Effects of methionine supplementation in a reduced protein diet on growth performance, oxidative status, intestinal health, oocyst shedding, and methionine and folate metabolism in broilers under Eimeria challenge

Background This study investigated effects of different methionine(Met)supplementation levels in a reduced protein diet on growth performance,intestinal health,and different physiological parameters in broilers under Eimeria challenge.A total of 600 fourteen-day-old Cobb500 male broilers were challenged with E.maxima,E.tenella,and E.acervulina,and randomly allocated in a 2×5 factorial arrangement.Birds received normal protein diets(20%crude protein,NCP)or reduced protein diets(17%crude protein,LCP),containing 2.8,4.4,6.0,7.6,and 9.2 g/kg of Met.Results On 6 and 9 days post inoculation(DPI),increasing Met level linearly improved the growth performance(P<0.05).Total oocyst shedding linearly increased as Met level increased(P<0.05).Duodenal villus height(VH):crypt depth(CD)in the LCP groups were higher on 6 DPI(P<0.01)while lower on 9 DPI(P<0.05)compared to the NCP groups.Jejunal CD and duodenal VH:CD changed quadratically as Met level increased(P<0.05).On 6 DPI,liver glutathione(GSH)and glutathione disulfide(GSSG)linearly increased as Met level increased(P<0.05).On 9 DPI,GSSG quadratically increased,whereas GSH:GSSG quadratically decreased as Met levels increased(P<0.05).The expression of amino acid transporters linearly decreased as Met level increased(P<0.05).The expression of zonula occludens 2 and claudin-1 linearly increased on 6 DPI whereas decreased on 9 DPI as Met level increased(P<0.05).The expressions of cytokines were lower in the LCP groups than the NCP groups(P<0.05).Interaction effects were found for the expression of IL-10 and TNFαon 6 DPI(P<0.05),where it only changed quadratically in the NCP group as Met level increased.The expression of Met and folate metabolism genes were lower in the LCP groups than the NCP groups on 9 DPI(P<0.05).The expression of these genes linearly or quadratically decreased as Met level increased(P<0.05).Conclusion These results revealed the regulatory roles of Met in different physiological parameters including oxidative status,intestinal health,and nutrient metabolism in birds fed reduced protein diet and challenged with Eimeria.

Improvement of glucose metabolism in middle-aged mice on a high-fat diet by whole-grain highland barley is related to low methionine levels

Methionine restriction(MR)is an effective dietary strategy to regulate energy metabolism and alleviate oxidative stress and inflammation in the body,especially in the middle-aged and elderly population.However,the high methionine content of meat products makes this dietary strategy impossible to combine with protein supplementation and MR.Highland barley(HB),a low-methionine cereal,not only provides the body with protein but also has improved glucose metabolism and antioxidant and anti-inflammatory properties.Therefore,this study evaluated the feasibility of HB as a source of methionine-restricted dietary protein and the potential mechanisms.Middle-aged C57BL/6J mice were fed a control diet(CON),a high-fat diet(HFD),a whole-grain HB high-fat diet(HBHF),or a HBHF+methionine diet(HBHFmet)for 25 weeks.The results showed that the HBHF could keep the body weight,fasting glucose,insulin,homeostasis model assessment of insulin resistance(HOMA-IR),blood lipids,inflammation,and oxidative stress of HFD mice at normal levels.Compared with the HFD groups,HBHF inhibited pancreatic cell apoptosis and improved insulin secretion while improving hepatic and skeletal muscle glucose metabolism.However,these efficacies were attenuated in HBHFmet group mice.These findings suggest that HBHF has an MR strategy.

