基于斑马鱼幼鱼的塑化剂肝毒性危害识别研究

目的 采用塑化剂邻苯二甲酸二乙基己酯（DEHP）和邻苯二甲酸二丁酯（DBP）作为阳性对照,建立斑马鱼幼鱼脂肪肝毒性风险评估模型进行塑化剂邻苯二甲酸丁酯苯甲酯（BBP）肝毒性危害识别。方法 分析DEHP和DBP暴露后斑马鱼肝脏脂肪信号强度及过氧化物酶体增殖物激活受体（PPAR）靶基因CD36的表达,初步构建斑马鱼幼鱼肝毒性模型;基于此模型对BBP进行肝毒性评价,此外采用肝脏表达绿色荧光转基因斑马鱼幼鱼进行肝密度分析、甘油三酯（TG）定量检测以及成鱼肝脏病理检查进一步验证模型的可靠性,应用基准剂量法（BMD）推导关键效应点。结果 DEHP、DBP表现出极显著诱发脂肪肝风险（均P<0.01）,显著上调CD36相对表达量（P<0.001,P<0.05）,提示造模成功。BBP（0.000 012 5%、0.000 025%、0.000 05%）诱发斑马鱼幼鱼显著的脂质沉积（P<0.05,P<0.001,P<0.001）、显著影响密度（P<0.05,P<0.01,P<0.01）、显著增加TG蓄积（P<0.05,P<0.05,P<0.01）、上调CD36基因表达。此外,BBP（≥0.000 012 5%）诱发斑马鱼成鱼肝脏部位脂质空泡形成、空泡间隙和数量减少,提示BBP具有明显的肝毒性。关键效应为肝脏脂肪信号强度（S）,基准剂量下限（BMDL）值为0.013 mg/L。结论 本研究成功构建了一种通过肝脏脂肪信号强度来识别塑化剂肝毒性的斑马鱼幼鱼模型,结合基准剂量法进一步为塑化剂肝毒性危害识别提供科学依据。     

基于斑马鱼模型评价发酵对麦麸多糖抗氧化活性的影响

为探讨发酵是否能提高麦麸多糖的抗氧化活性,本实验首先挑选受精后7～9 hpf(hours post fertilization, hpf)的斑马鱼受精卵,将其暴露在不同质量浓度(0、25、50、100、200、300μg/mL)的未发酵麦麸多糖(WBP)和发酵麦麸多糖(FWBP)溶液中,统计初孵仔鱼心率、测量其体长及卵黄囊大小,统计96 hpf时斑马鱼胚胎孵化率,评估WBP和FWBP对斑马鱼胚胎的安全性。在上述试验的基础上,将7～9 hpf的斑马鱼胚胎在不同质量浓度(0、50、100、200μg/mL)的WBP和FWBP溶液中预暴露到24 hpf,在72 hpf时通过荧光染色检测斑马鱼仔鱼活性氧(ROS)产生率、脂质过氧化率和细胞死亡率,分析过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性。结果表明：高质量浓度(>300μg/mL)WBP和FWBP导致斑马鱼胚胎死亡率提高、心率和孵化率降低,体长变短,对斑马鱼胚胎的发育具有毒性作用;50、100、200μg/mL的WBP和FWBP均能降低了斑马鱼体内的ROS产生率、脂质过氧化率和细胞死亡率,提...     

基于斑马鱼模型评价白茶的降糖作用

研究以白茶为对象,采用新型斑马鱼实验技术开展了白茶降糖比较研究。基于非胰岛素依赖型糖尿病（NIDDM）斑马鱼研究发现,50和100 μg/mL老白牡丹茶（AWP）孵育24 h葡萄糖（GLU）值水平分别降低了29.68%和20.51%,孵育48 h GLU值分别降低了42.16%和51.54%,差异均显著（P<0.05）;25、50和75 μg/mL白牡丹茶（WP）孵育24 h GLU值分别降低了40.18%、52.57%和62.11%,差异均显著（P<0.05）。另外,基于胰岛素依赖型糖尿病（IDDM）斑马鱼研究,20和30 μg/mL AWP孵育24 h GLU值分别降低了24.92%和23.66%,孵育48 h分别降低了54.35%和38.38%,差异均显著（P<0.05）;40 μg/mL WP孵育24 h GLU值降低了22.58%（P<0.05）,20 μg/mL WP孵育48 h降低了26.51%（P<0.05）;30 μg/mL茶多酚（TP）孵育48 h GLU值降低了24.53%（P<0.05）。重要的是,在同等生药量下,20 μg/mL WP和AWP对NIDDM斑马鱼的降糖效果显著优于TP;30 μg/mL AWP对IDDM斑马鱼的降糖效果略优于TP,再明显优于WP。结果表明白茶具有明显的降糖功效,TP也具有明确的降糖作用,但白茶的降糖效应并不仅限于依赖TP,具体何成分起关键作用尚值得关注和研究。     

