Analysis of the key networks,metabolic pathways,and regulation substances of hypoxia based on the omics and zebrafish model

OBJECTIVE Hypoxia is associated with many complicated pathophysiological and biochemical processes that integrated and regulated via the key gene,protein and endogenous metabolite levels.Up to date,the exact molecular mechanism of hypoxia still remains unclear.In this work,we further explore the molecular mechanism of hypoxia and adaption to attenuate the damage in zebrafish model that have potential to resist hypoxic environment.METHODS The hypoxic zebrafish model was established in different concentration of oxygen with 3%,5%,10%,21%in water.The brain tissue was separated and the RNA-seq was used to identify the differentially expressed genes.The related endogenous metabolites profiles were obtained by LC-HDMS,and the multivariate statistics was applied to discover the important metabolites candidates in hypoxic zebrafish.The candidates were searched in HMDB,KEGG and Lipid Maps databases.RESULTS The zebrafish hypoxic model was successfully constructed via the different concentration of oxygen,temperature and hypoxic time.The activities of the related hypoxic metabolic enzymes and factors including HIF-1a,actate dehydrogenase(LDH)and citrate synthase(CS)were evaluated.Significant differences(P<0.05 and fold change>2)in the expression of 422 genes were observed between the normal and 3% hypoxic model.Among them,201 genes increased depended on the lower concentration of oxygen.53 metabolites were identified that had significant difference between the hypoxia and control groups(P<0.05,fold change>1.5 and VIP>1.5).The ten key metabolites were increased gradually while six compounds were decreased.The endogenous hypoxic metabolites of phenylalanine,D-glucosamine-6P and several important lipids with the relevant hub genes had similar change in hypoxic model.In addition,the metabolic pathways of phenylalanine,glutamine and glycolipid were influenced in both the levels of genes and metabolites.CONCLUSION The up-regulation of phenylalanine,D-glucosamine-6P and lipid may have further understanding of protective effect in hypoxia.Our data provided an insight to further reveal the hypoxia and adaptation mechanism.

Establishment of an adult zebrafish model of retinal neurodegeneration induced by NMDA

AIM: To establish a model of retinal neurodegeneration induced by N-Methyl-D-aspartic acid(NMDA) in adult zebrafish.METHODS: We compared the effects of three different NMDA delivery methods on retinal neurodegeneration in adult zebrafish: immersion(I.M.), intravitreal injection(I.V.), and intraperitoneal injection(I.P.), and examined retinal pathology and degeneration by hematoxylin and eosin and TUNEL staining in the treated zebrafish. Effects of the NMDA receptor antagonist MK-801 and the natural product resveratrol on NMDA-induced retinal neurodegeneration were also assessed.RESULTS: The thickened inner retina was seen in histology with 100 μmol/L NMDA by I.M. administration. Significant apoptosis in the retinal ganglion cell layer and retinal thickness reduction occurred in 0.5 mol/L NMDA I.P. administration group.Seizure-like behavioral changes, but no retinal histological alteration occurred in 16 mg/kg NMDA I.P. administration group. Resveratrol and MK-801 prevented NMDA-induced retinal neurodegeneration in the zebrafish. CONCLUSION: Among the three drug administration methods, I.V. injection of NMDA is the most suitable for establishment of an acute retinal damage model inzebrafish. I.M. with NMDA is likely the best for use as a chronic retinal damage model. I.P. treatment with NMDA causes brain damage. Resveratrol and MK801 may be a clinically valuable treatment for retinal neurodegeneration.

c-myb is involved in CML progression and is a therapeutic target in the zebrafish CML model

Background:Despite the success of tyrosine kinase inhibitors in chronic myeloid leukemia(CML)therapy,CML still faces the challenges of drug resistance and progression to blast crisis.Twenty-five percent of patients have imatinib resistance and treatment difficulties due to heterogeneity after progression,but little is known about the mechanism.A key transcription factor in hematopoiesis,MYB,has been reported to increase abnormally in several types of aggressive blood disorders including CML.Methods:This study used a zebrafish model to explore the relationship between BCR/ABL1 and c-myb in CML progression.A CML zebrafish model was crossed with a c-myb hyperactivity transgenic line.Results:It was found that both exogenous BCR/ABL1 and c-myb could up-regulate the expression of neutrophil-related genes.More seriously,neutrophil accumulation was observed when BCR/ABL1 was combined with c-myb overexpression.Further studies showed that c-myb may be one of the downstream targets of BCR/ABL1 and the effect of BCR/ABL1 on neutrophils was c-myb dependent.Taking advantage of this inheritable in vivo model,it was shown that a combination of imatinib and flavopiridol,a cyclin-dependent kinase inhibitor targeting MYB,could more effectively alleviate the aggressive phenotype of the double transgene line.Conclusion:In summary,this study suggests that c-myb acts downstream of BCR/ABL1 and is involved in CML progression and is therefore a risk factor and a valuable target for the treatment of CML progression.The model used in the study could be helpful in high-throughput drug screening in CML transformation.