Determination of Carbon and Nitrogen Isotope Fractions in Asparagine, Aspartic Acid, Threonine and Methionine

The nomenclature for compounds that are modified with isotopes is growing every day. Compounds can be modified with isotopes either individually, in a functional group or groups, or completely with all atomic centers of the element. This diversity of isotope-modified compounds increases the range of researches that can be studied using them. Compounds modified with isotopes of carbon-13 or nitrogen-15 can be converted into carbon monoxide, carbon dioxide and molecular nitrogen. Currently, only the average value of carbon-13 or nitrogen-15 isotopes can be determined. However, by directly determining the atomic share of these isotopes in organic compounds modified with isotopes, information about the isotopic centers of the element can be obtained. The atomic fraction of an element is defined as a single carbon or nitrogen isotope-modified center or centers, or all centers that are isotope-modified with that element at the same time. Carbon-13 or nitrogen-15 isotopes’ atomic fraction can be determined molecularly or with fragment ions of different elemental content, or both. This makes the method self-verifying, increasing the accuracy and reliability of the results obtained. Amino acids, such as asparagine, aspartic acid, methionine, and threonine, are essential for the human body. This proposed method of isotopic analysis will increase the possibilities for scientific research using these compounds.

Feeding Practices and Use of Lysine and Methionine in Pigrationing on Intensified Pig Breeding in the West Center and Hauts Bassins Regions of Burkina Faso

The aim of the study was to assess feeding practices and the use of lysine and methionine in pig rationing on intensified and semi-intensive pig breeding in the Koudougou and Bobo-Dioulasso areas. To this end, a cross-sectional survey was carried out on 87 breeding in these towns. A Discriminant Factorial Analysis (DFA) confirming a k-means classification of the data collected was used to retain 71 breeding divided into three breeding classes: Class A (32.4% of breeding), Class B (14.08%) and Class C (53.52%). The results show that the majority of pig breeders were men between the ages of 36 and 59. Average herd sizes were 35 ± 28;79 ± 42 and 89 ± 21 pigs for Classes A, B and C respectively. The main breeds of pig found on the breeding were crossbred, Large white, local, Landrace and Duroc. Class A (26.1%), B (30%) and C (15.8%) breeders were familiar with both lysine and methionine. Class A breeders distributed feed staggered (65.2%) and in rations (34.8%). Lysine (13%) and methionine (8.7%) were purchased at 5250 FCFA/kg. Those in class B distributed feed staggered (50%) and in the form of rations (50%), in which they incorporated lysine (30%) and methionine (30%) purchased at a cost of 2500 FCFA/kg and 3000 FCFA/kg respectively. Rationing and staggered feeding were practiced by 23.7% and 76.3% of Class C breeders respectively. Only lysine purchased at 3400 FCFA/kg was incorporated into rations by 10.5% of breeders. The high cost of lysine and methionine was incriminated by Class A (100%), B (33.3%) and C (50%) breeders. In conclusion, intensive pig breeding, the practice of rationing and the incorporation of the amino acids lysine and methionine are of ascending importance from classes C, A to B. The high cost of feedstuffs, particularly lysine and methionine, compromises their use in rations, which could have a negative impact on expected breeding performance. The screening and use of feeds rich in and/or enriched with these amino acids, through the development or adaptation of technologies, could improve the efficiency of rations and the productivity of intensive pig breeding in Burkina Faso.

Effects of methionine treatment on storage quality and antioxidant activity of postharvest jujube fruit

Jujube fruits usually suffer from physiological disorders or infectious diseases during storage,leading to quality deterioration,softening,or rotting.Therefore,this study aims to investigate the effect of methionine soaking treatment on the postharvest jujube fruit decay rate and storage quality.Methionine treatment significantly reduced the decay rate of postharvest jujube fruit and effectively maintained fruit color and titratable acid.Methionine treatment effectively delayed the decrease of firmness,soluble solids,ascorbic acid,and lignin contents in jujube fruit.Methionine treatment reduced the content of alanine and phenylalanine,and increased the content of glycine in jujube fruit,but displayed no significant effect on total amino acid content.In addition,jujube fruits in the methionine treatment group had a higher total phenolic and flavonoid content and antioxidant capacity,both in free and bound forms.Compared with the control,methionine treatment also significantly increased the content of individual phenolic acid fractions(gallic acid,vanillic acid,and syringic acid)and flavonoid fractions(catechin,epicatechin,rutin)in free form as well as individual phenolic acid fractions in bound form(gallic acid and ferulic acid)in jujube fruits.Overall,this study suggested that the methionine treatment could be used as a prospective preservative to reduce the postharvest decay of jujube fruit and alleviate its nutritional quality deterioration during cold storage at 4°C.

