UPLC-MS/MS代谢组学评价归芪益母口服液对产后奶牛瘤胃的调节作用

doi:10.3864/j.issn.0578-1752.2023.02.013

摘要/Abstract

摘要：

【背景】在当前饲料禁抗的背景下，利用低残留、无毒副作用的中草药制剂进行奶牛围产期保健已备受广大养殖企业的青睐。前期研究发现，归芪益母口服液能够减轻产后奶牛炎性反应、增强免疫力和缓解能量负平衡，但其详细的作用机制尚不清楚。【目的】通过超高效液相色谱-串联质谱（UPLC-MS/MS）代谢组学技术评价归芪益母口服液对产后奶牛瘤胃代谢的调节作用，以期从新的视角揭示该配方的作用机制。【方法】试验选择18头体况相近、2—3胎次健康待产荷斯坦奶牛，随机分为对照组和试验组，每组各9头。试验组和对照组分别在产后0 d灌服归芪益母口服液和饮用水，连续灌服6 d。产后0 d未灌服前和产后7 d晨饲前采集瘤胃液，预处理后采用UPLC-MS/MS技术对瘤胃代谢物进行定性和定量分析。两组瘤胃液代谢物数据导入MetaboAnanlyst 5.0软件进行主成分分析（PCA）和正交偏最小二乘判别分析（OPLS-DA），考察两组奶牛瘤胃液代谢轮廓的变化，筛选差异代谢物进行聚类分析和通路富集分析。【结果】产后7 d，试验组瘤胃代谢轮廓较对照组发生明显改变，43种瘤胃代谢物含量发生了显著变化（VIP>1，P<0.05），其中8种代谢物含量显著上调（P<0.05），分别是9-氧代-10E，12Z-十八碳二烯酸、石榴酸、6-二甲基氨基嘌呤、L-脯氨酸、马来酸、N-乙酰基5-羟色胺、泛酰醇、刺芒柄花素，35种代谢物含量显著下调（P<0.05），包括有机酸及其衍生物、核苷酸及其代谢物、碳水化合物及其代谢物等；聚类分析结果显示差异代谢物能明显区分对照组与试验组奶牛瘤胃液；代谢通路富集分析显示归芪益母口服液明显改变了奶牛瘤胃内组氨酸代谢、戊糖和葡萄糖醛酸相互转化、精氨酸和脯氨酸代谢、色氨酸代谢、嘧啶代谢和嘌呤代谢通路。【结论】归芪益母口服液能促进产后奶牛瘤胃菌群代谢机能，是其缓解产后奶牛能量负平衡、增强免疫、抗炎和维持肠道稳态的机制之一。

关键词: 归芪益母口服液, 产后奶牛, 瘤胃液, UPLC-MS/MS代谢组学

Abstract:

【Background】Under the background of ban on antibiotics in animal feed at the current, the use of low residue, non-toxic Chinese herbal medicine preparations for perinatal health care has been favored by the majority of breeding enterprises. The previous studies found that Guiqi Yimu oral liquid could reduce inflammatory response, enhance immunity and relieve negative energy balance in postpartum cows, but the detailed mechanism of action was unclear. 【Objective】This study aimed to reveal the mechanism of action of Guiqi Yimu oral liquid from a new perspective through the regulatory effect of the prescription on rumen metabolism in postpartum cow with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) metabolomics technology. 【Method】 18 healthy parturient Holstein cows with similar body condition and 2-3 parities were selected and averagely divided into the control group and the test group. From Day 0 to Day 6 after birth, the cows of test group were administrated Guiqi Yimu oral liquid, and those of control group were orally administrated water oral. Rumen fluids of all cows were collected at postpartum Day 0 before administration and postpartum Day 7 before first meal. After pretreatment, the metabolites in rumen were qualified and quantified with UPLC-MS/MS technology. The obtained data was imported into MetaboAnanlyst 5.0 software to perform principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA), investigating alteration in metabolic profiles of rumen fluids between the two groups, and screening differential metabolites for cluster and metabolic pathway enrichment analysis. 【Result】 The rumen metabolic profile of the test group was obvious different with the control group at postpartum Day 7 due to the concentrations of 43 rumen metabolites changed significantly (VIP>1, P<0.05). Compared wtih the control group, 8 metabolites were up-regulated (P<0.05), including 9-oxoODE, punicic acid, 6-dimethylaminopurine, L-proline, maleic acid, N-acetyl-5-hydroxytryptamine, pantothenol, and formononetin, and 35 metabolites were down-regulated in the test group (P<0.05), including organic acids and their derivatives, nucleotides and their metabolites, carbohydrates and their metabolites. Metabolic pathway enrichment analysis showed Guijiyimu oral liquid obviously altered rumen histidine metabolism, pentose and glucuronic acid mutual conversion, arginine and proline metabolism tryptophan metabolism, pyrimidine metabolism and purine metabolism pathways in postpartum dairy cows. 【Conclusion】 Guiqi Yimu oral liquid promoted the metabolic function of rumen flora in postpartum dairy cows, which was one of the mechanisms to alleviate the negative energy balance, enhance immunity, anti-inflammation and maintain intestinal homeostasis in postpartum dairy cows.

