Isolation and Characterization of Efficient Active Compounds Using High-Performance Centrifugal Partition Chromatography (CPC) from Anti-Inflammatory Activity Fraction of Ecklonia maxima in South Africa

Ecklonia maxima is a brown seaweed, which is abundantly distributed in South Africa. This study investigated an efficient approach using high-performance centrifugal partition chromatography (HPCPC), which has been successfully developed for the isolation and purification of phlorotannins, eckmaxol, and dieckol from the ethyl acetate fraction of E. maxima (EEM). We evaluated EEM for its inhibitory effect against lipopolysaccharide (LPS)-induced inflammatory responses in zebrafish embryos. The separation of eckmaxol and dieckol from samples of EEM using HPCPC was found to be of high purity and yield under an optimal solvent system composed of n-hexane:ethyl acetate:methanol:water (2:7:3:7, v/v/v/v). To evaluate the anti-inflammatory efficacy of EEM containing active compounds, zebrafish embryos exposed to LPS were compared with and without EEM treatment for nitric oxide (NO) production, reactive oxygen species (ROS) generation, and cell death two days after fertilization. These evaluations indicate that EEM alleviated inflammation by inhibiting cell death, ROS, and NO generation induced by LPS treatment. According to these results, eckmaxol and dieckol isolated from brown seaweed E. maxima could be considered effective anti-inflammatory agents as pharmaceutical and functional food ingredients.     

NaF和PFOS对斑马鱼肝脏抗氧化能力、非特异性免疫及组织结构的影响

为探讨有机氟化物和无机氟化物单一及联合暴露对斑马鱼(Danio rerio)肝脏的毒性效应,本研究将雄性斑马鱼随机分为对照组、氟化钠(NaF,无机氟)暴露组、全氟辛烷磺酸(PFOS,有机氟)暴露组、NaF+PFOS(无机氟及有机氟联合)暴露组,经15、30 d暴露试验后,检测了血液硝基蓝四氮唑(NBT)阳性细胞数及肝脏...     

温度对斑马鱼胚胎发育的影响研究

观察了不同温度（22℃、24℃、26℃、28℃、30℃、32℃）下斑马鱼受精卵的死亡率、孵化周期、孵化率等指标的变化规律。结果表明：在22-28℃范围内,随温度升高,死亡率逐渐降低;在28-32℃范围内,随温度升高,死亡率逐渐增高,28℃为斑马鱼胚胎发育的最适温度。该研究结果为丰富硬骨鱼类繁殖生理学提供参考资料。     

Bioprospecting of Less-Polar Fractions of Ericaria crinita and Ericaria amentacea: Developmental Toxicity and Antioxidant Activity

Ericaria crinita and Ericaria amentacea from the Adriatic Sea (Croatia) were investigated with respect to the presence of less-polar compounds for the first time after fractionation by solid-phase extraction (SPE). The composition of less-polar fractions of freeze-dried E. crinita (FdEc) and E. amentacea (FdEa) were analyzed by high-performance liquid chromatography–high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). The major identified compounds were: amides of higher aliphatic acids (palmitoleamide, linoleamide, palmitamide, oleamide and erucamide) and related compounds, carotenoid (fucoxanthin), chlorophyll derivatives (pheophytin a and b and their derivatives) and higher terpenes (loliolide, isoamijiol with its oxidation product), β-stigmasterol and (3β,6α)-14-methylergosta-8,24(28)-diene-3,6-diol). The toxic effects observed on the less-polar fractions obtained from Ericaria species on zebrafish Danio rerio embryos could be associated with the high abundance of all five detected amides. The antioxidant activity of the fractions was evaluated by means of five independent assays, including the reduction of the radical cation (ABTS), the oxygen radical absorbance capacity (ORAC), ferric-reducing antioxidant power (FRAP), the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay and the Folin–Ciocalteu method. A higher antioxidant activity of E. amentacea in comparison to that of the E. crinita fractions was found with IC₅₀ concentrations of 0.072 and 1.177 mg/mL, respectively. The correlation between the activity and the chemical composition revealed that the synergistic effect of different compounds impacted their antioxidant response.     

斑马鱼幼鱼嗜中性粒细胞对副溶血弧菌清除的动态变化

副溶血弧菌(Vibrio parahaemolyticus)是人-兽-鱼共患的致病菌,严重威胁食品安全和人类的健康。为了研究宿主嗜中性粒细胞清除细菌感染的动态变化过程,利用红色荧光蛋白标记的副溶血弧菌Vp57~(RFP)菌株感染受精后48 h(hours post fertilization,hpf)的斑马鱼(Danio rerio)幼鱼耳泡,建立局部感染模型,并以大肠杆菌(Escherichia coli)Ec01菌株为对照,系统比较了副溶血弧菌和大肠杆菌的半数致死剂量(median lethal dose,LD_(50))、存活曲线以及和嗜中性粒细胞相互作用的动态过程,RT-qPCR验证炎症相关基因il1b和il10在感染前后的表达量变化。结果显示副溶血弧菌和大肠杆菌感染斑马鱼幼鱼的LD_(50)分别为7.90×10~8 CFU/mL和1.14×10~(11) CFU/mL,与感染大肠杆菌相比,斑马鱼感染副溶血弧菌后招募嗜中性粒细胞的速度更快,且数量更多。RT-qPCR结果发现斑马鱼感染副溶血弧菌后在相同时间点能够激活更高的il1b和il10基因的表达,引起强烈的炎症反应。基于以上研究,我们建立的副溶血弧菌-斑马鱼幼鱼耳泡局部感染模型,为进一步研究嗜中性粒细胞清除副溶血弧菌感染的动态过程,以及深入揭示天然免疫应答机制提供了依据。     