Lipid metabolism and m^(6)A RNA methylation are altered in lambs supplemented rumen-protected methionine and lysine in a low-protein diet

Background:Methionine or lysine has been reported to influence DNA methylation and fat metabolism,but their combined effects in N6-methyl-adenosine(m^(6)A)RNA methylation remain unclarified.The combined effects of rumen-protected methionine and lysine(RML)in a low-protein(LP)diet on lipid metabolism,m^(6)A RNA methylation,and fatty acid(FA)profiles in the liver and muscle of lambs were investigated.Sixty-three male lambs were divided into three treatment groups,three pens per group and seven lambs per pen.The lambs were fed a 14.5%crude protein(CP)diet(adequate protein[NP]),12.5%CP diet(LP),and a LP diet plus RML(LP+RML)for 60 d.Results:The results showed that the addition of RML in a LP diet tended to lower the concentrations of plasma leptin(P=0.07),triglyceride(P=0.05),and non-esterified FA(P=0.08).Feeding a LP diet increased the enzyme activity or m RNA expression of lipogenic enzymes and decreased lipolytic enzymes compared with the NP diet.This effect was reversed by supplementation of RML with a LP diet.The inclusion of RML in a LP diet affected the polyunsaturated fatty acids(PUFA),n-3 PUFA,and n-6 PUFA in the liver but not in the muscle,which might be linked with altered expression of FA desaturase-1(FADS1)and acetyl-Co A carboxylase(ACC).A LP diet supplemented with RML increased(P<0.05)total m^(6)A levels in the liver and muscle and were accompanied by decreased expression of fat mass and obesity-associated protein(FTO)and alk B homologue 5(ALKBH5).The m RNA expressions of methyltransferase-like 3(METTL3)and methyltransferase-like 14(METTL14)in the LP+RML diet group were lower than those in the other two groups.Supplementation of RML with a LP diet affected only liver YTH domain family(YTHDF2)proteins(P<0.05)and muscle YTHDF3(P=0.09),which can be explained by limited m^(6)Abinding proteins that were mediated in m RNA fate.Conclusions:Our findings showed that the inclusion of RML in a LP diet could alter fat deposition through modulations of lipogenesis and lipolysis in the liver and muscle.These changes in fat metabolism may be associated with the modification of m^(6)A RNA methylation.

SELECTION OF METHIONINE-RESISTANT VARIANT OF MEDICAGO SATIVA

A variant cell line of Medicago saliva L. resistant to methionine (100 mmol/L) was selected .from calli treated with 1.5 mmol/L sodium azide. Plants were regenerated from this selected cell line. After growing for 6 months on the medium devoid of selection pressure, the variant cell line still showed high level of resistance, which was 7.2-fold higher than that of the wild type. This indicated the stability of the resistance expression. The variant cell line also expressed a 3.3-fold higher cross-resistance to ethionine, than that of the wild type. The contents of methionine, lysine, threonine and isoleucine in the variant calli were respectively 2.0, 1.43, 1.71 and 1.57 times higher than those in the wild type. However, in the variant regenerated plantlets they respectively were 2.0, 1.33, 1.22 and 1.22 times higher than those in the wild type regenerants. Four new protein bands, viz. 65 kD, 23 kD, 20 kD and 18 kD, appeared in the SDS-PAGE pattern of soluble proteins in the variant calli. In the peroxidase isoenzyme electrophoresis pattern, two new bands were also observed in the variant calli.

