药学学报

No.	Gene name	Sequence (5′ to 3′)	T_m/℃
1	M-α-sma-F	GGCACCACTGAACCCTAAGG	20
2	M-α-sma-R	ACAATACCAGTTGTACGTCCAGA	23
3	M-Col1-F	TAAGGGTCCCCAATGGTGAGA	21
4	M-Col1-R	GGGTCCCTCGACTCCTACAT	20
5	M-Il-1β-F	GCAACTGTTCCTGAACTCAACT	22
6	M-Il-1β-R	ATCTTTTGGGGTCCGTCAACT	21
7	M-Il-6-F	TAGTCCTTCCTACCCCAATTTCC	23
8	M-Il-6-R	TTGGTCCTTAGCCACTCCTTC	21
9	M-Tnf-α-F	CCCTCACACTCAGATCATCTTCT	23
10	M-Tnf-α-R	GCTACGACGTGGGCTACAG	19
11	M-β-actin-F	CCACAGCTGAGAGGGAAATC	20
12	M-β-actin-R	AAGGAAGGCTGGAAAAGAGC	20

No.	Gene name	Sequence (5′ to 3′)	T_m/℃
1	M-α-sma-F	GGCACCACTGAACCCTAAGG	20
2	M-α-sma-R	ACAATACCAGTTGTACGTCCAGA	23
3	M-Col1-F	TAAGGGTCCCCAATGGTGAGA	21
4	M-Col1-R	GGGTCCCTCGACTCCTACAT	20
5	M-Il-1β-F	GCAACTGTTCCTGAACTCAACT	22
6	M-Il-1β-R	ATCTTTTGGGGTCCGTCAACT	21
7	M-Il-6-F	TAGTCCTTCCTACCCCAATTTCC	23
8	M-Il-6-R	TTGGTCCTTAGCCACTCCTTC	21
9	M-Tnf-α-F	CCCTCACACTCAGATCATCTTCT	23
10	M-Tnf-α-R	GCTACGACGTGGGCTACAG	19
11	M-β-actin-F	CCACAGCTGAGAGGGAAATC	20
12	M-β-actin-R	AAGGAAGGCTGGAAAAGAGC	20

