山羊慢性酒糟中毒血液代谢物分析及乙醇中毒通路关键基因mRNA转录水平变化

doi:10.11843/j.issn.0366-6964.2023.04.038

摘要/Abstract

摘要： 本试验旨在揭示慢性酒糟中毒对山羊血液代谢物及其显著性代谢通路——乙醇中毒代谢通路的影响，筛选潜在性诊断生物标志物，为该病神经系统紊乱发生机制及早期诊断提供参考资料。试验选取4月龄健康大足黑山羊与波尔山羊的杂交后代15只，随机分为3组，65%白酒糟组、75%白酒糟组、基础日粮对照组。每隔15 d，进行肝肾、钙磷及蛋白质代谢指标检测，结合临床症状判断山羊慢性酒糟中毒建模成功。采集血样，利用超高效液相色谱—四极杆飞行时间质谱联用技术进行差异代谢物分析并筛选差异代谢物代谢富集通路，研究差异代谢通路中关键基因的表达变化。代谢组学结果表明，慢性酒糟中毒引起山羊体内多巴胺（DA）和2-呋喃酰甘氨酸（2-FG）上调，前列腺素I₂（PGI₂）下调；突触小泡周期、多巴胺能突触、乙醇中毒、牛磺酸和亚牛磺酸代谢、组氨酸代谢、苯丙氨酸、酪氨酸和色氨酸生物合成、三羧酸循环、鞘脂信号通路、cAMP信号通路等代谢通路均差异显著（P<0.05）。其中，乙醇中毒代谢途径变化显著，且影响因子>0.2；乙醇中毒代谢通路中，75%白酒糟组TH、DAT表达量显著降低（P<0.05），DDC、MAOA表达量呈降低趋势（P>0.05）。综上所述，慢性酒糟中毒可引起山羊血液中有机酸类、氨基酸类、脂质与类脂分子类、胺类、醇类、脂肪酸类及类固醇类代谢物的显著性改变，DA、2-FG、PGI₂可作为慢性酒糟中毒病的潜在性诊断生物标志物。差异代谢物富集的共同通路为乙醇中毒代谢途径，途径中DAT、TH mRNA表达下降，使机体DA浓度升高，进而引起中毒山羊的神经功能紊乱。

关键词: 山羊, 代谢组学, 慢性酒糟中毒, 乙醇中毒通路

Abstract: This experiment aims at investigating the effects of chronic ethanol poisoning on goat blood metabolites and its important metabolic route, screen prospective diagnostic biomarkers, and comprehend some of the etiology of nervous system disorders of the disease and early detection. Fifteen healthy 4-month-old local goats were randomly assigned to three groups:65% distiller's grains, 75% distiller's grains, basic diet control group. Three sets of blood samples were obtained every 15 days during the trial for the detection of liver and kidney, calcium, and protein metabolism indicators. The model of goat distiller's grains chronic poisoning was deemed successful when combined with clinical symptoms. Collect blood samples and screen differential metabolites utilizing ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, analyze metabolic enrichment pathways of differential metabolites, and evaluate the expression of key genes on the significant differential metabolic pathways. The metabolome results showed that chronic distiller's grain poisoning caused up-regulation of dopamine (DA) and 2-Furoylglycine (2-FG) and down-regulation of prostaglandin I₂ (PGI₂) in goats; synaptic vesicle cycle, dopaminergic synapses, Metabolic pathways such as ethanol toxicity, taurine, and hypotaurine metabolism, histidine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tricarboxylic acid cycle, sphingolipid signaling pathway, and cAMP signaling pathway were Significant difference. Among them, the metabolic pathways of ethanol poisoning changed significantly, and the impact factor was >0.2. In the metabolic pathway of alcohol intoxication, the expressions of TH and DAT in group II were significantly decreased (P<0.05); the expressions of DDC and MAOA showed a decreasing trend (P>0.05). In summary, chronic lees intoxication can cause significant alterations in organic acids, amino acids, lipids, lipid-like molecules, amines, alcohols, fatty acids, and steroids metabolites in the blood of goats, and DA, 2-FG, and PGI₂ can be used as potential diagnostic biomarkers for chronic lees intoxication disease. The common pathway of differential metabolite enrichment is the ethanol intoxication metabolic pathway, in which DAT and TH mRNA expression is decreased, resulting in elevated DA concentrations in the organism, which in turn causes neurological dysfunction in intoxicated goats.