半胱氨酸、蛋氨酸对体外培养绒山羊次级毛囊生长及毛乳头细胞增殖的影响

旨在探讨半胱氨酸(Cys)、蛋氨酸(Met)对体外培养燕山绒山羊次级毛囊(SF)生长及毛乳头细胞(DPCs)增殖凋亡的影响。本研究选取1只1周岁左右健康雄性燕山绒山羊皮肤若干,完整分离的次级毛囊随机分成Cys试验组和Met试验组,每组各浓度3个重复,每个重复8根毛囊,Cys试验组添加浓度分别为0、40、80、120、160和200μg·mL^(-1);Met试验组添加浓度分别为0、10、15、20、25和30μg·mL^(-1)。毛囊培养7 d,每隔24 h观察毛囊生长形态并测量毛囊生长长度。从燕山绒山羊次级毛囊中提取、纯化DPCs,细胞计数并绘制DPCs生长曲线,将DPCs随机分为Cys试验组和Met试验组,每组各浓度6个重复,Cys试验组添加浓度分别为0、20、40、60、80、100μg·mL^(-1);Met试验组添加浓度分别为0、2、4、6、8、10μg·mL^(-1)。培养2 h后用CCK-8试剂盒进行细胞增殖活力测定,确定燕山绒山羊DPCs中Cys和Met的最适培养浓度,利用qRT-PCR技术检测不同浓度Cys和Met的DPCs增殖相关基因(PCNA、CCND1、CDC42、CDK 4)、凋亡相关基因(P21、P53、Bax、Caspase-3、Bcl-2)、皮肤细胞分化相关基因(IVL)及角蛋白相关基因(K10、K 14)mRNA表达水平。结果表明,与对照组相比,添加80和120μg·mL^(-1) Cys与10、15、20和25μg·mL^(-1) Met均可显著影响次级毛囊生长速率和累计生长长度(P<0.01),且120μg·mL^(-1) Cys和20μg·mL^(-1) Met促生长效果最好;添加20、40和60μg·mL^(-1) Cys可以显著影响DPCs增殖(P<0.05),添加40μg·mL^(-1) Cys显著上调PCNA、CCND1、CDK4、Bcl-2、K10、K 14和IVL基因mRNA表达量(P<0.05);添加6μg·mL^(-1) Met可以显著影响DPCs增殖(P<0.05),并显著上调PCNA、CCND1、CDK4、P21、Bcl-2、K 10和K 14基因mRNA表达量(P<0.05)。综上,添加Cys和Met可以显著促进长绒期燕山绒山羊次级毛囊生长,最适添加量分别为120μg·mL^(-1)和20μg·mL^(-1);添加Cys和Met可通过上调细胞增殖、分化、角蛋白等基因mRNA、下调细胞凋亡基因mRNA表达,促进乳头细胞增殖、抑制细胞凋亡进而促进燕山绒山羊次级毛囊生长。

无鱼粉无豆粕饲料中补充蛋氨酸对鲤生长性能、氮循环和氨基酸沉积率的影响及补充晶体氨基酸饲料的投喂策略

本试验旨在研究无鱼粉无豆粕饲料中补充蛋氨酸对鲤生长性能、氮循环和氨基酸沉积的影响,以及投喂频率和补充蛋氨酸对鲤餐后血浆氨基酸含量的影响。选取初始体重为27 g的鲤幼鱼540尾,随机分为2组(每组9个重复,每个重复30尾),分别投喂添加0(C组)和3 g/kg蛋氨酸(DL-蛋氨酸形式,纯度为98%)(M组)的无鱼粉无豆粕饲料,2种试验饲料中蛋氨酸含量分别为3.85和6.83 g/kg饲料(1.14和1.99 g/16 g N),进行为期8周的生长试验。生长试验结束后称重,采样后继续进行1周的投喂频率试验。投喂频率试验中,将C组和M组试验鱼分别随机分为3组,投喂频率分别为1、2和3次,共6组,每组3个重复,每个重复25尾。采集餐后不同时间点血浆样品,测定血浆游离氨基酸含量。结果显示:1)在无鱼粉无豆粕饲料中补充蛋氨酸显著提高了鲤的总摄食量、特定生长率和蛋白质效率(P<0.05),显著降低了摄食率和饲料系数(P<0.05);2)与C组相比,M组鲤氮摄入量从54.33 g/桶显著提高到60.73 g/桶(P<0.05),但氮排放量从41.08 g/桶显著降低到37.93 g/桶(P<0.05),各氨基酸以及总必需氨基酸和总非必需氨基酸沉积率均显著提高(P<0.05),血浆尿素氮和总胆固醇含量显著下降(P<0.05);3)投喂频率达到3次时,饲料中补充蛋氨酸可显著提高鲤血浆蛋氨酸、赖氨酸曲线下面积(AUC)(P<0.05)。综上所述,在无鱼粉无豆粕饲料中补充蛋氨酸至鲤需求量,在日投喂3次的养殖模式下,鲤的生长性能得到显著改善,饲料效率和氮沉积率显著提高,说明提高投喂频率是提高晶体氨基酸利用率的有效方法。