A novel zebrafish vascular injury model for asseessing drug efficancy of Yangxue Qingnao granules

OBJECTIVE To assess the efficancy of Yangxue Qingnao granules on simvastatininduced vascular injury model in zebrafish.METHODS Since statins can inhibit vascular development in zebrafish,in this study,we developed a novel animal model using 1 to 3 day post-fertilization larval zebrafish by optimizing the doses and duration of simvastatin exposure.Five pro-angiogenic drugs with a variety of mechanisms were tested to validate the newly developed zebrafish model.Zebrafish was treated with different concentration of Yangxue Qingnao granules(62.5,125 and 250 μg·mL-1) for 2 d then tested for the area of subintestinal vein vessels.RESULTS Vascular regeneration promoting effect of five pro-angiogenic drugs(calycosin,astragaloside,chlorogenic acid,ferulic acid and Panax Notoginseng Saponins) were 8-48%,24-51%,35-58%,28-75% and 37-69%,respectively.In 62.5,125 and 250 μg·mL-1 Yangxue Qingnao granules group,vascular regeneration promoting effect were 21%(P>0.05),84%(P<0.01) and 53%(P<0.01).CONCLUSION Our results demonstrate that the zebrafish vascular injury model validated in this study could be used for in vivo angiogenesis studies and drug screening and for assessing pro-angiogenic drugs with different mechanisms.Yangxue Qingnao granules could promote the vascular regeneration in zebrafish.

Comparison of Two Chemically-Induced Colitis-Models in Adult Zebrafish, Using Optical Projection Tomography and Novel Transcriptional Markers

Crohn’s disease and ulcerative colitis—inflammatory bowel diseases (IBD)—are chronic conditions with an inadequately understood pathogenesis. Employing a set of novel molecular markers in a gene expression assay (qPCR), we have used adult zebrafish to investigate two acute inflammatory models, induced by the haptenizing agents oxazolone and TNBS. In addition, goblet cell dynamics in the scales and intestine and 5-HT (serotonin) in intestinal tissues were investigated through optical projection tomography. Gene expression studies revealed a distinct and significant upregulation of proinflammatory cytokines, acute-phase reactants and metalloprotease 9 in both chemical models, primarily after 72 hours. In comparison, transcription factors and cytokines associated with Th1 and Th17 (Crohn’s) and Th2 (ulcerative colitis) were mainly not affected in this acute setting. However, elevated transcript levels were detected in Foxp3, IL-10 and T-bet, which are linked with tolerance and Tregs in mammals. Goblet cells in scales were depleted in both chemical models and in the intestine of oxazolone-treated fish. A marked 5-HT signal was noted in intestinal tissue of some chemically treated zebrafish. In conclusion, a distinct acute inflammatory reaction was induced in both chemical models. Further, oxazolone and TNBS did not result in clear-cut Th2 and Th1/Th17 pathway signaling at this early timepoint, but the applied molecular tools may provide further insight to the IBD pathogenesis and translational value of the IBD zebrafish model.

Novel MIP gene mutation causes autosomal-dominant congenital cataract

●AIM:To identify disease-causative mutations in families with congenital cataract.●METHODS:Two Chinese families with autosomaldominant congenital cataract(ADCC)were recruited and underwent comprehensive eye examinations.Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family.Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis.The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis.Major intrinsic protein(MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos.Zebrafish embryonic lens phenotypes were screened using confocal microscopy.●RESULTS:A novel heterozygous mutation(c.85G>A;p.G29R)in the MIP gene was identified in the proband of one family.A known heterozygous mutation(c.97C>T;p.R33C;rs864309693)in MIP was found in the proband of another family.In-silico prediction indicated that the novel mutation might affect the MIP protein function.Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP.●CONCLUSION:Two missense mutations in the MIP gene in Chinese cataract families are identified,and one of which is novel.These findings expand the genetic spectrum of MIP mutations associated with cataracts.The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

