哺乳动物硒蛋白的研究进展

硒是哺乳动物和人必需的微量元素，其生物学功能主要是以硒蛋白的形式表现的。近年来，关于硒蛋白作为饲料添加剂方面的报道层出不穷。作者就硒蛋白概况、分类、几种重要硒蛋白及其生物学功能、硒蛋白的合成及调控作一综述。     

GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

【目的】 研究硒蛋白谷胱甘肽过氧化物酶4（glutathione peroxidases 4,GPX4）失活如何参与调控脂多糖（lipopolysaccharide,LPS）诱导的RAW264.7巨噬细胞炎症反应及其潜在的分子机制。【方法】 体外培养RAW264.7巨噬细胞,以DMSO为对照,使用0.1~5.0 μmol/L GPX4抑制剂FIN56处理,通过CCK-8法检测细胞活力和Western blotting检测GPX4蛋白表达水平,确定抑制剂最适浓度。将RAW264.7巨噬细胞分为4组：对照组,添加DMSO培养24 h;FIN56（GPX4抑制剂）组,添加0.5 μmol/L FIN56培养24 h;DMSO-LPS组,DMSO培养24 h后使用LPS （100 ng/mL）刺激3 h;FIN56-LPS组,FIN56培养24 h后使用LPS刺激3 h。各组细胞经培养后,利用荧光探针2',7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorodi-hydrofluorescein diacetate,H₂DCFDA）检测细胞内活性氧（reactive oxygen species,ROS）水平,使用试剂盒法检测细胞内丙二醛（malondialdehyde,MDA）水平,分别使用ELISA和荧光定量PCR检测促炎细胞因子白细胞介素1β（interleukin-1β,IL-1β）、白细胞介素6（interleukin-6,IL-6）、肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）的分泌水平和基因表达量,使用Western blotting法检测Toll样受体4（toll like receptor 4,TLR4）信号通路相关蛋白表达水平。【结果】 与DMSO处理相比,0.5 μmol/L FIN56处理巨噬细胞24 h对细胞活性无显著影响（P>0.05）,且显著降低GPX4蛋白表达水平（P<0.05）,故选用0.5 μmol/L FIN56用于后续实验。与对照组相比,FIN56和LPS均显著增加了ROS积累（P<0.05）,而与DMSO-LPS组相比,FIN56-LPS组ROS水平显著升高（P<0.05）。与对照组相比,LPS可显著增加小鼠巨噬细胞IL-1β、IL-6和TNF-α的mRNA表达量和IL-6含量,以及c-Jun氨基末端蛋白激酶（c-Jun N-terminal protein kinase,JNK）和c-Jun磷酸化水平（P<0.05）,而FIN56可显著下调LPS诱导的IL-6的mRNA表达量和含量及JNK和c-Jun磷酸化水平（P<0.05）,但对p38蛋白（p38 MAPK,p38）、细胞外调节蛋白激酶（phosphorylate extracellular regulated protein kinases1/2,Erk1/2）蛋白表达水平无显著影响（P>0.05）。【结论】 GPX4失活可有效阻断JNK和c-Jun磷酸化,从而特异性抑制LPS诱导的巨噬细胞的炎症反应。该结果可为以GPX4为靶点研发抗炎治疗策略提供理论依据。     

动物硒蛋白功能、表达及其肉质调控机制研究进展

硒蛋白是微量元素硒(Se)在动物体内发挥生物学功能的主要形式,其表达调控与动物生长和畜禽肉质密切相关。介绍动物体内Se元素吸收代谢的过程及其主要存在形式,系统总结动物硒蛋白的结构、种类、表达分布、生物学功能、合成过程、影响因素与候选功能基因等,重点分析硒蛋白在活体肌肉中的作用,以及对畜禽肉质调控的影响。通过梳理上述方面的研究进展,以期为新饲料添加剂开发和畜禽肉质调控研究提供科学依据。     

Supranutritional dietary selenium depressed expression of selenoprotein genes in three immune organs of broilers

The objective of this study was to investigate the effects of supranutritional dietary selenium (Se) on selenoproteins expression in three immune organs of chickens. A total of 160 1‐day‐old male Cobb broilers were randomly divided into two groups and fed a Se‐deficient corn‐soybean basal diet supplemented with 0.3 (adequate) and 3.0 (excess) mg/kg Se for 42 days. Immune organs were collected, and effects of supranutritional Se on messenger RNA abundance of 23 selenoprotein genes and eight inflammation‐related genes were compared at day 42. Also enzyme activities were measured at days 14, 28 and 42. The results showed supranutritional dietary Se depressed growth performance of chicken and down‐regulated nine and three selenoprotein genes in thymus and spleen, respectively, and only Sepp1 was up‐regulated in the bursa of Fabricius. Also three, three and seven inflammation‐related genes were up‐regulated in three organs, respectively. Supranutritional Se elevated (P < 0.05) activities of superoxidase dismutase, total antioxidant capacity and glutathione peroxidase, mainly in early stages. In summary, supranutritional Se resulted in down‐regulation of selenoprotein genes and up‐regulation of inflammation‐related genes in three immune organs of chicken, which indicated potential roles of those selenoprotein genes in immune organs of the chicken.     