植酸-胞嘧啶对DL-蛋氨酸粉尘爆燃火焰抑制特性研究

饲料及其添加剂粉尘具有较高的燃烧热,在生产过程中存在一定的爆燃风险,威胁生命财产安全,预混抑制剂是目前应用最为广泛的一种抑爆手段,但传统抑制剂不可食用,无法加入至饲料类粉尘实现抑爆。因此,以饲料主要添加剂DL-蛋氨酸(DLM)粉尘为研究对象,采用自主合成的营养价值高、绿色可食用生物质基植酸-胞嘧啶(PA-CY),研究了PA-CY对DLM粉尘爆燃火焰传播特性的影响,通过高速摄影和可视化竖直管道记录爆燃火焰传播过程并计算火焰速度,采用热电偶监测火焰温度变化。结果表明:随PA-CY质量分数的升高DLM爆燃火焰亮度持续下降,严重破坏了火焰结构,加入20%PA-CY后火焰峰值速度、平均速度和峰值温度由27.66 m/s、14.39 m/s、1014℃分别下降至13.83 m/s、6.88 m/s、540℃,下降比重达50.0%、52.2%和46.7%,且PA-CY质量分数达30%后粉尘无法被点燃,说明PA-CY抑制效果显著。此结果可为饲料类粉尘爆燃的防治提供理论支持。

柱前衍生高效液相荧光检测法测定复方氨基酸注射液中甲硫氨酸亚砜的含量

目的建立测定复方氨基酸注射液中甲硫氨酸亚砜质量分数的高效液相色谱法。方法采用柱前衍生HPLCFLD法,色谱柱为Agilent Poroshell 120 EC-C18柱(3.0 mm×100 mm,2.7μm)或效能相当的色谱柱,以乙酸钠四氢呋喃溶液为流动相A,乙酸钠溶液-乙腈-甲醇(体积比20∶40∶40)为流动相B,流速为1.0 mL/min,采用荧光检测器,激发波长为233 nm,发射波长为441 nm。结果甲硫氨酸亚砜质量浓度在0.02605~2.605μg/mL范围内线性关系良好(r=0.9996),检测限为1.3×10^(-3)ng,平均回收率为100.1%,RSD为0.3%。结论建立的方法操作简单、准确、灵敏度高,适用于复方氨基酸注射液中甲硫氨酸亚砜的测定。

高含固污泥厌氧消化中甲硫氨酸分解抑制特性

高含固污泥热水解厌氧消化条件下,沼气中硫化氢体积分数较常规含固率污泥厌氧消化低。沼气中硫化氢主要来源于污泥中含硫氨基酸的降解,而硫化氢体积分数下降与甲硫氨酸分解抑制相关。在测定高含固厌氧消化过程中含硫氨基酸分解中间产物分布的基础上,以甲硫氨酸为基质对高含固厌氧消化污泥进行富集,分析富集后微生物种群结构特性及甲硫氨酸分解过程中挥发性有机硫分布,探讨高含固厌氧消化环境对甲硫氨酸分解过程的抑制特性。结果显示:高含固厌氧消化环境下,甲硫氨酸厌氧分解含硫产物包括甲硫醇、甲硫醚、二甲二硫和硫化氢。常规含固率及高含固率环境下,甲硫氨酸分解批次试验中硫化氢积累体积分数分别约为2417×10^(-6)和1418×10^(-6),表明高含固厌氧消化过程中硫化氢生成量下降可能由甲硫氨酸厌氧分解时中间产物积累导致。高含固污泥采用甲硫氨酸富集培养时,结果显示Pseudomonas相对丰度为26%,分解产物包括氨氮、甲硫醇和二甲二硫等。其中,二甲二硫先于甲硫醇达峰值,体积分数分别约为267.7×10^(-6)和164.9×10^(-6),推测反应前期甲硫醇快速转化为二甲二硫,后期速率减慢,甲硫醇开始累积。当氨氮质量浓度增至4000 mg/L时,二甲二硫生成速率降低16.79%,表明高含固系统的高氨氮环境可抑制甲硫氨酸分解产生二甲二硫。

