中国药学杂志

No.	Metabolite	Fold Change	P value	VIP	AH vs CUMS	CUMS vs Con
M1	(+/-)-Potassium citramalate monohydrate	4.186	0.000 0	1.357 4	↑	↓
M2	Acetyl CoA	0.056	0.000 0	1.348 4	↓	↑
M3	L-Threonine	0.068	0.000 0	1.720 0	↓	↑
M4	L-Proline	0.071	0.000 0	1.718 0	↓	↑
M5	L-Homoserine	0.074	0.000 0	1.717 6	↓	↑
M6	DL-Methionine sulfoxide	0.046	0.000 0	1.334 3	↓	↑
M7	L-Hydroxyproline	0.035	0.000 1	1.327 2	↓	↑
M8	Glycine	0.194	0.000 1	1.320 6	↓	↑
M9	Hypotaurine	0.045	0.000 1	1.315 5	↓	↑
M10	L-Histidine	2.511	0.000 1	1.308 1	↑	↓
M11	D-2-Hydroxyglutaric acid	8.174	0.000 1	1.309 5	↑	↓
M12	DL-2-Aminoadipic acid	3.832	0.000 1	1.309 6	↑	↓
M13	L-2-Aminoadipic acid	4.262	0.000 2	1.298 4	↑	↓
M14	3-Methyl-L-histidine	0.154	0.000 2	1.656 9	↓	↑
M15	O-Acetyl-L-serine hydrochloride	2.402	0.000 2	1.304 9	↑	↓
M16	Succinic acid	3.182	0.000 3	1.237 2	↑	↓
M17	N-Methyl-D-aspartic acid	2.453	0.000 3	1.296 5	↑	↓
M18	1-Methyl-L-histidine	0.164	0.000 3	1.644 7	↓	↑
M19	Methylmalonic acid	3.188	0.000 3	1.283 9	↑	↓
M20	Dodecanoic acid	0.354	0.000 3	1.307 1	↓	↑
M21	D-Alanine	0.329	0.000 5	1.257 5	↓	↑
M22	Sarcosine	0.370	0.000 6	1.234 0	↓	↑
M23	Alanine	0.374	0.000 7	1.230 9	↓	↑
M24	Ophthalmic acid	0.379	0.000 8	1.598 2	↓	↑
M25	Malic acid	2.809	0.000 9	1.238 6	↑	↓
M26	Citraconic acid	0.393	0.001 7	1.207 4	↓	↑
M27	D-Glucuronic acid sodium salt monohydrate	2.985	0.001 7	1.171 8	↑	↓
M28	(3-Carboxypropyl)trimethylammonium chloride	3.700	0.002 3	1.526 6	↑	↓
M29	Fumaric acid	2.209	0.003 5	1.178 3	↑	↓
M30	cis-4-Hydroxy-D-proline	0.280	0.003 6	1.166 7	↓	↑
M31	d-Desthiobiotin	0.492	0.003 9	1.150 3	↓	↑
M32	Methylguanidine hydrochloride	3.859	0.004 9	1.464 9	↑	↓
M33	sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt	2.519	0.004 9	1.161 3	↑	↓
M34	O-Acetyl-L-carnitine hydrochloride	0.374	0.005 2	1.149 7	↓	↑
M35	Stachyose hydrate from Stachys tuberifera	0.496	0.005 4	1.467 7	↓	↑
M36	L-Citrulline	2.088	0.005 4	1.456 8	↑	↓
M37	Citrulline	2.125	0.005 8	1.449 9	↑	↓
M38	D-Lactose monohydrate	2.529	0.006 3	1.424 8	↑	↓
M39	Shikimic acid	2.588	0.006 5	1.431 1	↑	↓
M40	D-(+)-Trehalose dihydrate	2.395	0.006 8	1.416 4	↑	↓
M41	Uridine 5'-diphospho-N-acetylgalactosamine disodium salt	0.156	0.006 9	1.438 6	↓	↑
M42	D-(+)-Maltose monohydrate	2.423	0.006 9	1.414 5	↑	↓
M43	Sucrose	2.489	0.007 6	1.404 7	↑	↓
M44	Palatinose hydrate	2.414	0.007 9	1.400 8	↑	↓
M45	D-(+)-Cellobiose	2.452	0.008 1	1.397 7	↑	↓
M46	D-(+)-Raffinose pentahydrate	0.447	0.010 5	1.014 3	↓	↑
M47	D-Glucosamine 6-sulfate	2.765	0.011 4	1.350 4	↑	↓
M48	L-Glutamic acid	2.308	0.014 2	1.340 2	↑	↓
M49	2,3-Pyridinedicarboxylic acid	2.715	0.014 7	1.322 1	↑	↓
M50	N-Acetyl-DL-serine	2.278	0.015 4	1.329 4	↑	↓
M51	L-Anserine nitrate salt	2.493	0.016 0	1.332 0	↑	↓
M52	Glycerophosphate disodium salt hydrate	2.043	0.017 2	1.311 4	↑	↓
M53	L-Methionine sulfoximine	0.451	0.018 5	1.310 5	↓	↑
M54	D-Allose	0.475	0.023 4	1.277 0	↓	↑
M55	D-(+)-Galactose	0.477	0.023 6	1.275 8	↓	↑
M56	N-Methyl-L-alanine	0.479	0.024 2	1.272 1	↓	↑
M57	D-(+)-Mannose	0.480	0.024 3	1.271 2	↓	↑
M58	Glutaryl-L-carnitine lithium salt	2.186	0.027 6	1.245 5	↑	↓