基于斑马鱼生物模型评价青钱柳的降糖作用

该研究以5 dpf斑马鱼为对象,系统研究了其对葡萄糖（GLU）和四氧嘧啶（ALX）耐受性的影响,研究了GLU与ALX联合建立糖尿病斑马鱼生物模型的可行性和有效性。以此为基础,研究了斑马鱼对青钱柳（CP）的耐受性并指导开展了CP的降糖作用评价。结果发现,以10 mg/mL GLU与3.60或7.20 μg/mL ALX联合诱导24 h,可显著增加斑马鱼GLU值,分别增加了51.52%（p<0.05）和68.32%（p<0.05）。经200、400和800 μg/mL阿卡波糖治疗处理后斑马鱼GLU值分别降低了27.28%、33.69%和38.75%,差异均显著（p<0.05）,表明以10 mg/mL GLU与3.60 μg/mL ALX联合孵育24 h可以有效建立糖尿病斑马鱼模型。另外,研究发现,400和800 μg/mL CP可以显著降低10 mg/mL GLU溶液诱导的斑马鱼GLU值,分别降低了37.59%（p<0.05）和38.57%（p<0.05）;200、400和800 μg/mL CP可以显著降低糖尿病斑马鱼GLU值,分别降低了27.43%、32.75%和29.20%,差异均显著（p<0.05）。结果表明,基于正常生理和病理生理的斑马鱼生物模型评价,CP具有明显的降糖效应,其在辅助降糖功能饮品开发上具有广阔的应用前景。     

石榴酒体外及斑马鱼体内抗氧化活性研究

探对石榴酒的抗氧化活性进行研究,采用分光光度法测定石榴酒对DPPH·、·OH和O_2~-自由基的清除能力和石榴酒的还原力,并利用皮肤荧光斑马鱼进行石榴酒的抗氧化活性试验。结果表明:石榴酒在一定范围加入量内对DPPH·、·OH和O_2~-有良好的清除作用,清除率最大分别为92.95%、82.76%和63.85%。在对皮肤荧光斑马鱼进行石榴酒的抗氧化活性体内试验中,石榴酒浓度在30、40、50μL/mL时,相对抗氧化能力分别达到了39.7%、60.3%、75.4%。     

基于斑马鱼模型的茶多酚及儿茶素单体降糖作用研究

斑马鱼是一种新型模式生物,已成为生命科学研究的新宠.研究以茶多酚及其儿茶素单体为对象,采用胰岛素依赖型糖尿病(IDDM)斑马鱼模型开展了茶多酚及儿茶素单体的降糖效应比较研究.结果发现,15～45μg/mL茶多酚、EGCG、EGC、EC和ECG(以生药量计)均能够降低斑马鱼葡萄糖值水平,但其降糖效应弱于糖尿病治疗药物阿卡...     

基于斑马鱼模型的藿香紫苏饮免疫调节功能研究

研究藿香紫苏饮对由酒石酸长春瑞滨所诱导的免疫力低下斑马鱼的免疫功能的调节作用。通过给斑马鱼静脉注射0.2 mg/mL酒石酸长春瑞滨诱导建立免疫力低下模型,将斑马鱼随机分为正常对照组、模型对照组、藿香紫苏饮低剂量组（500μg/mL）、藿香紫苏饮中剂量组（1 000μg/mL）、藿香紫苏饮高剂量组（2 000μg/mL）,每组30尾。使用荧光显微镜观察并分析各组斑马鱼巨噬细胞吞噬功能及T细胞减少的改善效果。结果表明,藿香紫苏饮高剂量组（2 000μg/mL）斑马鱼体内剩余荧光微球数量显著低于模型对照组（P<0.05）;藿香紫苏饮低、中、高剂量组斑马鱼T细胞荧光强度显著高于模型对照组（P<0.01）。藿香紫苏饮具有促进斑马鱼巨噬细胞吞噬、改善T细胞减少的功效。     