蛋氨酸及其小肽在奶牛生产中的应用研究进展

蛋氨酸是奶牛最重要的限制性氨基酸之一,其在奶牛日粮中的缺乏可导致蛋白质利用率降低、环境污染等问题。蛋氨酸小肽作为蛋氨酸补充剂其利用效率比游离蛋氨酸更高,因此其在奶牛生产中的饲用价值值得深入探讨并开发应用。本文简述了蛋氨酸及其小肽的结构、吸收代谢方式和生物学功能,总结了蛋氨酸及其小肽在奶牛生产中的应用进展,并重点阐述了蛋氨酸及其小肽影响奶牛生产性能的可能机制,以期为蛋氨酸及其小肽在奶牛养殖上的应用提供理论依据。

Effect of Low Temperature Stress on Sweet Potato S-Adenosyl Methionine Synthetase Gene Expression

[Objective] The mRNA expression level changes of S-adenosylmethionine synthetase （SAMS） under low temperature stress was studied. [Method] Total RNA were extracted from leaves, stem and earthnut of sweet potato 0,12,24,48 and 72 h after low temperature treatement, mRNA expression level was analyzed by reverse expression and Real-time PCR technique. [Result] The expression quality of the gene extracted from leaves, stem and earthnut increased and the expression quality reached the peak point 24,72 and 72 h after low temperature treatment respectively. The expression change of earthnut was the biggest. [Conclusion] Low temperature was good for increasing mRNA expression of relevart genes.

白杨素减轻非酒精性脂肪性肝炎小鼠脂肪变性及血脂异常

目的:探究白杨素对非酒精性脂肪性肝炎(NASH)的治疗作用。方法:将8周龄C57BL/6雄性小鼠随机分为对照组、模型组和白杨素组。除对照组给予普通饲料外,其余各组给予蛋氨酸胆碱缺乏(MCD)饲料喂养。造模5周后灌胃,白杨素组给予白杨素(20 mg/kg),对照组和模型组给予同体积生理盐水,连续给药6周。实验期间观察小鼠状态;处死后观察小鼠肝脏形态;检测体质量和肝脏湿重;用生化分析仪检测血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)含量、丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;用试剂盒检测肝脏组织中TG和TC含量;采用苏木素-伊红(HE)染色和F4/80免疫组化染色探究白杨素对NASH中肝细胞损伤和炎症的影响;用油红O染色探究肝脏脂质沉积的程度;Masson和天狼星红染色检测肝纤维化;免疫组化法检测纤维化相关分子的表达水平。结果:与对照组比较,模型组小鼠体重和肝脏湿重明显下降,肝脏体积减小,呈黄色,可见黄色脂肪斑,边缘钝;血清TG、LDL-C、ALT和AST水平,以及肝组织TG和TC水平显著升高,HDL-C水平降低;病理染色结果显示有明显的炎症细胞浸润、脂质沉积及肝脏纤维化。与模型组相比,白杨素组小鼠体重和肝重均有升高,肝脏红润,表面相对光滑,肝缘锐利;血清TG、LDL-C、AST和ALT水平,以及肝组织TG水平显著降低;白杨素可抑制炎症细胞浸润、脂质沉积及肝组织纤维化。结论:白杨素可抑制肝脏脂肪变性、炎症及纤维化,有潜力成为治疗NASH的候选药物。

玉米含硫氨基酸代谢机制研究进展

玉米是重要的粮食和饲料作物,为畜禽提供必需的代谢能和营养物质,饲料的营养价值与蛋白质及氨基酸组成比例密切相关。玉米籽粒蛋白质中氨基酸组成不平衡,添加豆粕可以补充玉米中缺乏的赖氨酸和色氨酸。然而,含硫氨基酸(半胱氨酸和蛋氨酸)也是大豆中的限制性氨基酸,其中蛋氨酸直接影响畜禽的机体蛋白质合成速率,进而影响肉蛋奶的产量。因此,提高玉米含硫氨基酸特别是蛋氨酸含量对畜牧业发展意义重大。我国玉米种质资源中缺乏高蛋氨酸种质,且育种进程缓慢。近年来,随着人们对植物硫元素的吸收和转运机制的深入研究,初步构建了含硫氨基酸代谢调控网络。本研究概括了近年来关于提高作物含硫氨基酸代谢机制的研究结果,提出将群体遗传学、比较基因组学及分子生物学相结合挖掘候选基因的新策略,并利用现代生物育种技术提高玉米籽粒蛋氨酸含量,为优质蛋白玉米遗传改良提供参考。