No.	Metabolite	HMDB ID	Ion mode	m/z	VIP	FC (GPE group vs CCl₄ group)
1	Montecristin	HMDB0029795	POS	575.503 7	1.348 98	5.733 3
2	PC(35∶3)	HMDB0007947	POS	770.567 6	1.770 6	2.669 1
3	PC(18∶3(6Z, 9Z, 12Z)/15∶0)	HMDB0008165	POS	742.536 1	1.877 32	3.242 5
4	PE(18∶1(11Z)/18∶2(9Z, 12Z))	HMDB0009027	POS	742.536 1	1.877 32	3.242 5
5	PC(18∶1(9Z)/P-16∶0)	HMDB0008126	POS	744.592 7	1.151 39	6.334 7
6	Ethyl glucuronide	HMDB0010325	NEG	221.066 5	19.714 5	158.64
7	Nobiletin	HMDB0029540	NEG	401.128 2	7.564 67	281.49
8	Ascorbic acid	HMDB0000044	NEG	175.024 8	2.733 84	9.929 2
9	N-Acetylmethionine	HMDB0011745	NEG	190.053 9	2.787 97	2.147 7
10	2, 2-Dimethylsuccinic acid	HMDB0002074	NEG	145.050 6	1.509 26	29.53
11	Pentadecanoic acid	HMDB0000826	NEG	241.216 7	1.293 2	1.742 6
12	4-Nitroaniline	HMDB0246529	NEG	137.0354	1.116 37	0.167 88
13	N-Acetylphenylalanine	HMDB0000512	NEG	206.082	1.647 33	2.715 6
14	PC(18∶0/18∶1(9Z))	HMDB0008038	POS	788.609 7	3.825 21	0.514 82
15	Acetylleucine	HMDB0011756	NEG	172.097 8	1.465 7	1.949 9
16	Cer(d18∶1/16∶0)	HMDB0004949	POS	520.507 2	1.187 38	0.591 63
17	Bathophenanthroline	HMDB0246617	POS	333.132 8	1.107 16	2.571 1
18	Doxycycline	HMDB0014399	NEG	443.140 3	2.578 83	178.88
19	Buprenorphine	HMDB0015057	POS	468.307 3	1.883 29	1.527 2
20	Glucose 6-phosphate	HMDB0001401	NEG	259.021 9	1.781 33	1.985 1
21	Mannose 6-phosphate	HMDB0001078	NEG	259.021 9	1.781 33	1.985 1
22	Fructose 1-phosphate	HMDB0001076	NEG	259.021 9	1.781 33	1.985 1
23	Mannose 1-phosphate	HMDB0006330	NEG	259.021 9	1.781 33	1.985 1
24	Sorbose	HMDB0246950	NEG	179.056 1	1.918 27	2.200 1
25	Mannose	HMDB0000169	NEG	179.056 1	1.918 27	2.200 1
26	Fructose	HMDB0000660	NEG	179.056 1	1.918 27	2.200 1
27	Castamollissin	HMDB0038888	POS	469.096 1	1.650 57	2.170 1
28	Trigoforin	HMDB0029495	POS	189.086 9	1.510 57	1.827 7
29	N-[(4E, 8E)-1, 3-dihydroxyoctadeca-4, 8-dien-2-yl]hexadecanamide	HMDB0035480	POS	536.504	1.134 28	0.503 58
30	N-Acetylglutamine	HMDB0006029	NEG	187.071 9	2.388 84	1.787 9
31	N-Acetylhistidine	HMDB0032055	NEG	196.072 6	1.082	1.694 4
32	Dehydroascorbic acid (oxidized vitamin C)	HMDB0001264	NEG	173.008 7	1.535 82	1.602 5
33	Ribitol	HMDB0000508	NEG	151.061	1.152 99	1.79
34	Tyrosine	HMDB0000158	POS	182.081 1	6.640 24	1.684 9
35	O-Tyrosine	HMDB0006050	POS	182.081 1	6.640 24	1.684 9
36	Heptadecanoic acid	HMDB0002259	NEG	269.247 8	1.259 36	1.516 6
37	Methylimidazoleacetic acid	HMDB0002820	POS	141.065 6	1.402 39	3.025 8
38	PC(14∶1(9Z)/16∶0)	HMDB0007902	POS	704.522 7	1.451 5	2.455 9
39	Mangiferdesmethylursanone	HMDB0036018	POS	429.372 5	1.182 26	0.591 78
40	Allantoin	HMDB0000462	NEG	157.036 2	3.727 13	0.607 9
41	Chromone	HMDB0032938	POS	147.044 1	1.069 52	2.184 2
42	Isovalerylglycine	HMDB0000678	NEG	158.082 1	1.133 26	0.518 85
43	2-Hydroxyethanesulfonic acid	HMDB0003903	NEG	124.991 3	1.966 18	0.297 07
44	Cytidine 5′-diphosphocholine (CDP-choline)	HMDB0001413	POS	489.113 5	1.082 65	1.648 2
45	Glutathione	HMDB0000125	NEG	306.076	4.332 64	1.860 4
46	Fagomine	HMDB0033453	POS	148.096 6	1.714 51	1.918 5
47	PC(20∶1(11Z)/15∶0)	HMDB0008297	POS	774.597 6	1.706 96	2.003 6