Key words: Guiqi Yimu oral liquid, postpartum dairy cows, rumen fluid, UPLC-MS/MS metabolomics

唐玉林, 张博, 任曼, 张瑞雪, 秦俊杰, 朱浩, 郭延生. UPLC-MS/MS代谢组学评价归芪益母口服液对产后奶牛瘤胃的调节作用[J]. 中国农业科学, 2023, 56(2): 368-378.

TANG YuLin, ZHANG Bo, REN Man, ZHANG RuiXue, QIN JunJie, ZHU Hao, GUO YanSheng. Evaluation of Regulatory Effect of Guiqi Yimu Oral Liquid on Rumen of Postpartum Dairy Cows Based on UPLC-MS/MS Metabolomics Technology[J]. Scientia Agricultura Sinica, 2023, 56(2): 368-378.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.02.013

https://www.chinaagrisci.com/CN/Y2023/V56/I2/368

图/表 7

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产后7 d奶牛瘤胃液下调差异代谢物"

差异代谢物 Differential metabolite	含量变化 Content change	重要变量投影值 Variable importance projection value	P值 P value
1-辛烯-3-酮 1-Octen-3-one	下调Down	2.54	3.64×10^-5
1,2-二氯乙烷 1,2-Dichloroethane	下调Down	2.53	4.16×10^-5
冰片 (+)-borneol	下调Down	2.52	4.81×10^-5
6-甲基-5-庚烯-2-酮 6-Methyl-5-hepten-2-one	下调Down	2.51	5.23×10^-5
(±)-萜烯-4-醇 p-Menth-1-en-4-ol	下调Down	2.50	6.17×10^-5
磷酸三乙酯 Triethyl phosphate	下调Down	2.48	7.72×10^-5
（±）9,10-二羟基-12Z-十八碳烯酸 9,10-DiHOME [(+-)9,10-dihydroxy-12Z-octadecenoic acid]	下调Down	2.29	4.91×10^-4
溶血卵磷脂酰胆碱16:0 Lysopc 16:0	下调Down	1.95	5.32×10^-3
2,6-二氨基庚二酸 2,6-Diaminooimelic Acid	下调Down	1.91	6.85×10^-3
吲哚-3-丙酸 3-Indolepropionic Acid	下调Down	1.89	7.40×10^-3
溶血磷脂酰胆碱 18:3 Lysopc 18:3	下调Down	1.88	7.95×10^-3
3-羟基-2-吡啶甲酸 3-Hydroxypicolinic acid	下调Down	1.88	8.04×10^-3
3-羟基 - DL-犬尿氨酸 4-3-Hydroxy-DL-kynurenine	下调Down	1.87	8.15×10^-3
脱氧鸟苷 Deoxyguanosine	下调Down	1.85	9.18×10^-3
胸腺嘧啶 Thymine	下调Down	1.78	1.30×10^-2
猪去氧胆酸 Hyodeoxycholic Acid	下调Down	1.76	0.014
2，2'-硫代二乙醇酸 Thiodiglycolic Acid	下调Down	1.71	0.018
尿刊酸 Urocanic Acid	下调Down	1.54	0.019
对苯二甲酸 Phthalic Acid	下调Down	1.70	0.019
L-胱氨酸 L-Cystine	下调Down	1.69	0.019
2,4-二羟基苯甲酸 2,4-Dihydroxybenzoic Acid	下调Down	1.67	0.021
溶血卵磷脂酰乙醇胺16:0 Lysope 16:0	下调Down	1.67	0.022
新蝶呤 Neopterin	下调Down	1.63	0.025
腐胺 Putrescine	下调Down	1.60	0.029
2-吲哚甲酸 Indole-2-Carboxylic Acid	下调Down	1.59	0.031
蛋氨酸亚砜 Methionine Sulfoxide	下调Down	1.58	0.032
乙基丙二酸Ethylmalonate	下调Down	1.57	0.032
土木香内酯 Alantolactone	下调Down	1.56	0.034
溶血卵磷脂酰胆碱18:2 Lysopc 18:2	下调Down	1.55	0.034
十八碳烯酸(反-9) Elaidic Acid (C18:1N9T)	下调Down	1.56	0.034
木糖 Xylose	下调Down	1.55	0.035
尿苷 Uridine	下调Down	1.55	0.036
9S-羟基-10E，12Z，15Z-十八碳三烯酸 9-HOTrE [9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid]	下调Down	1.50	0.044
苯乙酰甘氨酸 N-Phenylacetylglycine	下调Down	1.49	0.045
鸟苷 Guanosine	下调Down	1.48	0.046

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差异代谢物 Differential metabolite	含量变化 Content change	重要变量投影值Variable importance projection value	P值 P value
9-氧代-10E，12Z-十八碳二烯酸 9-oxoODE[9-oxo-10E,12Z-octadecadienoic acid]	上调Up	2.51	5.71×10^-5
石榴酸 Punicic Acid	上调Up	2.11	2.09×10^-3
6-二甲基氨基嘌呤 6-Dimethylaminopurine	上调Up	2.10	2.24×10^-3
L-脯氨酸 L-Proline	上调Up	1.88	7.83×10^-3
马来酸 Maleic Acid	上调Up	1.87	8.30×10^-3
N-乙酰基5-羟色胺 O-N-Acetyl-5-Hydroxytryptamine	上调Up	1.75	0.015
DL-泛酰醇 Pantothenol	上调Up	1.67	0.022
刺芒柄花素 Formononetin	上调Up	1.61	0.029