基于模式生物斑马鱼对口腔炎合剂的胚胎及肝肾发育的影响

目的利用斑马鱼为动物模型,初步分析口腔炎合剂对胚胎及肝肾发育的影响。方法待斑马鱼发育至受精后10 h(10 hpf)时以简单随机方法分组并添加梯度浓度的口腔炎合剂。药物作用72 h后在显微镜下观察口腔炎合剂对胚胎及肝肾发育,并记录图像数据。结果当口腔炎合剂稀释浓度高于1.625×10^(-5)g/mL时对10 hpf斑马鱼开始出现胚胎发育毒性,表现为发育迟缓、心腔狭窄等。当口腔炎合剂稀释浓度高于6.500×10^(-5)g/mL时对36 hpf斑马鱼产生肝肾发育毒性,主要表现为肝脏与周边组织分界线不太明显,肾脏和鱼鳔区域明显增大,并伴有水肿瘀滞现象。结论口腔炎合剂具有一定的胚胎及肝肾毒性,孕妇和哺乳期妇女,以及肝肾功能不全者原则上不得使用本品,必须使用时应严格限量,并在医生指导下谨慎使用。

斑马鱼模型在特发性脊柱侧凸研究中的应用

特发性脊柱侧凸(IS)指原因不明的脊柱发育异常,给患者带来身体和经济的双重负担。然而,目前IS的致病机制尚未完全明确,疾病的治疗通常以大创口的外科手术干预为主。为了探索IS的病因,并为新的诊疗方法提供理论基础,采用各种模式动物进行实验尤为必要。作为一种新兴的模式动物,斑马鱼繁殖周期短、培育成本低,且具备体外受精、体外发育和胚胎透明等特点,因此其遗传编辑和观测均较传统模式动物更容易。目前斑马鱼模型已成为研究IS的重要工具,其在揭示IS的分子机制和潜在治疗靶点方面展现出巨大潜力。

斑马鱼在肝病模型中的研究进展

肝脏是人体进行能量代谢的主要场所。肝病是严重影响人们生活质量的临床疾病之一,多种因素导致肝脏功能及结构异常。目前关于肝病模型的基础研究已取得一定进展,多种肝病模型用于肝病机制的研究,可有效复制肝病的临床特征及病变过程。采用大鼠、小鼠复制肝病模型均存在弊端,基础实验研究中采用斑马鱼复制肝病模型具有自身优势,在肝病治疗药物筛查中具有重要意义。全面了解斑马鱼在肝病模型中的作用,可以为相关疾病的治疗提供新思路。

棕榈酸诱导斑马鱼脂毒性损伤骨形成抑制模型的建立

目的 建立棕榈酸诱导的斑马鱼脂毒性损伤骨形成抑制模型。方法 将AB品系斑马鱼胚胎分为对照组、棕榈酸组(PA组)和辛伐他汀组(SIM组)。从3 dpf(days post fertilization, dpf)开始,PA组、SIM组给予PA造模。从5 dpf开始,SIM组给予SIM连续给药4 d。9 dpf通过钙黄绿素染色法、尼罗红染色法、甘油三酯及总胆固醇含量测定、q-PCR判断模型是否成功建立。结果 PA显著减少斑马鱼椎骨骨节数目、促进脂质堆积、增加甘油三酯及总胆固醇含量、促进成脂相关基因PPARγ、c/EBPα的表达并抑制成骨相关基因ALP、RUNX2的表达。SIM可以改善PA对斑马鱼的骨形成抑制效应。结论 采用PA给药的方法可成功造成与骨质疏松症(osteoporosis, OP)病理过程相似的脂毒性损伤骨形成抑制模型,该方法简便、灵敏、可控,可用于OP及相关疾病的药物筛选。