No.	Metabolite	HMDB ID	Ion mode	m/z	VIP	FC (GPE group vs CCl₄ group)
1	Montecristin	HMDB0029795	POS	575.503 7	1.348 98	5.733 3
2	PC(35∶3)	HMDB0007947	POS	770.567 6	1.770 6	2.669 1
3	PC(18∶3(6Z, 9Z, 12Z)/15∶0)	HMDB0008165	POS	742.536 1	1.877 32	3.242 5
4	PE(18∶1(11Z)/18∶2(9Z, 12Z))	HMDB0009027	POS	742.536 1	1.877 32	3.242 5
5	PC(18∶1(9Z)/P-16∶0)	HMDB0008126	POS	744.592 7	1.151 39	6.334 7
6	Ethyl glucuronide	HMDB0010325	NEG	221.066 5	19.714 5	158.64
7	Nobiletin	HMDB0029540	NEG	401.128 2	7.564 67	281.49
8	Ascorbic acid	HMDB0000044	NEG	175.024 8	2.733 84	9.929 2
9	N-Acetylmethionine	HMDB0011745	NEG	190.053 9	2.787 97	2.147 7
10	2, 2-Dimethylsuccinic acid	HMDB0002074	NEG	145.050 6	1.509 26	29.53
11	Pentadecanoic acid	HMDB0000826	NEG	241.216 7	1.293 2	1.742 6
12	4-Nitroaniline	HMDB0246529	NEG	137.0354	1.116 37	0.167 88
13	N-Acetylphenylalanine	HMDB0000512	NEG	206.082	1.647 33	2.715 6
14	PC(18∶0/18∶1(9Z))	HMDB0008038	POS	788.609 7	3.825 21	0.514 82
15	Acetylleucine	HMDB0011756	NEG	172.097 8	1.465 7	1.949 9
16	Cer(d18∶1/16∶0)	HMDB0004949	POS	520.507 2	1.187 38	0.591 63
17	Bathophenanthroline	HMDB0246617	POS	333.132 8	1.107 16	2.571 1
18	Doxycycline	HMDB0014399	NEG	443.140 3	2.578 83	178.88
19	Buprenorphine	HMDB0015057	POS	468.307 3	1.883 29	1.527 2
20	Glucose 6-phosphate	HMDB0001401	NEG	259.021 9	1.781 33	1.985 1
21	Mannose 6-phosphate	HMDB0001078	NEG	259.021 9	1.781 33	1.985 1
22	Fructose 1-phosphate	HMDB0001076	NEG	259.021 9	1.781 33	1.985 1
23	Mannose 1-phosphate	HMDB0006330	NEG	259.021 9	1.781 33	1.985 1
24	Sorbose	HMDB0246950	NEG	179.056 1	1.918 27	2.200 1
25	Mannose	HMDB0000169	NEG	179.056 1	1.918 27	2.200 1
26	Fructose	HMDB0000660	NEG	179.056 1	1.918 27	2.200 1
27	Castamollissin	HMDB0038888	POS	469.096 1	1.650 57	2.170 1
28	Trigoforin	HMDB0029495	POS	189.086 9	1.510 57	1.827 7
29	N-[(4E, 8E)-1, 3-dihydroxyoctadeca-4, 8-dien-2-yl]hexadecanamide	HMDB0035480	POS	536.504	1.134 28	0.503 58
30	N-Acetylglutamine	HMDB0006029	NEG	187.071 9	2.388 84	1.787 9
31	N-Acetylhistidine	HMDB0032055	NEG	196.072 6	1.082	1.694 4
32	Dehydroascorbic acid (oxidized vitamin C)	HMDB0001264	NEG	173.008 7	1.535 82	1.602 5
33	Ribitol	HMDB0000508	NEG	151.061	1.152 99	1.79
34	Tyrosine	HMDB0000158	POS	182.081 1	6.640 24	1.684 9
35	O-Tyrosine	HMDB0006050	POS	182.081 1	6.640 24	1.684 9
36	Heptadecanoic acid	HMDB0002259	NEG	269.247 8	1.259 36	1.516 6
37	Methylimidazoleacetic acid	HMDB0002820	POS	141.065 6	1.402 39	3.025 8
38	PC(14∶1(9Z)/16∶0)	HMDB0007902	POS	704.522 7	1.451 5	2.455 9
39	Mangiferdesmethylursanone	HMDB0036018	POS	429.372 5	1.182 26	0.591 78
40	Allantoin	HMDB0000462	NEG	157.036 2	3.727 13	0.607 9
41	Chromone	HMDB0032938	POS	147.044 1	1.069 52	2.184 2
42	Isovalerylglycine	HMDB0000678	NEG	158.082 1	1.133 26	0.518 85
43	2-Hydroxyethanesulfonic acid	HMDB0003903	NEG	124.991 3	1.966 18	0.297 07
44	Cytidine 5′-diphosphocholine (CDP-choline)	HMDB0001413	POS	489.113 5	1.082 65	1.648 2
45	Glutathione	HMDB0000125	NEG	306.076	4.332 64	1.860 4
46	Fagomine	HMDB0033453	POS	148.096 6	1.714 51	1.918 5
47	PC(20∶1(11Z)/15∶0)	HMDB0008297	POS	774.597 6	1.706 96	2.003 6