miR-144-3p参与高蛋氨酸饮食诱导Cbs^(+/-)小鼠的肝细胞自噬

背景:高蛋氨酸饮食可导致Cbs^(+/-)小鼠发生肝损伤,高同型半胱氨酸血症与肝脂肪变性、自身免疫性肝炎、酒精性脂肪肝等多种肝脏相关疾病的发生和进展有关。微小RNA(miRNAs)参与细胞存活、分化和细胞自噬等各种细胞过程,具有重要意义。目的:探讨miR-144-3p在高蛋氨酸饮食诱导Cbs^(+/-)小鼠肝细胞自噬中的关键作用。方法:①选取4周龄体质量相近的雄性胱硫醚β-合成酶基因正常(Cbs^(+/+))小鼠和单基因敲除(Cbs^(+/-))小鼠各10只,均饲以高蛋氨酸饮食,12周后处死,留取肝脏组织。②体外培养人源肝细胞(HL-7702),分为对照组(0μmol/L同型半胱氨酸)、同型半胱氨酸组(100μmol/L同型半胱氨酸)、mimic-NC组(转染mimic-NC)、mimic-NC+同型半胱氨酸组(转染mimic-NC+100μmol/L同型半胱氨酸)、miR-144-3p mimic组(转染miR-144-3p mimic)、miR-144-3p mimic+同型半胱氨酸组(转染miR-144-3p mimic+100μmol/L同型半胱氨酸)、inhibitor-NC组(转染inhibitor-NC)、inhibitor-NC+同型半胱氨酸组(转染inhibitor-NC+100μmol/L同型半胱氨酸)、miR-144-3p inhibitor组(转染miR-144-3p inhibitor)、miR-144-3p inhibitor+同型半胱氨酸组(转染miR-144-3p inhibitor+100μmol/L同型半胱氨酸)。采用荧光定量PCR检测肝组织和肝细胞中miR-144-3p的表达水平;转染miR-144-3p模拟物或抑制剂后,采用荧光定量PCR和Western blot分别检测miR-144-3p的转染效率及其对LC3B和p62蛋白表达的影响;酶联免疫法检测肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶的表达情况;Pearson相关性分析肝细胞miR-144-3p表达与肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶含量的相关性。结果与结论:①与Cbs^(+/+)组比较,Cbs^(+/-)组小鼠肝组织和同型半胱氨酸组肝细胞中miR-144-3p的表达水平降低(P<0.01);②转染miR-144-3p模拟物后,与mimic-NC比较,miR-144-3p mimic组中LC3B-Ⅱ/Ⅰ蛋白的表达水平降低,p62蛋白的表达水平升高(P<0.01);转染miR-144-3p inhibitor后,得到相反的结果(P<0.01);③与mimic-NC组相比,miR-144-3p mimic组肝细胞上清液中丙氨酸氨基转移酶和门冬氨酸氨基转移酶的含量降低(P<0.01);与inhibitor-NC组比较,得到相反的结果(P<0.01);④肝细胞中miR-144-3p的表达与肝细胞上清液中丙氨酸氨基转移酶(P<0.01,r=-0.8876)和门冬氨酸氨基转移酶(P<0.01,r=-0.8299)的含量呈负相关;⑤结果表明,同型半胱氨酸通过抑制miR-144-3p的表达促进肝细胞的自噬,进而加重肝损伤。