Key words: goat, metabolomics, chronic distiller's grain poisoning, ethanol poisoning pathway

中图分类号:

S859.82

郭灵君, 朱瑞, 罗怡茜, 张芝金, 张余蓬, 张德志, 李前勇. 山羊慢性酒糟中毒血液代谢物分析及乙醇中毒通路关键基因mRNA转录水平变化[J]. 畜牧兽医学报, 2023, 54(4): 1751-1765.

GUO Lingjun, ZHU Rui, LUO Yiqian, ZHANG Zhijin, ZHANG Yupeng, ZHANG Dezhi, LI Qianyong. Analysis of Blood Metabolites in Goat Chronic Distiller's Grain Poisoning and Expression Changes of Key Genes in Ethanol Poisoning Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1751-1765.

参考文献 30

[1]	DEROSE K, LIU F, DAVIS R W, et al. Conversion of Distiller's grains to renewable fuels and high value protein:integrated techno-economic and life cycle assessment[J]. Environ Sci Technol, 2019, 53(17):10525-10533.
[2]	IRAM A, CEKMECELIOGLU D, DEMIRCI A. Distillers' dried grains with solubles (DDGS) and its potential as fermentation feedstock[J]. Appl Microbiol Biotechnol, 2020, 104(14):6115-6128.
[3]	LIU Y Z, LIU S P, HUANG C H, et al. Chinese Baijiu distiller's grains resourcing:Current progress and future prospects[J]. Resour, Conserv Recycl, 2022, 176:105900.
[4]	POL K J V, LUEBBE M K, CRAWFORD G I, et al. Performance and digestibility characteristics of finishing diets containing distillers grains, composites of corn processing coproducts, or supplemental corn oil[J]. J Anim Sci, 2009, 87(2):639-652.
[5]	PANCINI S, SIMEONE A, BENTANCUR O, et al. Evaluation of sorghum dried distillers' grains plus solubles as a replacement of a portion of sorghum grain and soybean meal in growing diets for steers[J]. Livest Sci, 2021, 250:104564.
[6]	FLODMAN H R, BOYER E J, MUTHUKUMARAPPAN A, et al. Extraction of soluble fiber from Distillers' grains[J]. Appl Biochem Biotechnol, 2012, 166(4):1070-1081.
[7]	冀建军, 王俊萍, 赵书臣, 等. 白酒糟添加量对肉羊增重效果的影响[J]. 黑龙江畜牧兽医, 2016(12):83-84.JI J J, WANG J P, ZHAO S C, et al. Influence of the addition amount of liquor lees on the weight gain of mutton sheep[J]. Heilongjiang Animal Science and Veterinary Medicine, 2016(12):83-84. (in Chinese)
[8]	王丽. 日粮白酒糟水平对山羊生产性能和营养物质表观消化率影响[D]. 保定:河北农业大学, 2014.WANG L. Effects of white Distillers' grains diets on performance and nutrient apparent digestibility of goats[D]. Baoding:Hebei Agricultural University, 2014. (in Chinese)
[9]	王建华. 兽医内科学[M]. 4版. 北京:中国农业出版社, 2010:12.WANG J H. Veterinary internal medicine[M]. 4th ed. Beijing:China Agricultural Press, 2010:12. (in Chinese)
[10]	LIU K S. Chemical composition of distillers grains, a review[J]. Journal of Agricultural and Food Chemistry, 2011, 59(5):1508-1526.
[11]	冀建军, 王俊萍, 段新安, 等. 一例育肥羊白酒糟中毒的诊断与治疗[J]. 畜牧与兽医, 2016, 48(5):144.JI J J, WANG J P, DUAN X A, et al. Diagnosis and treatment of a case of fattening sheep poisoning with vinasses[J]. Animal Husbandry & Veterinary Medicine, 2016, 48(5):144. (in Chinese)
[12]	李丽. 浅析牛酒糟中毒的防治[J]. 山东畜牧兽医, 2018, 39(4):82.LI L. Analysis on the prevention and treatment of cattle distiller's grains poisoning[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2018, 39(4):82. (in Chinese)
[13]	WHITFIELD J B. Gamma glutamyl transferase[J]. Crit Rev Clin Lab Sci, 2001, 38(4):263-355.