绞股蓝改善CCl₄诱导的小鼠肝纤维化作用的肝脏代谢组学研究

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程含笑 ¹^,² , 胡林兰 ² , 吴剑坤 ³ , 何娴 ² , 赵鑫 ² , 鲁雅雯 ²^,^* , 王伽伯 ²^,^* , 李会芳 ¹^,^*

药学学报 | 研究论文 2025,60(4): 1001-1011

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药学学报 | 研究论文 2025, 60(4): 1001-1011

绞股蓝改善CCl₄诱导的小鼠肝纤维化作用的肝脏代谢组学研究

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程含笑¹^,², 胡林兰², 吴剑坤³, 何娴², 赵鑫², 鲁雅雯²^,^*, 王伽伯²^,^*, 李会芳¹^,^*

作者信息

¹ 山西中医药大学, 中药与食品工程学院, 山西晋中 030619

² 首都医科大学中医药学院, 中医药防治传染性重症肝病研究与转化实验室, 北京 100069

³ 首都医科大学附属北京中医医院, 北京 100010

通讯作者:

鲁雅雯，Tel: 86-351-3179903, E-mail: ywlu0411@163.com

王伽伯，E-mail: jiabo_wang@ccmu.edu.cn

李会芳，E-mail: lihuifangzy@sxtcm.edu.cn

Effects and mechanism investigation of Gynostemma pentaphyllum on alleviating CCl₄-induced liver fibrosis in mice via metabolomics

Han-xiao CHENG¹^,², Lin-lan HU², Jian-kun WU³, Xian HE², Xin ZHAO², Ya-wen LU²^,^*, Jia-bo WANG²^,^*, Hui-fang LI¹^,^*

Affiliations

¹College of Traditional Chinese Medicine and Food Engineering, Shanxi University of Chinese Medicine, Jinzhong 030619, China

²College of Traditional Chinese Medicine, Laboratory for Research and Translation of Traditional Chinese Medicine in Managing Severe Infectious Liver Diseases, Capital Medical University, Beijing 100069, China

³Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China

出版时间: 2025-04-12 doi: 10.16438/j.0513-4870.2024-1041

文章导航

摘要

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肝纤维化是慢性肝病进展的共同阶段, 然而全球范围内缺乏抗肝纤维化的临床药物。绞股蓝有“南方人参”之称, 民间常用于防治多种慢性肝病, 亦有其抗肝纤维化的报道, 但相关科学研究较少。本文采用LC-MS代谢组学分析方法, 在四氯化碳(carbon tetrachloride, CCl₄) 诱导肝纤维化小鼠模型上探究绞股蓝醇提物(Gynostemma pentaphyllum ethanol extract, GPE) 的作用及潜在机制。所有动物实验经首都医科大学实验动物伦理委员会批准(批准号: DWLLGZR202202204)。结果显示, GPE能显著减轻模型小鼠肝脏的炎性细胞浸润和胶原蓄积, 显著降低小鼠血清丙氨酸转氨酶和天冬氨酸转氨酶活力及羟脯氨酸水平, 可有效抑制胶原蛋白1A1 (collagen 1A1, COL 1A1) 和α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA) 的基因转录和蛋白质表达。肝脏非靶向代谢组学分析发现, GPE主要影响了47个差异代谢物; KEGG通路富集分析表明, 差异代谢物主要富集在果糖/甘露糖代谢和芳香氨基酸代谢通路。进一步采用靶向代谢组学检测验证了D-甘露糖、6-磷酸甘露糖、D-果糖、2-磷酸果糖、D-蔗糖、海藻糖、谷氨酸、苯丙氨酸、酪氨酸、L-2-氨基-3-氧代丁酸、乳酸、2-羟基丁酸和牛磺酸共13个差异代谢物, 可能是GPE抗肝纤维化相关的重要代谢物, 证实GPE抗肝纤维化作用可能与调控果糖/甘露糖代谢途径和芳香氨基酸代谢途径密切相关。