No.	Metabolite	Fold Change	P value	VIP	AH vs CUMS	CUMS vs Con
M1	(+/-)-Potassium citramalate monohydrate	4.186	0.000 0	1.357 4	↑	↓
M2	Acetyl CoA	0.056	0.000 0	1.348 4	↓	↑
M3	L-Threonine	0.068	0.000 0	1.720 0	↓	↑
M4	L-Proline	0.071	0.000 0	1.718 0	↓	↑
M5	L-Homoserine	0.074	0.000 0	1.717 6	↓	↑
M6	DL-Methionine sulfoxide	0.046	0.000 0	1.334 3	↓	↑
M7	L-Hydroxyproline	0.035	0.000 1	1.327 2	↓	↑
M8	Glycine	0.194	0.000 1	1.320 6	↓	↑
M9	Hypotaurine	0.045	0.000 1	1.315 5	↓	↑
M10	L-Histidine	2.511	0.000 1	1.308 1	↑	↓
M11	D-2-Hydroxyglutaric acid	8.174	0.000 1	1.309 5	↑	↓
M12	DL-2-Aminoadipic acid	3.832	0.000 1	1.309 6	↑	↓
M13	L-2-Aminoadipic acid	4.262	0.000 2	1.298 4	↑	↓
M14	3-Methyl-L-histidine	0.154	0.000 2	1.656 9	↓	↑
M15	O-Acetyl-L-serine hydrochloride	2.402	0.000 2	1.304 9	↑	↓
M16	Succinic acid	3.182	0.000 3	1.237 2	↑	↓
M17	N-Methyl-D-aspartic acid	2.453	0.000 3	1.296 5	↑	↓
M18	1-Methyl-L-histidine	0.164	0.000 3	1.644 7	↓	↑
M19	Methylmalonic acid	3.188	0.000 3	1.283 9	↑	↓
M20	Dodecanoic acid	0.354	0.000 3	1.307 1	↓	↑
M21	D-Alanine	0.329	0.000 5	1.257 5	↓	↑
M22	Sarcosine	0.370	0.000 6	1.234 0	↓	↑
M23	Alanine	0.374	0.000 7	1.230 9	↓	↑
M24	Ophthalmic acid	0.379	0.000 8	1.598 2	↓	↑
M25	Malic acid	2.809	0.000 9	1.238 6	↑	↓
M26	Citraconic acid	0.393	0.001 7	1.207 4	↓	↑
M27	D-Glucuronic acid sodium salt monohydrate	2.985	0.001 7	1.171 8	↑	↓
M28	(3-Carboxypropyl)trimethylammonium chloride	3.700	0.002 3	1.526 6	↑	↓
M29	Fumaric acid	2.209	0.003 5	1.178 3	↑	↓
M30	cis-4-Hydroxy-D-proline	0.280	0.003 6	1.166 7	↓	↑
M31	d-Desthiobiotin	0.492	0.003 9	1.150 3	↓	↑
M32	Methylguanidine hydrochloride	3.859	0.004 9	1.464 9	↑	↓
M33	sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt	2.519	0.004 9	1.161 3	↑	↓
M34	O-Acetyl-L-carnitine hydrochloride	0.374	0.005 2	1.149 7	↓	↑
M35	Stachyose hydrate from Stachys tuberifera	0.496	0.005 4	1.467 7	↓	↑
M36	L-Citrulline	2.088	0.005 4	1.456 8	↑	↓
M37	Citrulline	2.125	0.005 8	1.449 9	↑	↓
M38	D-Lactose monohydrate	2.529	0.006 3	1.424 8	↑	↓
M39	Shikimic acid	2.588	0.006 5	1.431 1	↑	↓
M40	D-(+)-Trehalose dihydrate	2.395	0.006 8	1.416 4	↑	↓
M41	Uridine 5'-diphospho-N-acetylgalactosamine disodium salt	0.156	0.006 9	1.438 6	↓	↑
M42	D-(+)-Maltose monohydrate	2.423	0.006 9	1.414 5	↑	↓
M43	Sucrose	2.489	0.007 6	1.404 7	↑	↓
M44	Palatinose hydrate	2.414	0.007 9	1.400 8	↑	↓
M45	D-(+)-Cellobiose	2.452	0.008 1	1.397 7	↑	↓
M46	D-(+)-Raffinose pentahydrate	0.447	0.010 5	1.014 3	↓	↑
M47	D-Glucosamine 6-sulfate	2.765	0.011 4	1.350 4	↑	↓
M48	L-Glutamic acid	2.308	0.014 2	1.340 2	↑	↓
M49	2,3-Pyridinedicarboxylic acid	2.715	0.014 7	1.322 1	↑	↓
M50	N-Acetyl-DL-serine	2.278	0.015 4	1.329 4	↑	↓
M51	L-Anserine nitrate salt	2.493	0.016 0	1.332 0	↑	↓
M52	Glycerophosphate disodium salt hydrate	2.043	0.017 2	1.311 4	↑	↓
M53	L-Methionine sulfoximine	0.451	0.018 5	1.310 5	↓	↑
M54	D-Allose	0.475	0.023 4	1.277 0	↓	↑
M55	D-(+)-Galactose	0.477	0.023 6	1.275 8	↓	↑
M56	N-Methyl-L-alanine	0.479	0.024 2	1.272 1	↓	↑
M57	D-(+)-Mannose	0.480	0.024 3	1.271 2	↓	↑
M58	Glutaryl-L-carnitine lithium salt	2.186	0.027 6	1.245 5	↑	↓

白术多糖对抑郁症小鼠动物模型的作用及机制研究

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伍春桃 ¹ , 陈磊 ¹ , 常祥兵 ¹ , 卢杨君 ² , 朱静坚 ² , 田薇 ¹^,^*

中国药学杂志 | 论著 2024,59(9): 789-800

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中国药学杂志 | 论著 2024, 59(9): 789-800

白术多糖对抑郁症小鼠动物模型的作用及机制研究

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伍春桃¹, 陈磊¹, 常祥兵¹, 卢杨君², 朱静坚², 田薇¹^,^*

作者信息

¹ 浙江农林大学食品与健康学院, 杭州 311300

² 缙云县农业农村局, 浙江丽水 323000

伍春桃,女,硕士研究生研究方向:中药功效成分合成生物学

通讯作者:

*田薇,女,博士,教授研究方向:中药学

Effect and Mechanism of Atractylodes macrocephala polysaccharide on CUMS Mouse Models

Chuntao WU¹, Lei CHEN¹, Xiangbing CHANG¹, Yangjun LU², Jingjian ZHU², Wei TIAN¹^,^*

Affiliations

¹ College of Food and Health, Zhejiang A&F University, Hangzhou 311300, China

² Agriculture and Rural Bureau of Jinyun County, Lishui 323000, China

出版时间: 2024-05-08 doi: 10.11669/cpj.2024.09.004

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摘要

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目的整合代谢组学技术和肠道菌群分析,探讨白术多糖(Atractylodes macrocephala polysaccharide,AMP)改善慢性不可预知性刺激(chronic unpredictable mild stress, CUMS)抑郁小鼠模型的作用机制。方法建立CUMS抑郁小鼠模型,AMP低、中、高剂量治疗组给药剂量分别为0.062 5、0.125、0.250 g·kg^-1,氟西汀组为0.015 g·kg^-1,对照组和模型组给予0.9%氯化钠溶液,所有组给药量均为5 mL·kg^-1·d^-1。酶联免疫法测定小鼠大脑中五羟色胺(5-hydroxytryptamine,5-HT)的含量,结合糖水偏好试验结果检验CUMS抑郁模型;采用16S rDNA扩增子测序技术对各组小鼠粪便中肠道菌群测序分析,液相色谱-质谱联用(LC-MS)技术对小鼠血清进行非靶向代谢组学测定。结果与对照组比较,CUMS模型小鼠糖水偏好率明显降低,小鼠大脑内的5-HT含量显著降低(P<0.01),提示CUMS抑郁模型建立成功。AMP治疗后,各组小鼠糖水偏好率和5-HT含量均增加(P<0.01);CUMS小鼠肠道菌群丰度具有回调效果;血清代谢物发生显著改变,58种代谢物显著回调;斯皮尔曼(Spearman)相关性分析显示,肠道菌群的改变与代谢物水平变化显著性相关。结论 AMP通过调节肠道菌群稳态,上调厚壁菌门与拟杆菌门的比值(F-B),进而调控代谢途径,对CUMS抑郁模型小鼠发挥干预效用。

