliminary verification of the model was conducted under a white-light optical microscope. Then, the model was verified by Tg (flk1: gfp; gata1: dsRed) double transgenic zebrafish, with vascular endothelial cells labeled by green fluorescent protein (GFP) and red blood cell labeled by fluorescent protein (dsRed), and thus the location of cerebral hemorrhage can be observed more clearly. Specifically, zebrafish embryos were microinjected with Control MO as the control group and those microinjected with col8a1 MO as the study group. Then the embryos were cultured until 48 hours post-fertilization to observe the leakage of red blood cells under the confocal laser scanning microscope. Finally, Tg (flk1: gfp) transgenic zebrafish was used to verify the model based on the blood-brain barrier (BBB). Through the leakage of dextran-rhodamine and DAPI dyes, the destruction of BBB and the occurrence of cerebral hemorrhage in zebrafish were further clarified, and quantitative statistics were carried out to verify the relationship between NCDCs development related genes and cerebral hemorrhage phenotype, which proved that the modeling was effective. Results: The zebrafish with col8a1, tfap2α, and msx1 mutations in the study group had apparent ICH compared with wildtype zebrafish, and the prevalence of ICH was 18.18% (52/286), 23.04% (62/251), and 35.94% (23/64), respectively. While, the zebrafish with msx2 and dkk1a mutations rarely had ICH, with the ICH prevalence of 1.03% (1/97) and 1.15% (1/87), respectively. The prevalence of red blood cells leakage in Tg (flk1:gfp; gata1:dsred) double transgenic zebrafish injected with Control Mo and col8a1 Mo was 0.37% (1/273) and 18.18% (52/286) (P<0.001). The number of DAPI positive nuclei of Tg (flk1: gfp) transgenic zebrafish injected with Control Mo and col8a1 Mo was 10.05±5.27 and 60.35±3.96 (P<0.001), and the fluorescent intensity of midbrain parenchymal induced by dextran-rhodamin leakage was 2.54±4.70 and 5.13±3.52 (P<0.001). Conclusion: This study successfully constructs the ICH zebrafish models, and ICH-related genes are screened out, such as col8a1, tfap2α, msx1, and so on.


Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Hemorragia Cerebral , Dextranos , Proteínas de Fluorescência Verde , Peixe-Zebra/genética
7.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 44(4): 693-698, 2022 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-36065704

RESUMO

Circular RNAs (circRNAs),a group of highly conserved small RNAs,are characterized by a closed circular structure from precursor linear RNA through reverse splicing.They are powerful regulators of the physiological and pathological processes in organisms at different development stages.Zebrafish can be used for the high-throughput drug screening with low cost.Thus,the circRNAs associated with development and inflammation can be mined from zebrafish.Recently,a variety of circRNAs in zebrafish have been identified and characterized.Studies have proved that circRNAs play a vital role in the development and inflammation of zebrafish.The paper summarizes the classification,characteristics,and biological functions of circRNAs,and reviews the research progress in zebrafish's circRNAs.


Assuntos
RNA Circular , Peixe-Zebra , Animais , Inflamação , RNA/genética , Peixe-Zebra/genética
8.
Adv Neurobiol ; 28: 87-107, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36066822

RESUMO

Homeostatic plasticity represents a set of compensatory mechanisms that are engaged following a perturbation to some feature of neuronal or network function. Homeostatic mechanisms are most robustly expressed during development, a period that is replete with various perturbations such as increased cell size and the addition/removal of synaptic connections. In this review we look at numerous studies that have advanced our understanding of homeostatic plasticity by taking advantage of the accessibility of developing motoneurons. We discuss the homeostatic regulation of embryonic movements in the living chick embryo and describe the spinal compensatory mechanisms that act to recover these movements (homeostatic intrinsic plasticity) or stabilize synaptic strength (synaptic scaling). We describe the expression and triggering mechanisms of these forms of homeostatic plasticity and thereby gain an understanding of their roles in the motor system. We then illustrate how these findings can be extended to studies of developing motoneurons in other systems including the rodents, zebrafish, and fly. Furthermore, studies in developing drosophila have been critical in identifying some of the molecular signaling cascades and expression mechanisms that underlie homeostatic intrinsic membrane excitability. This powerful model organism has also been used to study a presynaptic form of homeostatic plasticity where increases or decreases in synaptic transmission are associated with compensatory changes in probability of release at the neuromuscular junction. Further, we describe studies that demonstrate homeostatic adjustments of ion channel expression following perturbations to other kinds of ion channels. Finally, we discuss work in xenopus that shows a homeostatic regulation of neurotransmitter phenotype in developing motoneurons following activity perturbations. Together, this work illustrates the importance of developing motoneurons in elucidating the mechanisms and roles of homeostatic plasticity.