关键词

绞股蓝 / 肝纤维化 / 代谢组学 / 果糖/甘露糖代谢 / 芳香氨基酸代谢

Abstract

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Liver fibrosis is a common stage in the progression of chronic liver diseases, yet there is a lack of clinical drugs against liver fibrosis globally. Gynostemma pentaphyllum has the name of "Southern ginseng", commonly used in folk prevention and treatment of a variety of chronic liver disease, there are also reports of its anti-hepatic fibrosis. However, there are fewer relevant scientific studies. In this study, we used LC-MS metabolomics analysis to investigate the effects of Gynostemma pentaphyllum ethanol extract (GPE) on liver fibrosis in carbon tetrachloride (CCl₄)-induced mouse models and its potential mechanisms. All animal experiments were approved by the experimental animal ethics committee of Capital Medical University (DWLLGZR202202204). The results showed that GPE could significantly reduce the inflammatory cell infiltration and collagen accumulation in the liver of model mice, significantly reduce serum alanine transaminase and aspartate transaminase activity and hypoxanthine levels in mice, and could effectively inhibit the gene transcription and protein expression of collagen 1A1 (COL 1A1) and α-smooth muscle actin (α-SMA). Non-targeted metabolomics analysis of the liver showed that GPE mainly affected 47 differential metabolites; KEGG pathway enrichment analysis indicated that the differential metabolites were mainly enriched in the fructose/mannose metabolism and aromatic amino acid metabolism pathways. The targeted metabolomic assay was further used to validate a total of 13 differential metabolites of D-mannose, mannose 6-phosphate, D-fructose, fructose 2-phosphate, D-sucrose, trehalose, glutamate, phenylalanine, tyrosine, L-2-amino-3-oxobutyric acid, lactate, 2-hydroxybutyrate, and taurine, which may be important metabolites related to GPE's anti-fibrotic effects. This confirms that GPE's anti-liver fibrosis effects may be closely related to the regulation of the fructose/mannose metabolism pathway and the aromatic amino acid metabolism pathway.

Key words

Gynostemma pentaphyllum / liver fibrosis / metabolomics / fructose/mannose metabolism / aromatic amino acid metabolism

引用本文

程含笑, 胡林兰, 吴剑坤, 何娴, 赵鑫, 鲁雅雯, 王伽伯, 李会芳. 绞股蓝改善CCl₄诱导的小鼠肝纤维化作用的肝脏代谢组学研究. 药学学报, 2025 , 60 (4) : 1001 -1011 . DOI: 10.16438/j.0513-4870.2024-1041

Han-xiao CHENG, Lin-lan HU, Jian-kun WU, Xian HE, Xin ZHAO, Ya-wen LU, Jia-bo WANG, Hui-fang LI. Effects and mechanism investigation of Gynostemma pentaphyllum on alleviating CCl₄-induced liver fibrosis in mice via metabolomics[J]. Acta Pharmaceutica Sinica, 2025 , 60 (4) : 1001 -1011 . DOI: 10.16438/j.0513-4870.2024-1041