关键词

白术多糖 / 慢性不可预知性刺激 / 肠道菌群 / 代谢组学

Abstract

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OBJECTIVE To explore the mechanism of Atractylodes macrocephala polysaccharide (AMP) in improving chronic unpredictable mild stress (CUMS) depression in mice by integrating metabolomics technology and gut microbiota analysis. METHODS CUMS depression mouse model was established. Low, medium and high dose AMP treatment were given at 0.062 5, 0.125, 0.250 g·kg^-1, respectively, fluoxetine was given at the dosage of 0.015 g·kg^-1, control group and model group were given 0.9% saline solution, and all groups were given at 5 mL·kg^-1·d^-1 of drug. The content of 5-hydroxytryptamine(5-HT) in the brain of mice was determined by enzyme-linked immunosorbent assay (ELISA), and the CUMS depression model was tested by combining sucrose preference test (SPT) results. The 16S rDNA amplicon sequencing technology was used to analyze the gut microbiota in the feces of mice in each group. LC-MS technology was used to perform non-targeted metabolomics determination of mouse serum. RESULTS Compared with the control group, the sucrose preference rate of CUMS model mice was significantly lower, and the content of 5-HT in the brain of mice was significantly reduced (P<0.01), indicating that the CUMS depression model was successfully established. After AMP treatment, the sucrose preference rate and 5-HT content of mice in each group increased (P<0.01), the gut microbiota of CUMS mice had a regulatory effect, serum metabolites were significantly changed, and 58 metabolites were significantly adjusted. Spearman correlation analysis showed that the changes in gut microbiota were significantly associated with the changes in metabolite levels. CONCLUSION AMP exerts intervention effects on CUMS depression model mice by regulating the stability of gut microbiota, upregulating the F-B ratio, and thereby regulating metabolic pathways.

Key words

Atractylodes macrocephala polysaccharide / chronic unpredictable mild stress / intestinal flora / Metabolomics

引用本文

伍春桃, 陈磊, 常祥兵, 卢杨君, 朱静坚, 田薇. 白术多糖对抑郁症小鼠动物模型的作用及机制研究. 中国药学杂志, 2024 , 59 (9) : 789 -800 . DOI: 10.11669/cpj.2024.09.004

Chuntao WU, Lei CHEN, Xiangbing CHANG, Yangjun LU, Jingjian ZHU, Wei TIAN. Effect and Mechanism of Atractylodes macrocephala polysaccharide on CUMS Mouse Models[J]. Chinese Pharmaceutical Journal, 2024 , 59 (9) : 789 -800 . DOI: 10.11669/cpj.2024.09.004

正文

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抑郁症被认为是一种全身性疾病,是严重危害人类身心健康和生活质量的全球性疾病^[1-2],其发病机制非常复杂。目前,因临床上使用的多种抗抑郁药物存在起效缓慢、治愈率低、副作用明显等问题^[3-4],促使人们不断地寻求安全、高效的新型抗抑郁药物^[5]。现代药理学研究表明,抗抑郁作用是多种机制共同作用的结果,主要包括提高单胺类神经递质水平、抑制下丘脑-垂体-肾上腺轴(hypothalamo-pituitary-adrenal axis,HPA)轴亢进、增加神经营养因子(neurotrophic factor,NTF)表达、调节免疫炎症反应和调节肠道微生物-肠-脑轴等。研究发现肠道微生物组的破坏与几种神经性疾病相关,包括帕金森病、孤独症、精神分裂症和抑郁症^[6-8]。

随着对脑-肠轴的探究更深入,也有更多的研究表明脑-肠-微生物轴紊乱可能是抑郁症的病理机制^[9-10],Zhang等^[11]发现巴戟天寡糖可能通过影响肠道菌群代谢从而发挥抗抑郁作用。此外,临床研究还发现,抑郁症患者的肠道微生物群失调与脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)水平降低有关,例如,在益生菌介入后,抑郁症患者肠道中菌群Faecaliberium丰度降低的症状得到改善^[12]。现代药理研究发现巴戟天寡糖^[11]、远志寡糖酯^[13]、茯苓多糖^[14]、石菖蒲多糖^[15]、白芍多糖^[15]、银杏叶多糖^[16]、枸杞多糖^[17]、百合多糖^[18]、黄芪多糖^[19]、刺五加多糖^[20]及金银花多糖^[21]等中药寡糖与多糖均具有显著的抗抑郁活性,可以通过提高脑内单胺类神经递质水平、抑制HPA轴亢进、增加 NTF 表达、调节免疫炎症反应以及肠道微生物-肠-脑轴等多途径发挥抗抑郁作用。中药寡糖与多糖因作用途径/靶点多样、来源丰富等优势,成为抗抑郁药物开发的重要源泉。

白术(Atractylodes macrocephala Koidz.)属于菊科植物,在中药古方中常作为健脾养心药对抑郁症发挥治疗作用。白术对抑郁症^[22]、阿尔茨海默病^[23]和脑缺血损伤性疾病^[24]发挥多方面的治疗作用,但其发挥作用的机制长期以来研究未果^[25]。本研究发现白术多糖(Atractylodes macrocephala polysaccharide,AMP)对慢性不可预知性刺激(CUMS)模型小鼠具有显著的干预作用,但其确切机制尚不明确。以往的研究表明,中药寡糖与多糖抗抑郁作用机制主要有以下几类:提高脑内单胺类神经递质水平^[11,15,18]、调节神经可塑性,提高 BDNF 水平^[13,18]、调节肠道菌群结构^[16]、调节免疫炎症反应^[14,19,21]、降低皮质酮水平^[17]、对抗海马神经元损伤^[21]、调节氧化应激水平^[19-20]等。AMP是一种在肠内难以被吸收的天然多糖物质,揭示它在抗抑郁方面是否存在新的作用机制,将为白术抗抑郁药物的开发提供理论基础和科学依据。本研究整合代谢组学和肠道菌群, 探讨AMP改善CUMS小鼠失调的肠道菌群和血清代谢物的潜在作用机制,从而解析AMP通过体内代谢产物-肠道菌群介导的改善CUMS小鼠抑郁症状的机制。研究结果对AMP作为抗抑郁药物的开发和按照循证证据来科学探究我国传统中药具有重要意义。

1 材料

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1.1 实验动物

108只SPF级雄性美国癌症研究所(Institute of Cancer Research, ICR)小鼠,体质量18~20 g(浙江省医学科学院实验动物中心),动物生产许可证号为SCXK(浙)2019-0002。动物饲养于温度23~25 ℃、相对湿度50%~70%及通风良好的动物房里,每日光照12 h,自由进食饮水,适应性饲养 1周后开始实验。动物饲养管理均严格按照中华人民共和国国务院批准的《实验动物管理条例》执行。

