Effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

芜菁Br14-3-3的克隆及其在非生物胁迫下的表达分析

克隆了‘津田芜菁’（Brassica rapa subsp. rapifera‘Tsuda’）通用调节因子14-3-3基因cDNA序列，命名为Br14-3-3（GenBank登录号为MK896872）。该基因全长1 113 bp，开放阅读框全长774 bp，编码含有257个氨基酸的多肽。荧光定量PCR分析Br14-3-3在‘津田芜菁’不同组织中及其在温度、脱水、渗透、ABA和无机盐等非生物胁迫下幼苗中的表达，结果表明该基因在‘津田芜菁’的花中表达量最高，幼苗中次之；低温抑制了Br14-3-3的表达，其他非生物胁迫可诱导该基因表达，暗示Br14-3-3在非生物胁迫应答中发挥功能。     

不同营养水平日粮对陕北白绒山羊生长性能和小肠组织SLC7A7、SLC3A1及SLC15A1 mRNA表达的影响

本试验旨在研究日粮营养水平对断奶后2~6月龄陕北白绒山羊生长性能及小肠组织中与氨基酸转运吸收相关的SLC7A7、SLC3A1和SLC15A1 mRNA表达的影响。选取健康、日龄（（60±1.60）d）和体重（（10.73±1.03）kg）相近的雌性陕北白绒山羊羔羊36只，随机分为4组，分别饲喂4种试验日粮，其消化能和粗蛋白质水平分别为标准日粮的85%、100%、115%和130%。标准日粮营养水平参考肉羊饲养标准NY/T816-2004，依据生长阶段（10~19 kg、15~27 kg）和目标增重设置。试验期间，分别于120和180日龄称重，于180日龄，每个重复屠宰1只试验羊，采集十二指肠中上部、空肠中段、回肠末端组织样品，通过实时荧光定量PCR检测SLC7A7、SLC3A1和SLC15A1表达水平。结果表明：1）第一阶段（60~120 d）115%水平组平均日增重显著高于其他三组（P<0.05），第二阶段（121~180 d）115%水平组平均日增重显著高于85%和100%水平组（P<0.05），与130%水平组无明显差异。两阶段115%水平组羔羊干物质采食量极显著高于其他3组（P<0.01）。两阶段料重比115%水平组显著低于85%、100%水平组（P<0.05）。2）相同营养水平下，SLC7A7和SLC15A1 mRNA的表达丰度顺序均为回肠>空肠>十二指肠；3）随日粮营养水平的增加，SLC7A7、SLC3A1和SLC15A1 mRNA在小肠各段的相对表达量呈先上升后下降的趋势，且115%水平组表达量最高。115%水平组SLC7A7和SLC15A1 mRNA相对表达量显著高于其他3组（P<0.05）；115%水平组SLC3A1 mRNA的相对表达量显著高于85%和130%水平组组（P<0.05）。本试验条件下，与其他营养水平组相比，115%水平组陕北白绒山羊羔羊在2~6月龄生长性能最佳，SLC7A7、SLC3A1和SLC15A1 mRNA在小肠各段的表达量最高。     

胡椒4-香豆酸：辅酶A连接酶Pn4CL基因的克隆及表达分析

根据胡椒4-香豆酸：辅酶A连接酶（4-coumarate:coenzyme A ligase, 4CL）基因的部分序列设计引物,运用RACE方法获得其家族成员的1个全长cDNA,命名为Pn4cl,长度2130 bp,开放阅读框1638 bp,编码545个氨基酸。预测Pn4CL分子量为59.57 kDa,理论等电点为5.70。该基因含有AMP-binding（AMP-binding enzyme）、CaiC[Acyl-CoA synthetase (AMP-forming) /AMP-acid ligaseⅡ]、PLN02246、AFD-class I等结合域,具有植物4CL所共有的保守结构域。系统进化分析表明,Pn4CL与北细辛的同源性最高,同时与木兰分支类植物的4CL聚类在一起,与菊分支的进化距离较近,与蔷薇分支的进化距离较远。亚细胞定位表明,该蛋白定位在细胞膜上。Real-time RT-PCR结果表明,该基因受外援激素SA和MeJA诱导表达,同时接种辣椒疫霉菌后,Pn4CL基因的表达量在抗/感2种胡椒中均出现先增加后减少的现象,并且在抗病种质中表达量较高。研究结果为Pn4CL的功能研究提供了理论依据。     