正文

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据统计, 全球范围内每年死于肝脏相关疾病人群超过200万^{[1, 2]}, 我国人口众多, 有超过4亿人患有各种慢性肝病^[3], 其常见病因包括病毒性肝病、非酒精性脂肪性肝病、酒精性肝病等^{[4, 5]}。值得注意的是, 尽管病因迥异, 但慢性肝病的临床发展过程相似, 大多经历从肝炎到肝纤维化、肝硬化, 再到肝衰竭/肝癌等终末期肝病的发展过程^[6]。其中, 肝纤维化是慢性肝病进展期的转折点, 也是影响慢性肝病预后的重要环节^{[7, 8]}。肝纤维化被认为是一种可逆的病理现象, 因此, 早期识别并逆转肝纤维化进程是慢性肝病治疗的关键^{[9, 10]}。

目前, 全球范围内缺乏抗肝纤维化的临床药物。在抗肝纤维化药物开发中, 中医药优势日益凸显^[11]。多项高质量临床研究已经证实复方鳖甲软肝片、安络化纤丸、扶正化瘀胶囊等中药复方在抗肝纤维化方面疗效显著^[12-15], 对其中单味药研究发现, 绞股蓝、鳖甲、大黄等在抗肝纤维化应用方面均疗效确切^[16-18]。绞股蓝Gynostemma pentaphyllum (Thunb.) Makino是多年生草质藤本植物, 为葫芦科绞股蓝属, 又叫五叶参、七叶胆^[19], 是亚洲著名的食用和药用植物, 因在非五加科人参属的植物中含有人参皂苷类成分而被称为“南方人参”^[20]。研究表明, 绞股蓝具有抗炎、保肝和调节免疫力等作用^[21], 并通过调节细胞凋亡途径发挥保肝、抗肝纤维化作用^[22]。但绞股蓝相关研究主要集中在调节血糖、血脂、抗氧化等方面, 其抗肝纤维化的相关研究较少。

基于此, 本研究通过非靶向代谢组学于四氯化碳(carbon tetrachloride, CCl₄) 诱导的肝纤维化小鼠模型上筛选绞股蓝醇提物(Gynostemma pentaphyllum ethyl acetate, GPE) 改善肝纤维化作用相关代谢物及代谢通路, 靶向代谢组学对所筛选代谢通路中的相关代谢物进行验证, 以阐明GPE对CCl₄诱导肝纤维化小鼠模型的治疗作用和肝脏代谢轮廓的调节作用, 揭示GPE调节肝细胞稳态, 保护肝脏的潜在作用机制。

材料与方法

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实验动物 C57BL/6J雄性小鼠, 体质量16~18 g, 购自北京维通利华实验动物技术有限公司, 生产许可证号SCXK (京) 2021-0011。小鼠饲养于首都医科大学实验动物中心, 温度(25 ± 2) ℃, 相对湿度50%~60%, 12 h明暗交替, 小鼠自由进食饮水。在首都医科大学实验动物伦理委员会批准下进行, 批准编号DWLLGZR202202204。

药品与试剂 绞股蓝(批号: 20230615) 购自北京本草方源有限公司, 经首都医科大学吴剑坤副教授鉴定为绞股蓝Gynostemma pentaphyllum (Thunb.) Makino的干燥全草。四氯化碳(批号: 289116) 购自美国Sigma-Aldrich公司; 橄榄油(oil, 批号: 20170619) 购自中国医药集团有限公司; 乙腈(批号: 207296) 购自美国Thermo Fisher公司; 甲酸(批号: 80065518) 购自上海麦克林生化科技有限公司; 乙酸铵(批号: B2311112) 购自美国SIGMA-ALDRICH公司; 天冬氨酸转氨酶(aspartate aminotransferase, AST) 活力检测试剂盒(批号: BC1565)、丙氨酸转氨酶(alanine aminotransferase, ALT) 活力检测试剂盒(批号: BC1555) 和羟脯氨酸(hydroxyproline, HYP) 检测试剂盒(批号: BC0255) 购自北京索莱宝生命科学公司; 胶原蛋白1A1 (collagen 1A1, COL 1A1, 批号: A1352)、α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA, 批号: A17910) 和甘油醛-3-磷酸脱氢酶(recombinant glyceraldehyde-3-phosphate dehydrogenase, GAPDH, 批号: AC002) 抗体购自南京爱博泰克公司; L-2-氯苯丙氨酸(B25919) 购自上海源叶生物科技有限公司; L-缬氨酸-D8 (批号: HY-I1124) 和D-虫荧光素游离酸(批号: HY-12591A) 购自美国Med Chem Express公司。