[14]	刘琼, 杨歆, 唐简, 等. 慢性乙型肝炎患者外周血单个核细胞Tim-3和血清IL-33表达及临床意义[J]. 中华医院感染学杂志, 2020, 30(13):2014-2017.LIU Q, YANG X, TANG J, et al. Clinical significance of expression of Tim-3 on peripheral blood mononuclear cells and serum IL-33 in diagnosis of patients with chronic hepatitis B[J]. Chinese Journal of Nosocomiology, 2020, 30(13):2014-2017. (in Chinese)
[15]	ZHANG G M, GUO X X, ZHANG G M. Limiting the testing of urea:urea along with every plasma creatinine test?[J]. J Clin Lab Anal, 2017, 31(5):e22103.
[16]	SOLINAS M, BELUJON P, FERNAGUT P O, et al. Dopamine and addiction:what have we learned from 40 years of research[J]. J Neural Transm (Vienna), 2019, 126(4):481-516.
[17]	MIYAJIMA K, KAWAMOTO C, HARA S, et al. Tyrosine hydroxylase conditional KO mice reveal peripheral tissue-dependent differences in dopamine biosynthetic pathways[J]. J Biol Chem, 2021, 296:100544.
[18]	PETTERSEN J E, JELLUM E. The identification and metabolic origin of 2-furoylglycine and 2, 5-furandicarboxylic acid in human urine[J]. Clin Chim Acta, 1972, 41:199-207.
[19]	CHENG Y, AUSTIN S C, ROCCA B, et al. Role of prostacyclin in the cardiovascular, response to thromboxane A2[J]. Faseb J, 2003, 17(5):A1054-A.
[20]	RICCIOTTI E, FITZGERALD G A. Prostaglandins and inflammation[J]. Arterioscler, Thromb, Vasc Biol, 2011, 31(5):986-1000.
[21]	NORLANDER A E, BLOODWORTH M H, TOKI S, et al. Prostaglandin I2 signaling licenses Treg suppressive function and prevents pathogenic reprogramming[J]. J Clin Invest, 2021, 131(7):e140690.
[22]	ERICSON M, BLOMQVIST O, ENGEL J A, et al. Voluntary ethanol intake in the rat and the associated accumbal dopamine overflow are blocked by ventral tegmental mecamylamine[J]. Eur J Pharmacol, 1998, 358(3):189-196.
[23]	BLOMQVIST O, ERICSON M, ENGEL J A, et al. Accumbal dopamine overflow after ethanol:localization of the antagonizing effect of mecamylamine[J]. Eur J Pharmacol, 1997, 334(2-3):149-156.
[24]	ERICSON M, CHAU P, CLARKE R B, et al. Rising taurine and ethanol concentrations in nucleus accumbens interact to produce dopamine release after ethanol administration[J]. Addict Biol, 2011, 16(3):377-385.
[25]	FORD C P. The role of D2-autoreceptors in regulating dopamine neuron activity and transmission[J]. Neuroscience, 2014, 282:13-22.
[26]	KLEPPE R, WAHEED Q, RUOFF P. DOPA homeostasis by dopamine:a control-theoretic view[J]. Int J Mol Sci, 2021, 22(23):12862.
[27]	ELKOUZI A, VEDAM-MAI V, EISINGER R S, et al. Emerging therapies in Parkinson disease-repurposed drugs and new approaches[J]. Nat Rev Neurol, 2019, 15(4):204-223.
[28]	RASTEDT D E, VAUGHAN R A, FOSTER J D. Palmitoylation mechanisms in dopamine transporter regulation[J]. J Chem Neuroanat, 2017, 83-84:3-9.
[29]	MELCHIOR J R, JONES S R. Chronic ethanol exposure increases inhibition of optically targeted phasic dopamine release in the nucleus accumbens core and medial shell ex vivo[J]. Mol Cell Neurosci, 2017, 85:93-104.
[30]	HEALEY J C, WINDER D G, KASH T L. Chronic ethanol exposure leads to divergent control of dopaminergic synapses in distinct target regions[J]. Alcohol, 2008, 42(3):179-190.