1.2 药品与试剂

白术来源于浙江省丽水市缙云县新建镇,经浙江农林大学食健学院田薇教授鉴定为菊科植物白术(Atractylodes macrocephala Koidz.)的干燥根茎。

无水乙醇(批号20201210)、氟西汀(批号100513-201602)、乙酸铵(批号20210225)(国药集团化学试剂有限公司);色谱级甲醇(批号20125179)、乙腈(批号AS112-001)(美国天地有限公司);过氧化氢(批号130518,武汉博士德生物工程有限公司);粪便基因组DNA快速提取试剂盒(批号RK02011,北京百泰克生物科技有限公司);AxyPrep DNA 凝胶回收试剂盒(爱思进生物技术,美国);Quantus^TM荧光剂(普洛麦尔,美国);DNA建库试剂盒(上海普迈生物科技)。

1.3 仪器

Sephadex G-100柱(上海源叶生物科技有限公司);Prestige-21型傅立叶变换红外光谱仪(日本岛津);Nanodrop2000型核酸检测仪(美国Bio-Rad公司);BioRad CHEF型凝胶电泳仪(美国BioTke公司);Agilent 6545型UPLC-QTOF-MS仪(美国安捷伦科技公司);Miseq PE300平台(美国Illumina公司);BEH酰胺柱(2.1 mm×100 mm,1.7 μm,美国Waters公司);SIMCA多元数据分析软件(版本14.1,瑞典Umetrics公司)。

2 方法

收起

2.1 AMP的制备

将白术根茎粉碎成粉末(过100目筛),然后用超纯水在90℃下提取3 h,提取2次,过滤后旋转蒸发浓缩,然后加入4倍体积的乙醇沉淀多糖。静置4 h后,收集沉淀,体积分数95%乙醇洗脱,真空干燥,干燥后用Sevag法和过氧化氢除去蛋白质和色素。然后将上清浓缩并冻干,得到粗多糖。用Sephadex G-100柱与蒸馏水进一步分离得到AMP。最后对溶液进行400~200 nm扫描,检测蛋白是否被完全去除。用傅里叶变换红外光谱仪测定AMP的结构。

2.2 动物分组与给药

将108只小鼠随机分为6组(n=18):对照组(Con)、模型组(CUMS)、AMP低剂量组(AL)、AMP中剂量组(AM)、AMP高剂量组(AH)、氟西汀治疗组(Flx),其中AMP治疗组低、中、高剂量组给药剂量分别为0.062 5、0.125、0.250 g·kg^-1,Flx组为0.015 g·kg^-1,Con组和CUMS组给予0.9%氯化钠溶液,所有组给药量均为5 mL·kg^-1·d^-1。对除Con组小鼠外的所有动物进行60 d的CUMS应激。

2.3 CUMS模型的构建

按照文献[26]方法进行CUMS应激刺激。对小鼠每天进行两种不同的刺激,且连续两天不能给予同种刺激,应激刺激持续60 d。CUMS方法包含不可预知性的温和刺激:禁食(24 h)、禁水(24 h)、束缚(12 h)、湿笼(12 h)、夹尾(5 min)、45°倾斜(6 h)、摇晃(5 min)、冰水游泳(5 min)、昼夜颠倒等。

2.4 糖水偏好试验

糖水偏好试验前48 h,训练大鼠适应质量分数1%蔗糖水,第1天给小鼠放入两瓶质量分数1%蔗糖水,第2天换为1瓶质量分数1%蔗糖溶液和1瓶纯饮水,两瓶质量一致。适应结束后,进行24 h的禁水、禁食,给予小鼠事先定量好的两瓶水:1瓶质量分数1%蔗糖水,1瓶为纯饮水,记录2 h内小鼠摄入的质量分数1%蔗糖水和纯水体积,时间间隔为7 d。糖水偏好率计算见公式1。

公式(1)糖水偏好率(%)=

糖 水 消 耗 量 总 液 体 消 耗 量

×100%

2.5 样本收集

CUMS抑郁小鼠造模后,在无菌环境下收集药物治疗前1天及治疗后第14天的小鼠粪便样品。经液氮速冻后保存在-80 ℃备用。通过眼球取血,将血液收集至干净离心管中,37 ℃(或室温)静置凝固分层1 h,后经3 000 r·min^-1室温离心10 min,吸取上清液转至干净的离心管中,再12 000 r·min^-1 4 ℃离心10 min,小心吸取上清液分装至干净的1.5 mL离心管中,每管0.2 mL,液氮速冻后于-80 ℃保存备用。小鼠解剖后取大脑,用预冷的磷酸盐缓冲溶液(PBS)清洗,用滤纸擦干后装入离心管内,液氮冷冻后置于-80 ℃下保存。

2.6 大脑中五羟色胺(5-HT)的测定

采用酶联免疫法对小鼠大脑中5-HT含量进行测定。供试品溶液的制备:测定前,取大脑组织放入玻璃匀浆器中,按质量分数10%比例加入0.01 mol·L^-1 PBS,在冰上研磨匀浆,然后置于4 ℃离心机中,2 500 r·min^-1条件下离心20 min,取上清液,-20 ℃下保存待上机。按试剂盒说明书的操作进行表达水平测定。绘制5-HT的标准曲线,得回归方程y=-4.366 2+132.723 9x(r²=0.996 4)。

2.7 肠道菌群分析

使用粪便基因组DNA快速提取试剂盒对第0天和第14天的小鼠粪便样本中细菌提取总DNA。将同一样本的聚合酶链式反应(PCR)产物混合后使用质量分数2%琼脂糖凝胶回收PCR产物,利用AxyPrep DNA凝胶回收试剂盒进行回收产物纯化,质量分数2%琼脂糖凝胶电泳检测,并用Quantus^TM荧光剂对回收产物进行检测定量。使用DNA建库试剂盒进行建库:①接头链接;②使用磁珠筛选去除接头自连片段;③利用PCR扩增进行文库模板的富集;④磁珠回收PCR产物得到最终的文库。利用MiseqPE300平台进行测序。

2.8 代谢组学检测

用400 μL预冷的甲醇沉淀血清(100 μL),然后在-80 ℃下孵育2 h。随后将样品在4 ℃下以14 000 r·min^-1离心10 min,将上清液冷冻干燥并使用超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF/MS)进行代谢组学分析。使用安捷伦1290 Infinity系统在ACQUITY UPLC Glycan BEH酰胺色谱柱(2.1 mm×100 mm,1.7 μm)上对处理后的血清进行液相色谱分离。流动相A由体积分数0.3%氨水-15 mmol·L^-1乙酸铵组成,流动相B为乙腈(A∶B=9∶1)。流速设定为0.3 mL·min^-1。流动相A的梯度变化如下:首先,梯度在8 min内从10%上升到50%,并保持2 min,然后在0.5 min内从50%恢复到10%,并保持6.5 min。进样体积为3 μL。