Animal models of amyotrophic lateral sclerosis： a comparison of model validity

Animal models are necessary to investigate the pathogenic features underlying motor neuron degeneration and for therapeutic development in amyotrophic lateral sclerosis（ALS）. Measures of model validity allow for a critical interpretation of results from each model and caution from over-interpretation of experimental models. Face and construct validity refer to the similarity in phenotype and the proposed causal factor to the human disease, respectively. More recently developed models are restricted by limited phenotype characterization, yet new models hold promise for novel disease insights, thus highlighting their importance. In this article, we evaluate the features of face and construct validity of our new zebrafish model of environmentally-induced motor neuron degeneration and discuss this in the context of current environmental and genetic ALS models, including C9 orf72, mutant Cu/Zn superoxide dismutase 1 and TAR DNA-binding protein 43 mouse and zebrafish models. In this mini-review, we discuss the pros and cons to validity criteria in each model. Our zebrafish model of environmentally-induced motor neuron degeneration displays convincing features of face validity with many hallmarks of ALS-like features, and weakness in construct validity. However, the value of this model may lie in its potential to be more representative of the pathogenic features underlying sporadic ALS cases, where environmental factors may be more likely to be involved in disease etiology than single dominant gene mutations. It may be necessary to compare findings between different strains and species modeling specific genes or environmental factors to confirm findings from ALS animal models and tease out arbitrary strain-and overexpression-specific effects.

骨髓增生异常综合征在斑马鱼中的研究进展

骨髓增生异常综合征(myelodysplastic syndrome,MDS)是一种常见的血液疾病,其发病率呈现逐年上升的趋势。斑马鱼作为一种生物医学研究的模式生物,广泛应用于各类疾病的研究中。本文关注了近年来血液疾病在斑马鱼中的研究,MDS在小鼠、大鼠和斑马鱼中的研究及动物模型的应用,揭示了斑马鱼模型在其中发挥的重要作用。

成年斑马鱼脊髓损伤模型的制备和评估

目的:建立一种成年斑马鱼脊髓损伤(SCI)模型。方法:将成年斑马鱼随机分为正常组、假手术组和SCI组。SCI组进行脊髓完全横断手术,假手术组对斑马鱼的脊髓只暴露而不切断,正常组不做特殊处理。通过测试处理后斑马鱼的自由游泳能力和顺行轴突追踪来评估神经再生的情况。结果:假手术组与正常组斑马鱼5 min游泳路径的平均距离比较,差异无统计学意义(P>0.05),与第1周相比,SCI组斑马鱼第6周的5 min游泳路径的平均距离显著增加(P<0.05)。顺行神经示踪显示SCI组斑马鱼在损伤后2周出现可观察的轴突恢复。结论:成年斑马鱼具有显著的SCI恢复能力,本实验建立的成年斑马鱼SCI模型技术成熟、容易复制,可应用于SCI的相关研究。

TPI缺乏症斑马鱼模型的构建及分析

磷酸丙糖异构酶缺乏症(triosephosphate isomerase deficiency,TPI DF)是一种严重的多系统退行性疾病,通常表现为溶血性贫血、神经肌肉功能障碍和易感染,患者多于起病5年内死亡。目前尚不清楚TPI DF的具体发病机制,缺乏有效的临床治疗方法。本研究选取TPI DF患者中最常见的突变位点TPI1^(E105D),构建了表达人源性TPI1^(E105D)(hTPI1^(E105D))的转基因斑马鱼(Danio rerio)模型[Tg(Ubi:TPI1^(E105D)-eGFP)]。功能分析表明,过表达TPI1^(E105D)影响红系及髓系细胞发育、导致神经以及肌肉发育异常。综上所述,本研究构建了磷酸丙糖异构酶缺乏症的斑马鱼疾病模型,并能够复现TPI DF患者的大部分临床表型,该模型为后续研究TPI DF的发病机制及药物筛选提供了新的实验动物模型。

桔梗改善皮肤色素沉着的研究

目的:探究桔梗提取物对皮肤色素沉着的功效及其作用机制。方法:利用自由基清除实验研究桔梗提取物对DPPH自由基的清除作用,通过体外酪氨酸酶抑制实验和斑马鱼黑素模型实验研究桔梗提取物改善皮肤色素沉着的功效,最后结合网络药理学探究桔梗活性成分改善皮肤色素沉着的作用机制。结果:桔梗提取物具有显著的抗氧化作用和酪氨酸酶抑制活性,对斑马鱼色素沉着也有显著的改善功效。网络药理学研究发现,桔梗提取物可能通过调节AKT1、TYR和IL-6等细胞靶点及调控PI3K-AKT和脂质代谢等机制通路改善色素沉着。结论:桔梗提取物具有改善色素沉着的功效。