黄颡鱼硒蛋白基因的克隆与分析

为探讨黄颡鱼硒蛋白selenow2a、selenop2和selenot2基因之间的关系，采用3′/5′ RACE PCR克隆得到3个基因的cDNA全长，分别为891、1 998和1 432 bp，其中ORF长度分别为288、828和600 bp，编码95、275和219个氨基酸。在线工具SECISerach3对3个基因的cDNA序列分析结果显示，它们都含有可以编码硒代半胱氨酸的终止密码子，以及在3′非编码区存在SECIS元件。通过氨基酸序列比对和系统发育树分析，发现selenow2a、selenop2和selenot2基因预测得到的氨基酸序列与斑马鱼（Danio rerio）氨基酸相似性分别为82.24%、66.19%和79.45%，而与斑点叉尾鮰（Ictalurus punetaus）的氨基酸相似性分别为94.74%、68.50%和90.95%，在发育树上则显示为树杈相接近。采用实时荧光定量PCR检测3个硒蛋白基因的mRNA在黄颡鱼心脏、肝脏、肌肉、脑、肠、脾脏、精巢和卵巢组织中的表达，结果显示其mRNA表达水平呈现组织特异性。表明3个基因拥有硒蛋白家族的特征，但在组织表达上具有特异性。     

有机硒在猪营养中的研究进展

硒为动物必需微量元素,缺乏和过量对机体都可产生不利影响,甚至死亡。硒的形式多种多样,其中有机硒具有诸多优点,成为近年来硒研究的热点。本文重点综述有机硒在猪体内的代谢和对母猪、公猪繁殖性能、生长育肥猪生产性能及肉质品质的影响。     

硒蛋白x基因沉默和过氧化氢处理对人正常肝脏细胞中硒蛋白基因表达和抗氧化酶活性的影响

本试验旨在探讨硒蛋白x(Selx)基因沉默和过氧化氢(H2O2)处理对人正常肝脏细胞(LO2)中硒蛋白基因表达和抗氧化酶活性的影响,为探索Selx与其他硒蛋白之间的调控关系及Selx的功能奠定基础。对本实验室保存的Selx基因稳定沉默LO2细胞株(si Selx)和对照细胞株(CK),利用噻唑兰(MTT)法比较其生长速率;si Selx和CK用200μmol/L H2O2处理2.5 h后,采用荧光定量PCR技术考察25个硒蛋白基因的表达量;同时比较Selx基因沉默以及H2O2处理对LO2细胞株中谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性及总抗氧化能力(T-AOC)的影响。结果显示:1)Selx基因沉默显著降低了LO2细胞株的生长速率(P0.05);2)Selx基因沉默显著上调了G px6、Txnrd2、Seli基因的表达量(P0.05),显著下调了Dio2、Dio3、Selm、Sepw1基因的表达量(P0.05);H2O2处理显著上调了Dio1基因的表达量(P0.05),显著下调了G px2基因的表达量(P0.05);Selx基因沉默后H2O2处理显著上调了G px6、Txnrd2、Dio1基因的表达量(P0.05),显著下调了Txnrd3、Dio2、Selm、Sepw1、Selv基因的表达量(P0.05);3)Selx基因沉默显著提高了LO2细胞株GSH-Px活性(P0.05),显著提高了SOD的活性(P0.05);Selx基因沉默后H2O2处理显著提高了GSH-Px活性(P0.05)。由此可见,Selx基因沉默降低了LO2细胞株的生长速率,Selx基因沉默后H2O2处理影响部分硒蛋白基因在细胞中的表达量,同时也影响了GSH-Px、SOD活性。     

富硒螺旋藻,酵母,平菇的培养及其含硒蛋白的提取分离纯化

用含不同浓度硒的Ｚａｒｒｏｕｋ培养液培养螺旋藻,用含不同浓度硒的豆芽汁葡萄糖培养酵母,并用含不同浓度硒的了皮培养基培养平菇,试验结果表明：在适宜的培养条件下,螺旋藻,酵母及平菇生长最适宜硒浓度分别为６０ｍｇ．Ｌ＾－１,１０ｍｇ．Ｌ＾－１和２００ｍｇ．Ｌ＾－１,硒富集系数分别为１９．２,１４．４和２２．６。     