体外发酵法评估呼伦贝尔羊瘤胃对过瘤胃蛋氨酸的生物利用率及过瘤胃蛋氨酸对瘤胃微生物的影响

为了探究呼伦贝尔羊瘤胃体外发酵体系对过瘤胃蛋氨酸(RPMet,由玉米淀粉和棕榈油包被)的生物利用率及过瘤胃蛋氨酸对瘤胃微生物类群的影响,取4只装有瘘管的雌性呼伦贝尔羊的瘤胃液进行体外发酵试验,在发酵起始阶段与发酵后2 h、4 h、6 h、8 h、12 h、24 h测定发酵体系中的RPMet降解率。每个发酵时间节点设置5个平行处理,在以上7个时间节点取发酵液并进行16S rDNA测序分析。结果表明,在体外发酵体系中,RPMet在发酵后8 h的降解率小于30.0%,说明该产品的包被效果较好。测序结果显示,发酵24 h后的Simpson指数显著低于发酵12 h后的Simpson指数(P<0.05)。主坐标分析(PCoA)能够显示物种的β-多样性,本研究结果显示,各时间点的聚类结果皆存在显著差异(P<0.05)。拟杆菌门、瘤胃球菌属、普雷沃氏菌属菌的相对丰度在发酵后8 h内显著上升,发酵12 h后显著下降(P<0.05)。厚壁菌门、广古菌门、甲烷杆菌属、解琥珀酸菌属、颤螺菌属菌的相对丰度在发酵后12 h内显著下降,之后显著上升(P<0.05)。综上所述,研究中所用的RPMet的包被效果较好,且发酵前期主要是普雷沃氏菌属、瘤胃球菌属微生物降解玉米淀粉包被层,而RPMet的过度降解显著提高了甲烷杆菌属微生物的相对丰度。

基于生物信息学对比分析高脂饮食和蛋氨酸胆碱缺乏饮食构建的非酒精性脂肪性肝病小鼠模型

目的比较高脂饮食(high-fat diet,HFD)饮食和蛋氨酸胆碱缺乏(methionine-choline-deficient,MCD)饮食对C57小鼠非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)模型的影响,通过生物信息学方法揭示其差异表达基因(GEO-data-based analysis of differentially expressed genes,DEGs)并进行功能分析。方法从GEO数据库中获取GSE78170、GSE123354的表达数据。使用R语言分别比较C57小鼠对照组与MCD饮食组、C57小鼠对照组与HFD饮食组数据获得DEGs。对DEGs进行进一步的差异分析、功能富集分析、构建基因和蛋白互作网络及重叠DEGs对比分析。结果筛选出HFD诱导的上调基因44个,下调基因16个;MCD诱导的上调基因43个,下调基因73个;分析富集分析排名前12位的生物学过程,提示HFD与肝脏单纯脂肪积累相关,MCD则能全方位促进NAFL进一步转变为NASH。结论HFD诱导的基因主要涉及肝脏脂肪积累,而MCD更倾向于全方位促进NAFL向NASH的进展。

蛋氨酸铬对母猪乳成分与泌乳相关激素及免疫指标的影响

为了研究蛋氨酸铬对母猪乳成分、泌乳相关激素和免疫指标的影响,选取18头健康杜洛克×长白母猪随机分为2组,即对照组(基础日粮)和试验组(基础日粮中添加5 g/t蛋氨酸铬),试验于产前7 d开始至产后21 d结束,在产后1、11、21 d采集母猪乳样,检测乳中乳糖、蛋白质、灰分、非脂固形物、脂肪的含量和乳的密度,胰岛素(INS)和催乳素(PRL)等泌乳相关激素的含量和免疫球蛋白A(IgA)、免疫球蛋白G(IgG)和免疫球蛋白M(IgM)等免疫指标的含量。结果显示,与对照组相比,产后1、11 d试验组乳中各乳成分指标差异均不显著(P>0.05),产后21 d试验组乳中乳糖、非脂固形物、蛋白质和脂肪的含量显著增加(P<0.05);各时间点试验组乳中INS的含量无显著差异(P>0.05),PRL含量显著升高(P<0.05);产后1 d试验组乳中IgG的含量无显著差异(P>0.05),IgG和IgM含量显著升高(P<0.05),产后11 d和21 d试验组IgA、IgG和IgM含量显著升高(P<0.05)。表明在基础日粮中添加蛋氨酸铬可提高母猪乳中营养成分的含量;提高母猪乳中PRL的含量增强泌乳能力;同时增加乳中IgA、IgG和IgM的含量,增强母猪和仔猪的免疫力。