使用ESI⁺和ESI^-模式在6545 Q/TOF-MS系统上进行质谱分析,检测参数设置如下:毛细管电压3 500 V;喷嘴电压120 V;雾化器241 kPa;干燥气体流量,8 L·min^-1;气体温度350 ℃。使用高分辨率模式(扩展动态范围2 GHz)在m/z 50~1 200的质量范围内执行全面扫描。使用定性工作流程和Profinder 10.0处理代谢组学数据,然后引入SIMCA(版本14.1)进行偏最小二乘判别分析(PLS-DA)。处理变量重要性指标(VIP)>1和P<0.05的潜在标志物用以下数据库:Metabo Analyst 5.0(https://www.metaboanalyst.ca/)和京都基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)(http://www.kegg.com/)。

2.9 统计学分析

使用SPSS 16.0软件对数据进行统计分析,所有数据均表示为平均值±标准差($\bar{x}±s$)。P<0.05被认为是具有统计学意义的差异。使用GraphPad Prism 8进行统计分析。

3 结果

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3.1 AMP的纯化和结构

AMP反复去除蛋白后,用分光光度计在400~200 nm处扫描纯化的AMP。结果表明,在260和280 nm处没有出现吸收峰,见图1A,说明AMP中蛋白质已被去除,总多糖含量达到95.16%,蛋白质含量占1.36%,其中总糖中醛酸含量占15.47%。

红外光谱仪在400~4 000 cm^-1范围内对AMP进行扫描,见图1B。结果表明,AMP具有典型的多糖结构。具体表现为:O—H在3 435 cm^-1处的伸缩振动、C—H在2 925 cm^-1处的伸缩振动、C=O在1 746和1 617 cm^-1处的不对称振动、C—O—C在1 144和1 103 cm^-1处的伸缩振动。

3.2 糖水偏好实验测试结果

造模60 d后,CUMS模型组小鼠糖水偏好率降低,且显著低于Con组小鼠,经AMP治疗14 d后,各组小鼠糖水偏好率均增加,接近于Con组,其中AM组和AH组显示出较Flx组更好的效用(P<0.01),见图2。结果表明,CUMS造模后小鼠出现抑郁样行为症状,表现为糖水偏好率显著降低,提示CUMS抑郁模型建立成功,分别给予AM、AH和Flx组治疗14 d后,显著减轻CUMS小鼠模型的抑郁样行为。综上所述,AMP对CUMS小鼠模型具有干预作用,其中AM组表现出最好效用。

3.3 各组小鼠大脑中5-HT含量的变化

现代医学认为抑郁症与脑内单胺类物质显著相关,采用酶联免疫法对小鼠大脑中5-HT含量进行测定,结果见图3。CUMS造模组小鼠大脑内5-HT含量显著降低,治疗14 d后,AMP治疗组和Flx组均可调节小鼠5-HT的水平。其中AL组的5-HT水平与Flx组相近,AM组和AH组的5-HT含量显著高于CUMS组,且与Con组没有明显差异,表示这两组对5-HT的回调程度更显著。由此可知,AMP可以调节抑郁小鼠大脑中5-HT含量,改善抑郁小鼠单胺类物质的异常变化。

3.4 16S rDNA测序结果与分析

通过对各组小鼠的粪便DNA进行微生物测序,探究AMP通过调节肠道菌群对抑郁模型小鼠的干预作用。各组小鼠粪便菌群中的优势菌门丰度进行对比,厚壁菌门和拟杆菌门为微生物群组成的主要菌门,诱发慢性不可预知温和应激抑郁后,抑郁小鼠的厚壁菌门和放线菌门丰度明显降低,拟杆菌门的丰度明显升高,厚壁菌门(Firmicutes)与拟杆菌门(Bacteroidetes)的比值,即F-B比值(Con∶CUMS=3.82∶1.96)显著降低,与CUMS组相比,AL组和AM组的菌群丰度没有显著变化,AH组中小鼠拟杆菌门的丰度显著降低,放线菌门的丰度升高,达到一定程度的回调。见图4。

各组小鼠菌群在属水平上的丰度进行对比,与Con组相比,双歧杆菌属(Bifidobacterium)、罗姆布茨菌属(Romboutsia)丰度显著降低,而Lachnospiraceae_NK4A136_group、拟杆菌属(Bacteroides)、Unclassified_f_Oscillospiraceae、拟普雷沃菌属(Alloprevotella)、Eubacterium_xylanophilum_group相对丰度有上升趋势。相比CUMS组, Bifidobacterium、Romboutsia在AH治疗组中丰度升高,恢复至Con相同水平,而AM组和Flx组相对恢复了Romboutsia的菌属丰度,AL组丰度没有显著改变。在CUMS组丰度上升的菌属中,AL组相对降低了Bacteroides、Alloprevotella的丰度,Flx组相对降低了Alloprevotella、Eubacterium_xylanophilum_group的丰度,而AH组对Lachnospiraceae_NK4A136_group、Bacteroides、Unclassified_f_Oscillospiraceae、Alloprevotella、Eubacterium_xylanophilum_group均有所降低,使菌属分布恢复趋近于Con组相似水平,见图5。这些结果表明AMP可以通过调节小鼠的肠道菌改善抑郁。Con、CUMS和AH组中Rikenellaceae_RC9_gut_group、Romboutsia、Alistipes、Prevotellaceae_UCG-001菌属的相对丰度变化,见图6。相比Con组,其中Rikenellaceae_RC9_gut_group、Alistipes、Prevotellaceae_UCG-001在CUMS抑郁造模后显著升高,在AH组回调,相反变化的Romboutsia 在CUMS抑郁模型中的丰度显著降低,AH给药后与Con组无差异。

3.5 小鼠血清样品的代谢组学分析

为了鉴定与抑郁相关的潜在生物标志物,使用UPLC-Q-TOF/MS对小鼠血清进行了非靶向代谢组学分析。正负两种模式下血清代谢物相对定量的PCA分析见图7。结果显示,CUMS抑郁模型组小鼠和Con组小鼠的代谢组存在明显差异,AL、AM、AH组都趋近于正常Con组小鼠。为筛选出与CUMS抑郁模型相关的潜在生物标志物,采用OPLS-DA对Con组和CUMS组的代谢物进一步分析,由正负离子两种模式下OPLS-DA得分图(图8A、B)可以看出两组的代谢特征存在明显差异,S-plot载荷图(图8C、D)显示抑郁引起代谢物变化,CUMS组有71种代谢物被明显上调、46种被抑制。当使用AMP干预后,有68种代谢物被观察到发生显著变化,其中58种差异代谢物在AMP干预后显著回调,见表1。

为评估58种差异代谢物的意义,使用Metabo Analyst 5.0进行了通路拓扑分析。差异代谢物共富集了36种代谢途径,主要包括氨酰-tRNA(aminoacyl-tRNA biosynthesis)、丙氨酸、天门冬氨酸和谷氨酸代谢(alanine, aspartate and glutamate metabolism)、精氨酸生物合成(arginine biosynthesis)、丁酸代谢(butanoate metabolism)、组氨酸代谢(histidine metabolism)、精氨酸和脯氨酸代谢(arginine and proline metabolism)等,见图9。