Assuntos
Plasticidade Neuronal , Peixe-Zebra , Animais , Embrião de Galinha , Homeostase/fisiologia , Neurônios Motores , Junção Neuromuscular/fisiologia
9.
Adv Neurobiol ; 28: 259-280, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36066829

RESUMO

This chapter reviews recent work showing that vertebrate motoneurons can trigger spontaneous rhythmic activity in the developing spinal cord and can modulate the function of several different central pattern generators later in development. In both the embryonic chick and the fetal mouse spinal cords, antidromic activation of motoneurons can trigger bouts of rhythmic activity. In the neonatal mouse, optogenetic manipulation of motoneuron firing can modulate the frequency of fictive locomotion activated by a drug cocktail. In adult animals, motoneurons have been shown to regulate swimming in the zebrafish, and vocalization in fish and frogs. We discuss the significance of these findings and the degree to which motoneurons may be considered a part of these central pattern generators.


Assuntos
Geradores de Padrão Central , Animais , Geradores de Padrão Central/fisiologia , Locomoção/fisiologia , Camundongos , Neurônios Motores , Medula Espinal , Peixe-Zebra
10.
Anal Chim Acta ; 1226: 340192, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068051

RESUMO

Viscosity is an essential microenvironmental parameter, which is related to various diseases such as acute inflammation. So it is necessary to develop a probe to monitor viscosity changes during the inflammatory progression in vivo. Herein, a HPQ (2-(2'-hydroxyphenyl)-4(3H)-quinazolinone)-based fluorescent probe named HPQ-BI-V is prepared for detecting viscosity in biological systems. The introduction of benzindole groups extends the π conjugation of HPQ, resulting in far-red emission wavelength at 610 nm. When the viscosity raises from 3.11 cP to 567.1 cP, the fluorescence signal increases 711 times, indicating the high sensitivity of the probe. Furthermore, this probe displays excellent selectivity for viscosity in comparison with other interfering analytes. Furthermore, the probe has excellent photostability and outstanding response capability in the physiological pH range. Given these advantages, HPQ-BI-V can be applied for detecting viscosity changes in HepG2 cells and zebrafish. In particular, the probe can successfully visualize viscosity changes in acute inflammatory mice induced by LPS and the assessment of anti-inflammatory drug.


Assuntos
Corantes Fluorescentes , Peixe-Zebra , Animais , Modelos Animais de Doenças , Células HeLa , Humanos , Inflamação/induzido quimicamente , Camundongos , Mitocôndrias , Viscosidade
11.
Anal Chim Acta ; 1226: 340288, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36068069

RESUMO

As a member of reactive sulfur molecules, hydrogen polysulfide (H2Sn) plays a vital role in cell protection, anti-oxidative stress and regulation of redox signaling. The highly selective and sensitive detection of H2Sn was still challenging due to its special nucleophilic and electrophilic reactivity. By incorporating phenyl 2-(benzoylthio) benzoate into semi-naphthofluorescein, we developed a novel red emissive fluorescent probe SNAFL-H2Sn for the detection of a representative H2Sn (e. g. H2S2). The addition of H2S2 would rapidly trigger SNAFL-H2Sn to produce significant turn-on fluorescence signal changes at 626 nm with a linear response over a range of 2-30 µM and a detection limit of 16 nM. SNAFL-H2Sn was capable of mapping exogenous and endogenous H2S2 in living cells and zebrafish. Moreover, SNAFL-H2Sn was applied to detect endogenous H2S2 under atorvastatin stimulation. The present study demonstrated that SNAFL-H2Sn potentially served as a promising tool for interrogating H2Sn functions in biological systems.


Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Animais , Dissulfetos , Hidrogênio , Sulfeto de Hidrogênio/metabolismo , Sulfetos/metabolismo , Regulação para Cima , Peixe-Zebra
12.
Ecotoxicol Environ Saf ; 241: 113825, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36068752

RESUMO

Ambient fine particulate matter (PM2.5) is a major environmental health problem worldwide, and recent studies indicate that maternal PM2.5 exposure is closely associated with congenital heart diseases (CHDs) in offspring. We previously found that supplementation with folic acid (FA) or Resveratrol (RSV) could protect against heart defects in zebrafish embryos exposed to extractable organic matter (EOM) from PM2.5 by targeting aryl hydrocarbon receptor (AHR) signaling and reactive oxygen species (ROS) production respectively. Thus, we hypothesized that FA combined with RSV may have a synergistic protective effect against PM2.5-induced heart defects. To test our hypothesis, we treated zebrafish embryos with EOM in the presence or absence of FA, RSV or a combination of both. We found that RSV and FA showed a clear synergistic protection against EOM-induced heart defects in zebrafish embryos. Further studies showed that FA and RSV suppressed EOM-induced AHR activity and ROS generation respectively. Although only RSV inhibited EOM-induced apoptosis, FA enhanced the inhibitory effect of RSV. Moreover, vitamin C (VC), a typical antioxidant, also exhibits a synergistic inhibitory effect with FA on EOM-induced apoptosis and heart defects. In conclusion, supplementation with FA and RSV have a synergistic protective effect against PM2.5-induced heart defects in zebrafish embryos by targeting AHR activity and ROS production respectively. Our results indicate that, in the presence of antioxidants, FA even at a low concentration level could protect against the high risk of CHDs caused by air pollution.


Assuntos
Cardiopatias Congênitas , Material Particulado , Animais , Antioxidantes/farmacologia , Ácido Fólico/farmacologia , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/prevenção & controle , Material Particulado/toxicidade , Espécies Reativas de Oxigênio , Resveratrol/farmacologia , Peixe-Zebra
13.
Ecotoxicol Environ Saf ; 241: 113838, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36068762

RESUMO

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous and persistent contaminant in aquatic ecosystems. Chronic toxicity information for aquatic organisms is limited, therefore we conducted chronic PFOS toxicity tests for four model organisms commonly used for freshwater toxicology assays: Chironomus dilutus (midge), Ceriodaphnia dubia (water flea), Hyalella azteca (amphipod) and Danio rerio (zebrafish). The 16-day survival test with C. dilutus resulted in the lowest PFOS exposure concentrations to cause significant impacts, with reduced survival at 1 µg/L, a LC50 of 7.5 µg/L, and a growth EC10 of 1.5 µg/L. D. rerio was the next most sensitive species, with a 30-day LC50 of 490 µg/L and reduced growth at 260 µg/L. Effects for C. dubia and H. azteca occurred at concentrations a thousand-fold higher than for C. dilutus. H. azteca had a 42-day LC50 of 15 mg/L, an EC50 of 3.8 mg/L for reproduction (neonates per female) and an EC50 of 4.7 mg/L for growth. C. dubia was similarly tolerant of PFOS, with a 6-day LC50 of 20 mg/L for survival and an EC50 of 7 mg/L for reproduction (neonates per adult). H. azteca, C. dubia, and, to a lesser extent, D. rerio, appear tolerant of PFOS concentrations typically found in the environment. However, in agreement with previous studies, C. dilutus was particularly sensitive to PFOS exposure, with lethal and sublethal effects occurring at concentration levels present at highly contaminated sites.