斑马鱼二型糖尿病模型的建立

目的 为系统研究二型糖尿病(T2DM)的发生发展机制,构建二型糖尿病斑马鱼模型,并对模型构建情况进行评价。方法 本研究采用葡萄糖浸泡法,将两组野生型蓝斑马鱼养在相同的环境中,光照、喂食、水源等条件一致,实验组斑马鱼隔天浸入2%的葡萄糖溶液中,历时28 d,构建斑马鱼二型糖尿病模型。通过观察形态学变化、体质量曲线绘制以及糖脂代谢生化检测等指标来评价模型的构建情况。结果 经过28 d,实验组斑马鱼体质量增长曲线明显高于对照组。在诱导过程中,实验组斑马鱼表现出游动速度加快、对环境刺激更敏感、排出白色代谢物等行为学改变,而对照组蓝斑马鱼保持正常游速且排出棕色代谢物。实验组斑马鱼的眼睛呈现出红肿的外观。同时,实验组斑马鱼组织葡萄糖水平、三酰甘油水平以及总胆固醇水平明显高于对照组(P<0.05)。结论 隔天葡萄糖浸泡法历经28 d可成功建立二型糖尿病斑马鱼模型,采用行为学观察、体质量增长曲线绘制和糖脂含量生化检测3种手段可以评价斑马鱼的疾病状态。

Housing Environment Affects Baseline Anxiety in Zebrafish

Neuroscience researchers that wish to address compelling questions in psychopharmacology would benefit from the use of zebrafish, which have a behavioral repertoire that is rich and complex and that reflects many fundamental processes in humans, such as those that evoke anxiety. Zebrafish behavior is easily quantified in diverse test environments, but it is important to consider the conditions used to house the fish, prior to the behavioral testing, when designing experiments. Studies show that fish housed at high densities exhibit elevated cortisol levels as compared with fish housed at lower densities. The literature is less clear about the behavioral consequences of housing and handling. We examined the effects of housing (group versus paired) on several behavioral parameters. We observed that group-housed fish exhibited an anxious phenotype as evidenced by tank diving and meandering. Other more general indicators of movement and spatial navigation were unaffected by housing. This study focused on two specific housing environments, and the results support the need for continued research in this area to further elucidate the contributions of housing and handling on the subtleties of zebrafish behavioral markers.

Efficacy Assessment of Whitening Cosmetics in Animal Models

Animal skin is generally preferred to evaluate the efficacy and safety:several animal models have been used,e.g.,the guinea pig,the mouse,and the zebrafish.The epidermis of guinea pigs displays a moderate number of melanocytes and melanosomes distributed in a similar way to human skin.Guinea pig and human skins have given the close morphologic and functional similarities(similar epidermis thickness,similar epidermal cells turnover time,etc.).The zebrafish presents several advantages,including easy maintenance and handling of the animals,short generation times,and high efficiency of drug penetration through the skin.To establish the animal model and to assess whitening efficacy and safety for whitening products.

基于斑马鱼模型及网络药理学研究云南金花茶多酚的抗炎作用

为研究云南金花茶多酚(Camellia fascicularis polyphenols,CFP)发挥抗炎作用的分子机制,采用斑马鱼模型对CFP进行抗炎活性评价,利用超高效液相色谱-四极杆-飞行时间串联质谱(ultra-performance liquid chromatography combined with quadrupole-time-of-flight mass/mass spectrometry,UPLC-Q-TOF-MS/MS)技术分析其化合物组成,通过SwissTargetPrediction、DisGeNET等数据库获得有效成分和炎症的作用靶点,借助STRING数据库构建蛋白-蛋白互作网络(protein-protein interaction,PPI),通过DAVID数据库进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,使用AutoDockTools和PyMOL软件对关键成分和核心蛋白进行分子对接及可视化。结果显示,从CFP中鉴定出21个酚类化合物,筛选出32个交集靶点,GO功能和KEGG通路富集分析显示CFP可能通过细胞增殖的正向调节、细胞质、三磷酸腺苷(adenosine triphosphate,ATP)结合等生物学过程作用于甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,PTGS2)、基质金属蛋白酶2(matrix metallopeptidase 2,MMP2)等核心靶点,进一步调控花生四烯酸代谢、低氧诱导因子-1(hypoxia inducible factor-1,HIF-1)、血小板活化等信号通路来发挥抗炎作用。分子对接结果表明,云南金花茶多酚类关键成分与炎症靶点间存在良好的结合活性。本研究可为云南金花茶多酚类成分抗炎产品的开发提供理论参考。