3.6 Spearman相关性分析

为探究血清中代谢物的变化与小鼠生理指标和肠道菌群变化的相关性,采用表1的58种差异代谢物与生理指标和高丰度菌属分别进行Spearman相关分析。抑郁相关生理指标与58种差异代谢物都有一定的相关性,见图10。例如,在CUMS抑郁模型小鼠中显著上调的代谢物L-苏氨酸(M3)、甘氨酸(M8)、亚牛磺酸(M9)、D-丙氨酸(M21)等,与小鼠糖水偏好、5-HT的表达呈负相关;相反地,在CUMS抑郁模型中显著下调的代谢物L-组氨酸(M10)、乙酰丝氨酸盐酸盐(M15)、N-甲基-D-天冬氨酸(M17)、D-葡萄糖醛酸(M27)、瓜氨酸(M37)、L-谷氨酸(M48)、N-乙酰基-DL-丝氨酸(M50)等与小鼠糖水偏好呈显著正相关,其中L-组氨酸(M10)、乙酰丝氨酸盐酸盐(M15)、N-甲基-D-天冬氨酸(M17)、L-谷氨酸(M48)、N-乙酰基-DL-丝氨酸(M50)与5-HT的变化呈正相关。

除Bacteroides和Blautia外,其他进行相关性分析的小鼠肠道菌属与差异代谢物都有一定的相关性,见图11。例如,在CUMS抑郁小鼠中显著上调的代谢物L-苏氨酸(M3)、L-高半胱氨酸(M5)、甘氨酸(M8)、亚牛磺酸(M9)与Rikenellaceae_RC9_gut_group、Prevotellaceae_UCG-001、Alistipes的丰度变化呈正相关,相反地,与Faecalibaculum和Romboutsia呈负相关;在CUMS抑郁小鼠中显著下调的代谢物乙酰丝氨酸盐酸盐(M15)、N-甲基-D-天冬氨酸(M17)、蔗糖(M43)、L-谷氨酸(M48)、N-乙酰基-DL-丝氨酸(M50)与Faecalibaculum、Coriobacteriaceae_UCG-002呈正相关,而与Rikenellaceae_RC9_gut_group、Prevotellaceae_UCG-001、Alistipes呈负相关。这些代谢物的丰度在AMP治疗后均得到回调,分析结果说明这些差异代谢物与抑郁生理指标和小鼠肠道菌群都显著相关。

为进一步探究肠道菌群与抑郁样症状的关系,将高丰度的10类菌属与小鼠的生理指标进行Spearman相关性分析,见图12。Bifidobacterium、Bacteroides、Blautia与生理指标显示没有相关性。只有Romboutsia的丰度变化与小鼠的糖水偏好呈显著正相关。与5-HT变化显著相关的菌属中Faecalibaculum、Romboutsia、Coriobacteriaceae_UCG-002的丰度变化与5-HT呈正相关,Lachnospiraceae_NK4A136_group、Rikenellaceae_RC9_gut_group丰度变化与5-HT呈负相关。这些结果说明小鼠肠道菌属丰度的变化与生理指标的变化也具有一定的相关性。

4 讨论

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4.1 AMP改善肠道菌群

厚壁菌门是肠道微生物的主要组成部分,其中的大多数菌属成员属于有益菌,可帮助生物体的能量吸收。本研究中CUMS模型小鼠厚壁菌门和双歧杆菌丰度显著降低,AMP治疗显著上调了Bifidobacterium、Romboutsia等有益菌丰度,这与Zhu等^[27]、Aizawa等^[28]的研究结果一致。新的研究表明Rikenellaceae、拟杆菌门和谱氏菌科的相对丰度与焦虑和抑郁的严重程度呈正相关^[29-31],AMP治疗后调节了CUMS抑郁小鼠肠道菌稳态,下调了Rikenellaceae_RC9_gut_group、Alistipes、Prevotellaceae_UCG-001等有害菌丰度。

4.2 AMP调控代谢通路

研究发现,在抑郁患者中精氨酸和脯氨酸代谢、谷胱甘肽代谢通路是主要的差异代谢途径,其中谷氨酸可以在醛脱氢酶 18 家族成员 A1(ALDH18A1)的调控下代谢为1-吡咯烷-5-羧酸盐,再由吡咯啉-5-羧酸还原酶-1(PYCR1)代谢为脯氨酸,且抑郁模型中检测到PYCR1和ALDH18A1的水平显著上调,说明可能是由于酶活性增强使谷氨酸更多地代谢为脯氨酸,从而造成抑郁组中谷氨酸水平降低而脯氨酸水平升高。本研究还发现CUMS抑郁模型小鼠脯氨酸显著上调,谷氨酸显著下调,与以往研究结果一致^[32]。精氨酸先代谢为瓜氨酸,再由瓜氨酸代谢为琥珀酸^[33]。本研究检测到CUMS抑郁小鼠中瓜氨酸和琥珀酸的显著下调,与以往的研究在抑郁患者和CUMS大鼠中发现精氨酸显著上调不同,抑郁模型小鼠的精氨酸代谢通路发生了异常。本研究检测到CUMS造模后D-葡萄糖醛酸显著下调,D-葡萄糖醛酸的代谢来源为D-葡萄糖,与先前报道的抑郁症患者体内D-葡萄糖异常升高一致^[34]。在AMP的干预下D-葡萄糖醛酸在小鼠血清中其水平显著上调,说明AMP可能参与调控CUMS抑郁小鼠的糖代谢。总之,AMP可能通过调节代谢物水平来调节相关代谢通路,从而改善小鼠抑郁症状。

4.3 AMP通过改善肠道菌群调控代谢物水平

本研究显示CUMS模型组小鼠中L-苏氨酸、甘氨酸、亚牛磺酸、D-丙氨酸等水平显著上调与抑郁症状(糖水偏好)的严重性呈负相关,与Geng等^[35]报道的结果一致。AMP干预下调了这些氨基酸代谢物水平,糖水偏好率上调。肠道菌与代谢组的相关性分析显示氨基酸水平与肠道菌Rikenellaceae_RC9_gut_group、另枝菌属(Alistipes)、Prevotellaceae_UCG-001的丰度呈正相关,与肠道菌Romboutsia的丰度呈负相关,与Wang等^[36]、Bojovic' K等^[37]报道的结果一致。AMP干预后增加了Romboutsia丰度,从而回调上述代谢物水平。另外,本研究还发现,Alistipes、Prevotellaceae_UCG-001、Rikenellaceae_RC9_gut_group与代谢物乙酰辅酶A呈正相关,而与延胡索酸呈负相关。研究表明大部分氨基酸是通过乙酰辅酶A和延胡索酸进入三羧酸循环中,CUMS模型动物体内处于能量代谢高水平状态时,会导致三羧酸循环出现障碍,中间代谢产物显著降低^[38]。AMP的干预可能通过上调延胡索酸水平,下调乙酰辅酶A代谢水平,从而干预三羧酸循环障碍。AMP可能通过影响肠道菌群进而回调血清中氨基酸和5-HT以及与能量代谢的相关代谢物水平,对小鼠的抑郁症状发挥干预作用。