Assuntos
Anfípodes , Chironomidae , Cladóceros , Poluentes Químicos da Água , Ácidos Alcanossulfônicos , Animais , Ecossistema , Feminino , Fluorcarbonetos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra
14.
Biomed Pharmacother ; 153: 113420, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076542

RESUMO

Betulin is the primary anti-inflammatory component of Betula platyphylla suk. cortex (birch bark), a time-honored Traditional Chinese Medicine (TCM) for healing trauma and tissue regeneration. However, the tissue regeneration effects and underlying molecular mechanism of betulin remain unknown. Therefore, it is necessary to investigate the wound repair effects and validate the mechanism of betulin in an appropriate model. In the present study, we evaluated the effects of tissue regeneration, melanin scavenging, and reactive oxygen species (ROS) inhibition of betulin using a zebrafish model. The mechanism of target genes and pathways were confirmed using quantitative polymerase chain reaction and western blotting in vivo, while molecular docking, absorption, distribution, metabolism, excretion, and toxicity investigations in-silico were conducted. Betulin significantly promoted the regeneration of zebrafish caudal fin length and area and alleviated melanin aggregation, as well as ROS generation. The relative mRNA expression of IL-1ß, TNF-α, p38α, ERK1/2, and Caspase3, and the relative protein expression of p38α, ERK1/2, Caspase3, phosphorylated proteins of p-p38α, p-ERK1/2, and p-p65 were down-regulated following betulin administration. Meanwhile, the protein ratios of p-p38α/p38α, p-ERK/ERK, and p-p65/p65 were significantly decreased. In an in-silico study, binding affinities between betulin and P38α, ERK1, ERK2, and Caspase3, and the pharmacokinetic profile of betulin were predicted. The findings suggest that the tissue regeneration mechanism of betulin is based on the inhibition of excessive inflammatory responses, melanin aggregation, and the pro-apoptotic factor, Caspase3, during the proliferation phase via the ROS/MAPKs/NF-ĸB signaling axis. Our results suggest betulin as a potential candidate for tissue regeneration.


Assuntos
NF-kappa B , Peixe-Zebra , Animais , Melaninas , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triterpenos , Peixe-Zebra/metabolismo
15.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076906

RESUMO

A retrospective study of 200 psoriasis patients and 100 healthy donors in a Spanish cohort was carried out to study the comorbidities associated with psoriasis and their association with the response to phototherapy. The results showed a higher incidence of psychiatric disease, liver disease, kidney disease, hypertension, heart disease, vascular disease, diabetes, gastrointestinal disease, autoimmune and infectious diseases, dyslipidemia, and psoriatic arthritis in patients with psoriasis than in the control group. The incidence of comorbidities was higher in psoriasis patients over 40 years old than in the control individuals of the same age, which could be indicative of premature aging. Phototherapy was seen to be an effective treatment in cases of moderate-severe psoriasis, total whitening being achieved in more than 30% of patients, with women showing a better response than men. Narrow-band ultraviolet B was found to be the most effective type of phototherapy, although achievement of PASI100 was lower in patients with liver disease, hypertension, heart disease, vascular disease, or diabetes. Strikingly, liver disease and anemia comorbidities favored therapeutic failure. Finally, zebrafish and human 3D organotypic models of psoriasis point to the therapeutic benefit of inhibiting the glucose transporter GLUT1 and the major regulator of blood glucose dipeptidyl peptidase 4. Our study reveals that specific comorbidities of psoriasis patients are associated to failure of phototherapy and, therefore, need to be considered when planning treatment for these patients.


Assuntos
Hipertensão , Psoríase , Terapia Ultravioleta , Adulto , Animais , Feminino , Humanos , Masculino , Fototerapia/métodos , Psoríase/tratamento farmacológico , Psoríase/terapia , Estudos Retrospectivos , Terapia Ultravioleta/métodos , Peixe-Zebra
16.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076925

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.


Assuntos
Displasia Arritmogênica Ventricular Direita , Adulto , Animais , Displasia Arritmogênica Ventricular Direita/genética , Heterozigoto , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Peixe-Zebra/genética
17.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076956

RESUMO

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (INa, ICaL, ICaT, IKr, IKs) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium IKr step current density in the range of testing potentials from 0 to +30 mV, IKr tail current density in the range of testing potentials from +10 to +70 mV, and IKs current density in the range of testing potentials from -10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (INa) and L- and T-type calcium currents (ICaL and ICaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K+ current (IKr and IKs). This mechanism could be favorable for cardiac protection.