饲料中维生素A对青鱼幼鱼生长、血清生化指标和肝脏糖脂代谢酶活性及基因表达的影响

为研究饲料中维生素A(VA)对青鱼幼鱼生长、血清生化指标和肝脏糖脂代谢相关酶活性及基因表达的影响,实验选取360尾初始体质量为(6.10±0.10) g的青鱼幼鱼,随机分配至3个实验组中,每个实验组设置3个平行。采用单因素实验设计,以无维酪蛋白和明胶为蛋白源、菜籽油为脂肪源、糊精为糖源,同时添加矿物质混合物和维生素混合物(无VA添加)配制成3组实验饲料,分别以饲料1 (Diet1)、饲料2 (Diet2)和饲料3 (Diet3)表示。在饲料1、饲料2和饲料3中分别添加0、2 200和20 000 IU/kg VA醋酸酯(500 000IU/g),经高效液相色谱法(Agilent-1100, Agilent,美国)检测后实验饲料中VA的实际含量分别为178.2、2 058.9和18 436.2 IU/kg,养殖周期为8周。结果显示:饲料中VA缺乏会显著降低青鱼幼鱼的增重率(WGR)和特定生长率(SGR);VA缺乏会显著降低血清血糖(GLU)、甘油三酯(TG)和低密度脂蛋白(LDL)浓度,增加总胆固醇(TCH)浓度。饲料中添加2 058.9IU/kg VA能显著提高肝脏己糖激酶(HK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)活性,促进葡萄糖转运蛋白-2 (GLUT-2)、HK、葡萄糖激酶(GK)、PFK和葡萄糖-6-磷酸酶(G6Pase)基因表达。当饲料中VA含量为2 058.9 IU/kg时,对肝脏脂肪酸转运蛋白-1(FATP-1)基因表达无显著影响,但显著影响肉碱棕榈酰转移酶-1 (CPT-1)和肉碱棕榈酰转移酶-2 (CPT-2)基因表达。当饲料中VA缺乏时,CPT-1和CPT-2基因表达受到显著性抑制;当饲料中添加过量VA时,乙酰辅酶A羧化酶-2 (ACC-2)和脂蛋白酯酶(LPL)基因表达受到抑制;同时,VA过量组中肝脏过氧化物酶体增殖物激活受体-γ(PPAR-γ)基因表达显著下降。研究表明,在饲料中添加2 058.9 IU/kg VA可以促进青鱼幼鱼生长,提高肝脏对葡萄糖的转运能力,促进糖酵解和糖异生代谢平衡,同时促进脂肪酸合成和转运。     

Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years, the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment. Until now, traditional RT-PCR, fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV, but these methods require high-level instruments and experimental conditions, not suitable for the rapid detection in field and farms. In order to develop a rapid, sensitive and practical method to detect and identify AIV subtypes, 4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established. Using this method, the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein, without cross reaction with other susceptible avian viruses. In addition, the detection limit of the common H1, H5, H7, and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit), which was 10 times more sensitive than that using the routine RT-PCR. Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR. All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast, convenient and practical method for the clinic test and epidemiological investigation of AIV.     

microRNA-124-3p调控H1N1亚型猪流感病毒致小鼠肺损伤的研究

为研究microRNA-124-3p（miR-124-3p）对H1N1亚型猪流感病毒（swine influenza virus，SIV）感染小鼠所致肺损伤的调控作用，本试验构建miR-124-3p腺病毒表达载体，通过小鼠尾部静脉注射法构建miR-124-3p差异表达小鼠模型，试验分3组：过表达组、抑制组和对照组。48 h后，各组小鼠鼻腔接种H1N1亚型SIV，每只10⁵ EID₅₀（50 μL）。连续观察14 d，计算小鼠平均体重变化率、观察病理切片并测定相关炎症因子IL-1β、TNF-α和IL-6 mRNA相对表达量。结果显示，已成功将pre-miR序列及其sponge序列插入腺病毒的穿梭质粒，并将其共转染293A细胞。实时荧光定量PCR检测证实，与对照组相比，过表达组和抑制组小鼠黑色素瘤细胞miR-124-3p表达水平分别极显著升高（P<0.01）和显著降低（P<0.05），表明成功构建腺病毒表达载体。过表达组、抑制组和对照组小鼠体重变化率分别为－5.5%、－12.4%和－8.6%。抑制组和对照组均可见肺泡壁增厚，其间有多量淋巴细胞浸润，部分肺泡内出现纤维蛋白渗出，且抑制组病理变化更为严重，肺泡中还有大量的红细胞浸润；而过表达组仅有少量的淋巴细胞浸润，肺脏组织较正常。与对照组相比，过表达组检测的炎症因子IL-1β、TNF-α和IL-6 mRNA表达水平均显著降低（P<0.05）；抑制组炎症相关炎症因子mRNA表达水平均显著升高（P<0.05）。本试验结果表明，miR-124-3p对H1N1亚型SIV感染小鼠所致的肺脏炎症因子的表达具有抑制作用，同时能减轻肺脏病理损伤。     

Marker‐assisted introgression of rare allele of β‐carotene hydroxylase (crtRB1) gene into elite quality protein maize inbred for combining high lysine,tryptophan and provitamin A in maize

Vitamin A deficiency in humans is a widespread health problem. Quality protein maize (QPM) is a popular food rich in lysine and tryptophan, but poor in provitamin A (proA). Here, we report the improvement of an elite QPM inbred, HKI1128Q for proA using marker‐assisted introgression of crtRB1‐favourable allele. HKI1128 was one of the parental lines of three popular hybrids in India and was converted to QPM in our earlier programme. Severe segregation distortion for crtRB1 was observed in BC₁F₁, BC₂F₁ and BC₂F₂. Background selection by 100 SSRs revealed mean recovery of 91.07% recurrent parent genome varying from 88.78% to 93.88%. Across years, introgressed progenies possessed higher mean β‐carotene (BC: 9.22 µg/g), β‐cryptoxanthin (BCX: 3.05 µg/g) and provitamin A (proA: 10.75 µg/g) compared to HKI1128Q (BC: 2.26 µg/g, BCX: 2.26 µg/g and proA: 3.38 µg/g). High concentration of essential amino acids, viz. lysine (mean: 0.303%) and tryptophan (0.080%) in endosperm, was also retained. Multi‐year evaluation showed that introgressed progenies possessed similar grain yield (1,759–1,879 kg/ha) with HKI1128Q (1,778 kg/ha). Introgressed progenies with higher lysine, tryptophan and proA hold immense potential as donors and parents in developing biofortified hybrids.