4.4 AMP可能参与调控能量代谢

大脑是一个能量需求极高的器官,大脑能量代谢的微小扰动均可能会造成其功能活动的改变进而导致情绪或精神障碍。三羧酸循环是能量代谢的核心代谢通路,葡萄糖等能量底物主要在线粒体内通过三羧酸循环和氧化磷酸化产生ATP,参与应激反应过程^[39]。本研究发现CUMS模型组小鼠延胡索酸、L-谷氨酸水平显著降低。L-谷氨酸作为细胞内多个代谢途径的枢纽参与能量代谢,Rong等^[40]发现重度抑郁患者血浆的谷氨酸显著降低,认为谷氨酸可能是抑郁症的临床特征标志物。来自人类代谢组、蛋白组学的研究已经发现大脑皮层如前额叶以及外周血样本中三羧酸循环过程相关代谢产物如延胡索酸、枸橼酸、异枸橼酸等的水平降低,提示能量代谢损伤可能是抑郁症临床症状产生的神经生物学基础^[41]。L-谷氨酸水平显著降低,可能会导致三羧酸循环障碍,ATP生成不足,能量供应匮乏。AMP治疗后上述代谢物的水平显著上调,推测AMP可能改善抑郁小鼠能量代谢紊乱的状态,干预CUMS模型小鼠的抑郁状态。

5 结论

收起

白术在中药古方中常作为主要配方药用于治疗抑郁症^[22,42]。AMP是白术中的有效成分,目前的研究发现它具有降血糖^[43]、降血脂^[44]、抗炎^[45]、抗肿瘤^[46]及治疗胃肠道疾病的作用^[47],对神经系统^[23]和免疫系统^[48]均有广泛的影响,但未见治疗抑郁症的作用机制报道。

本研究表明,抑郁与细胞糖代谢异常和ATP生成减少密切相关,结合能量状态与肠道菌和代谢组的研究结果,认为AMP可能会成为一个“脑-肠对话”调节能量变化的化学物质。而AMP通过调节肠道菌群的稳态,改善能量代谢障碍,可能成为一种抗抑郁作用的新机制。

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2024年第59卷第9期

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doi: 10.11669/cpj.2024.09.004

接收时间：2023-11-10
首发时间：2025-11-25
出版时间：2024-05-08

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收稿日期：2023-11-10

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¹ 浙江农林大学食品与健康学院, 杭州 311300

² 缙云县农业农村局, 浙江丽水 323000

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*田薇,女,博士,教授研究方向:中药学

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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No.	Metabolite	Fold Change	P value	VIP	AH vs CUMS	CUMS vs Con
M1	(+/-)-Potassium citramalate monohydrate	4.186	0.000 0	1.357 4	↑	↓
M2	Acetyl CoA	0.056	0.000 0	1.348 4	↓	↑
M3	L-Threonine	0.068	0.000 0	1.720 0	↓	↑
M4	L-Proline	0.071	0.000 0	1.718 0	↓	↑
M5	L-Homoserine	0.074	0.000 0	1.717 6	↓	↑
M6	DL-Methionine sulfoxide	0.046	0.000 0	1.334 3	↓	↑
M7	L-Hydroxyproline	0.035	0.000 1	1.327 2	↓	↑
M8	Glycine	0.194	0.000 1	1.320 6	↓	↑
M9	Hypotaurine	0.045	0.000 1	1.315 5	↓	↑
M10	L-Histidine	2.511	0.000 1	1.308 1	↑	↓
M11	D-2-Hydroxyglutaric acid	8.174	0.000 1	1.309 5	↑	↓
M12	DL-2-Aminoadipic acid	3.832	0.000 1	1.309 6	↑	↓
M13	L-2-Aminoadipic acid	4.262	0.000 2	1.298 4	↑	↓
M14	3-Methyl-L-histidine	0.154	0.000 2	1.656 9	↓	↑
M15	O-Acetyl-L-serine hydrochloride	2.402	0.000 2	1.304 9	↑	↓
M16	Succinic acid	3.182	0.000 3	1.237 2	↑	↓
M17	N-Methyl-D-aspartic acid	2.453	0.000 3	1.296 5	↑	↓
M18	1-Methyl-L-histidine	0.164	0.000 3	1.644 7	↓	↑
M19	Methylmalonic acid	3.188	0.000 3	1.283 9	↑	↓
M20	Dodecanoic acid	0.354	0.000 3	1.307 1	↓	↑
M21	D-Alanine	0.329	0.000 5	1.257 5	↓	↑
M22	Sarcosine	0.370	0.000 6	1.234 0	↓	↑
M23	Alanine	0.374	0.000 7	1.230 9	↓	↑
M24	Ophthalmic acid	0.379	0.000 8	1.598 2	↓	↑
M25	Malic acid	2.809	0.000 9	1.238 6	↑	↓
M26	Citraconic acid	0.393	0.001 7	1.207 4	↓	↑
M27	D-Glucuronic acid sodium salt monohydrate	2.985	0.001 7	1.171 8	↑	↓
M28	(3-Carboxypropyl)trimethylammonium chloride	3.700	0.002 3	1.526 6	↑	↓
M29	Fumaric acid	2.209	0.003 5	1.178 3	↑	↓
M30	cis-4-Hydroxy-D-proline	0.280	0.003 6	1.166 7	↓	↑
M31	d-Desthiobiotin	0.492	0.003 9	1.150 3	↓	↑
M32	Methylguanidine hydrochloride	3.859	0.004 9	1.464 9	↑	↓
M33	sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt	2.519	0.004 9	1.161 3	↑	↓
M34	O-Acetyl-L-carnitine hydrochloride	0.374	0.005 2	1.149 7	↓	↑
M35	Stachyose hydrate from Stachys tuberifera	0.496	0.005 4	1.467 7	↓	↑
M36	L-Citrulline	2.088	0.005 4	1.456 8	↑	↓
M37	Citrulline	2.125	0.005 8	1.449 9	↑	↓
M38	D-Lactose monohydrate	2.529	0.006 3	1.424 8	↑	↓
M39	Shikimic acid	2.588	0.006 5	1.431 1	↑	↓
M40	D-(+)-Trehalose dihydrate	2.395	0.006 8	1.416 4	↑	↓
M41	Uridine 5'-diphospho-N-acetylgalactosamine disodium salt	0.156	0.006 9	1.438 6	↓	↑
M42	D-(+)-Maltose monohydrate	2.423	0.006 9	1.414 5	↑	↓
M43	Sucrose	2.489	0.007 6	1.404 7	↑	↓
M44	Palatinose hydrate	2.414	0.007 9	1.400 8	↑	↓
M45	D-(+)-Cellobiose	2.452	0.008 1	1.397 7	↑	↓
M46	D-(+)-Raffinose pentahydrate	0.447	0.010 5	1.014 3	↓	↑
M47	D-Glucosamine 6-sulfate	2.765	0.011 4	1.350 4	↑	↓
M48	L-Glutamic acid	2.308	0.014 2	1.340 2	↑	↓
M49	2,3-Pyridinedicarboxylic acid	2.715	0.014 7	1.322 1	↑	↓
M50	N-Acetyl-DL-serine	2.278	0.015 4	1.329 4	↑	↓
M51	L-Anserine nitrate salt	2.493	0.016 0	1.332 0	↑	↓
M52	Glycerophosphate disodium salt hydrate	2.043	0.017 2	1.311 4	↑	↓
M53	L-Methionine sulfoximine	0.451	0.018 5	1.310 5	↓	↑
M54	D-Allose	0.475	0.023 4	1.277 0	↓	↑
M55	D-(+)-Galactose	0.477	0.023 6	1.275 8	↓	↑
M56	N-Methyl-L-alanine	0.479	0.024 2	1.272 1	↓	↑
M57	D-(+)-Mannose	0.480	0.024 3	1.271 2	↓	↑
M58	Glutaryl-L-carnitine lithium salt	2.186	0.027 6	1.245 5	↑	↓