基于斑马鱼功效模型的钙果饮补钙效果评价

建立斑马鱼功效模型,验证钙果饮的补钙效果。选择发育健康且生长阶段一致的3 dpf野生型斑马鱼,加入不同浓度(3.125μg·mL^-1、6.25μg·mL^-1、12.5μg·mL^-1、25.0μg·mL^-1、50.0μg·mL^-1、100.0μg·mL^-1)钙果饮,同时设置正常对照组,每孔容量为3 mL,确定钙果饮对正常斑马鱼的最大耐受浓度;取3个浓度梯度组(3.125μg·mL^-1、6.25μg·mL^-1、12.5μg·mL^-1)、阳性对照组和正常对照组,用钙黄绿素进行染色,计算斑马鱼的椎骨荧光强度。结果表明,钙果饮对正常斑马鱼的最大耐受浓度为12.5μg·mL^-1;钙果饮的补钙效果随浓度的增加而增加,与空白对照组相比,不同钙果饮浓度处理的斑马鱼骨骼荧光强度增强,说明钙果饮具有显著的补钙效果。     

拟除虫菊酯类农药对斑马鱼幼鱼的神经毒性作用研究

摘要：目的 探究四种拟除虫菊酯类农药（溴氰菊酯、丙烯菊酯、高效氯氰菊酯、顺式氰戊菊酯）对斑马鱼神经毒性的作用,并与大鼠毒性结果进行比较,以期为斑马鱼在食品安全评价中的应用提供参考。方法 对四种农药采用静态暴露方法,分析其在高中低三种剂量下对斑马鱼幼鱼的形态、神经行为的影响;同时以活性氧（Reactive oxygen,ROS）、超氧化物歧化酶（(superoxide dismutase,SOD）、乙酰胆碱酯酶（Acetylcholinesterase,AchE）为指标,研究各农药在高中低剂量下对斑马鱼幼鱼神经化学的影响。结果 四种拟除虫菊酯类农药均对斑马鱼幼鱼造成了体长变短、脊柱弯曲等形态变化;除丙烯菊酯外,其他三种农药均使斑马鱼幼鱼出现显著的运动活性降低趋势;在神经化学方面,与空白组和对照组相比,四种农药随着浓度增加均使斑马鱼幼鱼体内的AchE活性显著下降、SOD活性显著升高;但四种农药对ROS活性变化各有差异：溴氰菊酯与高效氯氰菊酯对斑马鱼幼鱼的ROS影响无显著性差异,而丙烯菊酯和顺式氰戊菊酯在高中剂量下均可导致斑马鱼幼鱼ROS显著增加。结论 拟除虫菊酯农药对斑马鱼幼鱼产生了显著的神经毒性作用,首次发现形态变化和AchE活性的变化与大鼠相关结果体现出较好的一致性,有望替代大鼠用于更加简便快捷的食品安全风险评估;其余指标与大鼠呈现不同程度的差异,可能与两者生物学特性的差别有关。     

基于黄嘌呤氧化酶活性抑制和斑马鱼高尿酸血症模型的降尿酸功效食药材筛选

结合体外(黄嘌呤氧化酶活性抑制)和体内(斑马鱼高尿酸血症模型)方法,对15种食药材乙醇粗提物的降尿酸活性进行筛选.体外实验设置空白组、酶反应组、抑制剂组、对照组,酶标仪295 nm测定吸光度,计算15种食药材对黄嘌呤氧化酶(XOD)活性的抑制率;体内实验随机选取受精后第5 d(5dpf)的斑马鱼,设置空白组、模型组(2...     

Antioxidants to prevent bovine neutrophil-induced mammary epithelial cell damage

Activated neutrophils are able to produce a large quantity of bactericidal molecules such as reactive oxygen species that have been associated with tissue damage in several inflammation models. The protective effects of antioxidants in a context of neutrophil-induced damage to mammary epithelial cells were first evaluated in vitro using a coculture model of activated bovine neutrophils and a bovine mammary epithelial cell line (MAC-T cells). Cell damage was determined by quantifying the release of lactate dehydrogenase by MAC-T cells in culture medium. Morphological observation of cells stained with acridine orange was used to visualize the extent of cell damage. When incubated with neutrophils activated by lipopolysaccharides and phorbol 12-myristate 13-acetate, MAC-T cells released large amounts of lactate dehydrogenase indicating significant cell damage. The addition of dimethylthiourea or bathocuproine disulfonic acid did not reduce the damage whereas catechin, deferoxamine or glutathione ethyl ester significantly reduced neutrophil-induced cytotoxicity in a dose-dependent manner. The effect of deferoxamine, an iron chelator, on the growth of Escherichia coli and the ability of bovine neutrophils to phagocytose these bacteria were then assessed in vitro. Our data showed that deferoxamine did not interfere with the phagocytic activity of neutrophils but inhibited growth of the bacteria. Overall, our results suggest that antioxidants may be effective tools for protecting mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.     