弗氏柠檬酸杆菌感染诱导斑马鱼皮肤免疫相关基因的差异表达

从草鱼肠道中分离到1株细菌,通过形态学观察、生理生化特征和16S rDNA序列分析等鉴定为弗氏柠檬酸杆菌,动物试验结果表明,该菌对斑马鱼有致病性;从感染诱导的斑马鱼皮肤组织中提取总RNA,经Biotin荧光标记与拥有15 617个cDNA片段的斑马鱼基因芯片(affymetrix)杂交筛选分析,获得斑马鱼皮肤免疫相关差异表达基因88个,其中有74个上调表达基因和14个下调表达基因;进一步根据基因功能聚类(GO)分析,初步将88个差异表达基因分为8个生物学功能,其中主要参与补体激活、急性期反应、应激防御反应、细胞凋亡、抗原加工提呈、细胞迁移粘附、凝血因子和血小板激活等免疫应答过程;同时进行信号通路(pathway)分析,结果表明MAPK(hsp70、daxx、nfkbiab)、JAK/STAT(ifn1)和TGF-β(thbs1)等信号通路参与斑马鱼皮肤抗弗氏柠檬酸杆菌感染免疫应答。采用基因芯片方法筛选与鱼类皮肤免疫相关的功能基因,为将来以斑马鱼为模型研究鱼类皮肤局部免疫应答的分子机制提供科学依据。     

维生素E对毒死蜱胁迫下斑马鱼血清CAT和SOD的保护作用

通过对斑马鱼肌肉注射不同浓度毒死蜱以及维生素E,研究斑马鱼血清中过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活力变化情况以及维生素E的抗氧化作用。试验分为5组,依次为对照组和低、中、高浓度毒死蜱组(0.002、0.02、0.2 mg/kg)以及高浓度联合维生素E(0.2 mg/kg+VE)治疗组。结果表明,毒死蜱不同浓度组短时间(24 h)作用均使两种酶活性升高(P﹤0.01),加维生素E组活性增高更显著;长时间(168 h)作用则明显抑制酶活性(P﹤0.01),维生素E组酶活性变化较平缓。表明毒死蜱对斑马鱼机体抗氧化系统损伤作用明显,维生素E对CAT和SOD活力有保护作用。     

乐果、铜和锌对水生生物的联合毒性研究

为通过不同生物等级的模式生物研究乐果、铜和锌的联合毒性,以明亮发光杆菌和斑马鱼胚胎为受试模式生物,通过混合毒性指数法（MTI法）评价了乐果、铜和锌的联合毒性效应。结果表明：明亮发光杆菌毒性测试显示,暴露测试15 min时,乐果、铜和锌的EC₅₀值分别为123、0.53 mg·L^-1和1.71 mg·L^-1;暴露测试30 min时,EC₅₀值分别为122、0.50 mg·L^-1和1.54 mg·L^-1。乐果-铜和乐果-锌暴露15 min测得的MTI分别为0.69和0.60。斑马鱼胚胎毒性测试显示,暴露72 h后乐果、铜和锌的LC50值分别为0.31、1.67 mg·L^-1和369 mg·L^-1。乐果-铜和乐果-锌暴露72 h后测得的MTI分别为0.70和0.75。研究表明,乐果和铜、锌分别混合后的联合毒性均有所增强,整体毒性表现为部分相加作用,因此两类物质在环境中的残留会对水生生物及其他生物造成潜在危害。     

斑马鱼在鱼类病原免疫学研究中的应用

斑马鱼全基因组测序已经完成,建立了多个专门的在线数据库,作为模式生物在遗传学、发育生物学等领域已得到了广泛的应用.本文介绍了斑马鱼用于鱼类病原免疫学研究的优点,包括个体发育过程透明、免疫系统较成熟,具有先天性和获得性2种免疫系统;多种鱼类来源的细菌和病毒在成年斑马鱼和胚胎中都能感染和复制,使其成为研究鱼类病原的入侵机制和宿主免疫应答的模式生物.同时,对当前已建立的鱼类细菌和病毒病原斑马鱼感染模型以及在疫苗方面的研究现状作简要概述.     

斑马鱼报警反应产生发育阶段的研究

鱼类在遭受捕食后,受损的表皮会释放大量的化学信号物质,同种其它个体嗅到这些化学信号后会作出躲避反应,称为报警反应(alarm response),报警反应通常出现在一定的发育阶段。采用斑马鱼(Dinio rerio)同种碾碎提取物(处理组,质量浓度为10-6g·L-1)作为报警物质,并设3个对照组:罗非鱼碾碎提取物组、双蒸水组、空白对照组,通过暴露处理实验,检测沉底累积时间(time of bottom)、呆滞累积时间(time of freezing)两项指标,研究了28 dpf(days post fertilization,受精后天数)、35 dpf、42 dpf 3个不同发育时期幼鱼的报警反应。结果显示:28 dpf时,处理组与3个对照组相比,在沉底累积时间、呆滞累积时间两项指标上均无显著差异,这个时期处理组有2 ind斑马鱼出现"沉底",其中1 ind出现"呆滞";35 dpf时,处理组与3个对照组相比,在沉底累积时间上有显著差异(P0.05),在呆滞累积时间上无显著差异,这个时期有5 ind斑马鱼出现"沉底",其中1 ind出现"呆滞";42 dpf时,处理组与对照组相比,在沉底累积时间、呆滞累积时间两项指标均有显著差异(P0.05),这个时期有8 ind斑马鱼出现"沉底",且8 ind全部出现"呆滞",与此对应的是这个时期大部分个体出现明显的报警反应。研究表明,斑马鱼报警反应产生的发育阶段为35~42 dfp之间,结合已知的野外斑马鱼生活史,推测斑马鱼在特定的阶段产生报警反应可能与栖息地的迁移有关。