No.

Metabolite

Fold Change

P value

VIP

AH vs CUMS

CUMS vs Con

(+/-)-Potassium citramalate monohydrate

4.186

0.000 0

1.357 4

↑

↓

Acetyl CoA

0.056

0.000 0

1.348 4

↓

↑

L-Threonine

0.068

0.000 0

1.720 0

↓

↑

L-Proline

0.071

0.000 0

1.718 0

↓

↑

L-Homoserine

0.074

0.000 0

1.717 6

↓

↑

DL-Methionine sulfoxide

0.046

0.000 0

1.334 3

↓

↑

L-Hydroxyproline

0.035

0.000 1

1.327 2

↓

↑

Glycine

0.194

0.000 1

1.320 6

↓

↑

Hypotaurine

0.045

0.000 1

1.315 5

↓

↑

M10

L-Histidine

2.511

0.000 1

1.308 1

↑

↓

M11

D-2-Hydroxyglutaric acid

8.174

0.000 1

1.309 5

↑

↓

M12

DL-2-Aminoadipic acid

3.832

0.000 1

1.309 6

↑

↓

M13

L-2-Aminoadipic acid

4.262

0.000 2

1.298 4

↑

↓

M14

3-Methyl-L-histidine

0.154

0.000 2

1.656 9

↓

↑

M15

O-Acetyl-L-serine hydrochloride

2.402

0.000 2

1.304 9

↑

↓

M16

Succinic acid

3.182

0.000 3

1.237 2

↑

↓

M17

N-Methyl-D-aspartic acid

2.453

0.000 3

1.296 5

↑

↓

M18

1-Methyl-L-histidine

0.164

0.000 3

1.644 7

↓

↑

M19

Methylmalonic acid

3.188

0.000 3

1.283 9

↑

↓

M20

Dodecanoic acid

0.354

0.000 3

1.307 1

↓

↑

M21

D-Alanine

0.329

0.000 5

1.257 5

↓

↑

M22

Sarcosine

0.370

0.000 6

1.234 0

↓

↑

M23

Alanine

0.374

0.000 7

1.230 9

↓

↑

M24

Ophthalmic acid

0.379

0.000 8

1.598 2

↓

↑

M25

Malic acid

2.809

0.000 9

1.238 6

↑

↓

M26

Citraconic acid

0.393

0.001 7

1.207 4

↓

↑

M27

D-Glucuronic acid sodium salt monohydrate

2.985

0.001 7

1.171 8

↑

↓

M28

(3-Carboxypropyl)trimethylammonium chloride

3.700

0.002 3

1.526 6

↑

↓

M29

Fumaric acid

2.209

0.003 5

1.178 3

↑

↓

M30

cis-4-Hydroxy-D-proline

0.280

0.003 6

1.166 7

↓

↑

M31

d-Desthiobiotin

0.492

0.003 9

1.150 3

↓

↑

M32

Methylguanidine hydrochloride

3.859

0.004 9

1.464 9

↑

↓

M33

sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt

2.519

0.004 9

1.161 3

↑

↓

M34

O-Acetyl-L-carnitine hydrochloride

0.374

0.005 2

1.149 7

↓

↑

M35

Stachyose hydrate from Stachys tuberifera

0.496

0.005 4

1.467 7

↓

↑

M36

L-Citrulline

2.088

0.005 4

1.456 8

↑

↓

M37

Citrulline

2.125

0.005 8

1.449 9

↑

↓

M38

D-Lactose monohydrate

2.529

0.006 3

1.424 8

↑

↓

M39

Shikimic acid

2.588

0.006 5

1.431 1

↑

↓

M40

D-(+)-Trehalose dihydrate

2.395

0.006 8

1.416 4

↑

↓

M41

Uridine 5'-diphospho-N-acetylgalactosamine disodium salt

0.156

0.006 9

1.438 6

↓

↑

M42

D-(+)-Maltose monohydrate

2.423

0.006 9

1.414 5

↑

↓

M43

Sucrose

2.489

0.007 6

1.404 7

↑

↓

M44

Palatinose hydrate

2.414

0.007 9

1.400 8

↑

↓

M45

D-(+)-Cellobiose

2.452

0.008 1

1.397 7

↑

↓

M46

D-(+)-Raffinose pentahydrate

0.447

0.010 5

1.014 3

↓

↑

M47

D-Glucosamine 6-sulfate

2.765

0.011 4

1.350 4

↑

↓

M48

L-Glutamic acid

2.308

0.014 2

1.340 2

↑

↓

M49

2,3-Pyridinedicarboxylic acid

2.715

0.014 7

1.322 1

↑

↓

M50

N-Acetyl-DL-serine

2.278

0.015 4

1.329 4

↑

↓

M51

L-Anserine nitrate salt

2.493

0.016 0

1.332 0

↑

↓

M52

Glycerophosphate disodium salt hydrate

2.043

0.017 2

1.311 4

↑

↓

M53

L-Methionine sulfoximine

0.451

0.018 5

1.310 5

↓

↑

M54

D-Allose

0.475

0.023 4

1.277 0

↓

↑

M55

D-(+)-Galactose

0.477

0.023 6

1.275 8

↓

↑

M56

N-Methyl-L-alanine

0.479

0.024 2

1.272 1

↓

↑

M57

D-(+)-Mannose

0.480

0.024 3

1.271 2

↓

↑

M58

Glutaryl-L-carnitine lithium salt

2.186

0.027 6

1.245 5

↑

↓