Deferoxamine reduces tissue damage during endotoxin-induced mastitis in dairy cows

The protective effects of 3 antioxidants on polymorphonuclear neutrophil-induced damage to mammary cells were evaluated in vivo using an endotoxin-induced mastitis model. Fifteen healthy, midlactation cows with no history of clinical Escherichia coli mastitis were randomly assigned to 1 of the 3 treatment groups corresponding to each modulator to be evaluated, that is, deferoxamine, catechin, and glutathione ethyl ester. Each cow had 1 quarter infused with saline and 1 quarter infused with the selected modulator; a third quarter was infused with lipopolysaccharides (LPS), whereas the fourth quarter received a combination of LPS and the modulator. Infusion of LPS caused acute mastitis as determined by visual observations and by large increases in milk somatic cell count, BSA, and proteolytic activity. These parameters were not affected by antioxidant administration. The extent of cell damage was evaluated by measuring milk levels of lactate dehydrogenase and N-acetyl-β-_D-glucosaminidase activity. Levels of these parameters were several times higher after LPS administration. Intramammary infusions of catechin or glutathione ethyl ester did not exert any protective effect, whereas infusion of deferoxamine, a chelator of iron, decreased milk lactate dehydrogenase and NA-Gase activity, suggesting a protective effect against neutrophil-induced damage. The protective effect of deferoxamine was also evidenced by a lower milk level of haptoglobin. The proteolytic activity of mastitic milk was not influenced by the presence of deferoxamine. Overall, our results suggest that local infusion of deferoxamine may be an effective tool to protect mammary tissue against neutrophil-induced oxidative stress during bovine mastitis.     

虾青素对活性氧所致细胞膜损伤的保护作用研究

目的研究虾青素保护活性氧(ROS)所致细胞膜损伤的作用。方法用ROS建立细胞膜损伤模型,测定细胞膜谷胱甘肽(GSH)含量;细胞膜ATP酶活性;亚硝酸盐法测定细胞膜一氧化氮(NO)含量;细胞内ROS含量变化。结果虾青素可有效地提高损伤细胞膜的GSH含量、恢复ATP酶活性并降低NO和ROS含量。结论虾青素有保护ROS所致细胞膜损伤的作用,其机制与抗氧化作用有关。     

活性氧对河岸葡萄叶圆片抗氧化酶活性的影响

研究了离体处理下活性氧对河岸葡萄叶圆片抗氧化酶活性的影响.结果表明,外源H2O2和Fe2++H2O2处理4 h可引起河岸葡萄叶圆片细胞膜透性明显增加,引起APOD,GPOD和SOD活性明显下降,但低浓度活性氧可不同程度引起叶圆片CAT活性增加;细胞膜透性与活性氧浓度呈显著正相关.     

Improving effect of a probiotic mixture on memory and learning abilities in d-galactose–treated aging mice

The aim of this study was to evaluate the antiaging effect of a probiotic mixture using an in vivo mouse model in which aging was induced with d-galactose. Results of the Morris water maze test indicated that long-term administration of the probiotic mixture improved memory and learning abilities and ameliorated the apoptosis pattern in the hippocampus of aging mice treated with d-galactose. An antioxidation experiment indicated that administration of the probiotic mixture could restore activities of the antioxidant enzymes superoxide dismutase and catalase and inhibit the production of malondialdehyde. The antioxidant-related proteins nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were upregulated in liver after treatment of d-galactose–treated aging mice with probiotics. Finally, the probiotic treatment did affect the production of short-chain fatty acids in d-galactose–treated aging mice. Our results highlighted a possible antioxidative effect triggered by short-chain fatty acids that contributed to improving the memory and learning abilities following treatment with the probiotic mixture and suggested that probiotics could serve as a therapy to modulate physiological function.     

黑木耳多糖清除活性氧及保护线粒体

目的:观察黑木耳多糖(AAP)是否具有清除超氧阴离子(O-2•)、抗氧化、保护线粒体作用。方法:以Fe2+-L-半胱氨酸(L-Cys)体系体外诱发鼠肝、脑线粒体及匀浆脂质过氧化并导致线粒体肿胀。以还原型辅酶I-吩嗪硫酸甲酯-氮蓝四唑(NADH-PMS-NBT)系统产生O-2•,用分光光度法测定脂质过氧化产物丙二醛(MDA)含量、线粒体肿胀度和O-2•的清除情况。结果:黑木耳多糖可明显抑制MDA生成及线粒体肿胀,并能明显清除O-2•,且呈剂量效应关系。结论:黑木耳多糖具有清除超氧阴离子、抗氧化及保护线粒体功能。