Assuntos
Inibidores do Transportador 2 de Sódio-Glicose , Peixe-Zebra , Potenciais de Ação , Animais , Compostos Benzidrílicos , Glucosídeos , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Canais de Potássio , Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Peixe-Zebra/metabolismo
18.
Int J Mol Sci ; 23(17)2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36076969

RESUMO

Lead (Pb) is an important raw material for modern industrial production, they enter the aquatic environment in several ways and cause serious harm to aquatic ecosystems. Lead ions (Pb2+) are highly toxic and can accumulate continuously in organisms. In addition to causing biological deaths, it can also cause neurological damage in vertebrates. Our experiment found that Pb2+ caused decreased survival, delayed hatching, decreased frequency of voluntary movements at 24 hpf, increased heart rate at 48 hpf and increased malformation rate in zebrafish embryos. Among them, the morphology of spinal malformations varied, with 0.4 mg/L Pb2+ causing a dorsal bending of the spine of 72 hpf zebrafish and a ventral bending in 120 hpf zebrafish. It was detected that spinal malformations were mainly caused by Pb2+-induced endoplasmic reticulum stress and apoptosis. The genetic changes in somatic segment development which disrupted developmental polarity as well as osteogenesis, resulting in uneven myotomal development. In contrast, calcium ions can rescue the series of responses induced by lead exposure and reduce the occurrence of spinal curvature. This article proposes new findings of lead pollution toxicity in zebrafish.


Assuntos
Curvaturas da Coluna Vertebral , Poluentes Químicos da Água , Animais , Ecossistema , Embrião não Mamífero/anormalidades , Desenvolvimento Embrionário/genética , Chumbo/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética
19.
Int J Mol Sci ; 23(17)2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36076983

RESUMO

Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that hdac6, hdac8, and nfkbiaa expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of hdac6, hdac8, or nfkbiaa with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in hdac6-depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.


Assuntos
Injúria Renal Aguda , Pronefro , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Mamíferos/metabolismo , Camundongos , NF-kappa B , Pronefro/metabolismo , Pronefro/patologia , Proteínas Repressoras , Peixe-Zebra/metabolismo
20.
Front Endocrinol (Lausanne) ; 13: 985304, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36120446

RESUMO

Low-density Lipoprotein Receptor-related Protein 5 (LRP5) functions as a co-receptor for Wnt ligands, controlling expression of genes involved in osteogenesis. In humans, loss-of-function mutations in LRP5 cause Osteoporosis-Pseudoglioma syndrome, a low bone mass disorder, while gain-of-function missense mutations have been observed in individuals with high bone mass. Zebrafish (Danio rerio) is a popular model for human disease research, as genetic determinants that control bone formation are generally conserved between zebrafish and mammals. We generated lrp5- knock-out zebrafish to study its role in skeletogenesis and homeostasis. Loss of lrp5 in zebrafish leads to craniofacial deformities and low bone mineral density (total body and head) at adult ages. To understand the mechanism and consequences of the observed phenotypes, we performed transcriptome analysis of the cranium of adult lrp5 mutants and siblings. Enrichment analysis revealed upregulation of genes significantly associated with hydrolase activity: mmp9, mmp13a, acp5a. acp5a encodes Tartrate-resistant acid phosphatase (TRAP) which is commonly used as an osteoclast marker, while Matrix metalloprotease 9, Mmp9, is known to be secreted by osteoclasts and stimulate bone resorption. These genes point to changes in osteoclast differentiation regulated by lrp5. To analyze these changes functionally, we assessed osteoclast dynamics in mutants and observed increased TRAP staining, significantly larger resorption areas, and developmental skeletal dysmorphologies in the mutant, suggesting higher resorptive activity in the absence of Lrp5 signaling. Our findings support a conserved role of Lrp5 in maintaining bone mineral density and revealed unexpected insights into the function of Lrp5 in bone homeostasis through moderation of osteoclast function.


Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Osteoclastos , Animais , Humanos , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mamíferos , Metaloproteinase 9 da Matriz , Fosfatase Ácida Resistente a Tartarato , Peixe-Zebra/genética
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