Effect of dietary organic selenium on survival,growth, antioxidation,immunity and gene expressions of selenoproteins in abalone Haliotis discus hannai

No studies have been focused on the inclusion of organic selenium in formulated feed of abalone when organic minerals are accepted by aquaculture feed industry. This experiment evaluated the effect of organic selenium on the growth, antioxidation, immunity and gene expressions of selenoproteins in abalone. Graded levels of organic selenium were included to the basal diet at 0.00, 0.15, 0.30 and 0.60 ppm providing 157.83, 322.51, 437.59 and 596.59 ug/kg of selenium in the diet. Accordingly, the diets were named as Se‐0.00, Se‐0.15, Se‐0.30 and Se‐0.60 respectively. Results had shown that survival rate, weight gain rate and shell length increase rate were lowest for Se‐0.60 group (p < 0.05). The concentration of selenium in the whole body generally increased with increasing level of dietary organic selenium (p < 0.05). The enzyme activities related to antioxidation increased initially with low levels of dietary selenium and decreased with higher levels, with the highest value in Se‐0.30 group (p < 0.05). The enzyme activities of immunity increased with the addition of dietary selenium. With the increase of dietary selenium, Myostain expression decreased initially and increased afterwards, with lowest value for Se‐0.30 group (p < 0.05). However, the expressions of other genes related to growth, antioxidation and selenoproteins firstly increased and then decreased, with the highest value recorded for Se‐0.15 group. In conclusion, the inclusion rate of 0.15 – 0.30 ppm Se in diet could promote growth, antioxidation, immunity and gene expressions of selenoproteins in abalone; however, excess selenium with 0.60 ppm inclusion lever in diet had toxic effects.     

Effect of maternal organic selenium supplementation during pregnancy on sow reproductive performance and long-term effect on their progeny

Selenium (Se) is an essential trace element for animals and exists in nature in both inorganic and organic forms. Although organic Se is more bioavailable than inorganic Se, there are inconsistent reports on the effect of organic Se on the reproductive performance of sows. This study was conducted to investigate the effect of maternal organic Se (2-hydroxy-4-methylselenobutanoic [HMSeBA]) supplementation on reproductive performance and antioxidant capacity of sows, and the long-term effect on the growth performance and antioxidant capacity of their offspring with or without lipopolysaccharide (LPS) challenge. The experimental design used in this study was a completely randomized design; 45 Landrace × Yorkshire sows were randomly allocated to receive one of the following three diets during gestation: control diet (Control, basal diet, n = 15), sodium selenite (Na₂SeO₃)-supplemented diet (Na₂SeO₃, basal diet + 0.3 mg Se/kg Na₂SeO₃, n = 15), and HMSeBA-supplemented diet (HMSeBA, basal diet + 0.3 mg Se/kg HMSeBA, n = 15). On day 21 of age, male offspring from each group were injected with LPS or saline (n = 6). As compared with the control group, maternal HMSeBA supplementation increased the number of total born piglets, while decreased birth weight (P < 0.05). In the first week of lactation, maternal HMSeBA supplementation increased litter weight gain compared with the Na₂SeO₃ group (P < 0.05) and increased the average daily gain of piglets compared with the control group and Na₂SeO₃ group (P < 0.05). Meanwhile, maternal HMSeBA supplementation decreased piglet birth interval as compared with the control group and Na₂SeO₃ group (P < 0.05). Besides, plasma glutathione peroxidase (GSH-Px) activity was higher in the HMSeBA group on farrowing 0 min and 90 min, while malondialdehyde (MDA) concentration was lower on farrowing 0, 90, and 135 min than those in the control group (P < 0.05). In addition, maternal HMSeBA supplementation increased the concentration of selenoprotein P (SELENOP) in colostrum compared with the control group (P < 0.05). Further study revealed that the LPS-challenged HMSeBA group had higher GSH-Px and total antioxidant capacity and lower MDA in weaning piglets compared with the LPS-challenged control group (P < 0.05). Taken together, maternal HMSeBA supplementation increased the number of total born piglets, shortened the duration of farrowing, improved the antioxidant capacities of sows and their offspring, and improved the growth performance of suckling pigs at the first week of lactation. Thus, HMSeBA supplementation during gestation has the potentiality to produce more kilogram of meat.