仪器 Vanquish型超高效液相、Orbitrap Exploris 120高分辨质谱和Heraeus Fresco17型离心机(美国Thermo Fisher Scientific公司); BSA124S-CW型天平(德国Sartorius公司); PS-60AL型超声仪(深圳市雷德邦电子有限公司); JXFSTPRP-24型匀浆机(上海净信科技有限公司); LGJ-10C冷冻干燥机(四环福瑞科仪科技发展有限公司); Infinite M1000型酶标仪(德国TECAN公司); TQXS三重串联四级杆液质联用仪(美国Waters公司)。

数据库及软件 京都基因和基因组百科全书(KEGG: Kyoto Encyclopedia of Genes and Genomes, https://www.genome.jp); 人类代谢组学数据库(HMDB, https://hmdb.ca/); SIMCA 14.1 (瑞典Umetrics公司)。

动物分组、造模与给药 将30只C57/6J雄性小鼠适应性喂养1周后, 随机分为3组, 空白对照组、CCl₄模型组和GPE给药组。CCl₄模型组和GPE给药组采用CCl₄腹腔注射造模法注射5% CCl₄/Oil溶液诱导肝纤维化模型^[23], 前两周每天注射一次, 后四周每两天注射一次。GPE给药组从造模开始每日以500 mg·kg^-1的生药剂量灌胃1次, 空白对照组、CCl₄模型组小鼠灌胃等量羧甲基纤维素钠, 连续给药6周。

生物样本采集与处理 末次给药后, 使用异氟烷将小鼠麻醉后左心室心尖取外周血, 将血液放在4 ℃冰箱静置1 h后, 离心机条件设置为3 500 r·min^-1, 4 ℃离心15 min, 取上层血清, 分装, -80 ℃冷冻保存。取血后, 快速断颈椎处死, 剪开小鼠腹部, 剪取完整的肝脏组织, 用预冷的PBS洗去肝脏表面血迹, 使用纱布吸净液体, 对其进行拍照称重。随即将肝脏分成三份, 两份冻存于-80 ℃冰箱, 一份使用4%多聚甲醛固定于4 ℃保存。

肝组织病理学观察 小鼠肝组织经4%多聚甲醛固定, 乙醇脱水, 石蜡包埋, 切片, 用苏木素-伊红(hematoxylin and eosin staining, H&E#38; E) 和马松(Masson) 染色, 在光学显微镜下观察肝脏组织的病理变化。

生化指标检测 取-80 ℃冰箱保存的血清及肝脏组织样本在4 ℃冰箱解冻, 采用蛋白印迹法(Western blot) 测定肝组织中COL 1A1和α-SMA指标, 参照试剂说明书的规定操作。采用生化试剂盒检测血清中ALT、AST及HYP指标, 参照试剂盒的规定操作。采用实时荧光定量法(RT-qPCR) 检测肝组织中Col 1a1、α-sma、白细胞介素-1β (interleukin-1β, Il-1β)、白细胞介素-6 (interleukin-6, Il-6) 及肿瘤坏死因子α (tumor necrosis factor-alpha, Tnf-α) 指标, 参照试剂盒的规定操作。Col 1a1、α-sma、β-actin、Il-1β、Il-6和Tnf-α引物购自上海生工生物工程股份有限公司, 序列见表 1。

非靶向代谢组学检测

肝脏样品制备解冻肝组织后称取20 mg, 加入200 μL裂解液, 使用研磨仪研磨, 离心取上清, 加入3倍含0.1%甲酸的乙腈(含L-缬氨酸-D8、L-2-氯苯丙氨酸和D-虫荧光素游离酸的混合内标, 0.1 mg·mL^-1) 沉淀蛋白2 h, 4 ℃、12 000 r·min^-1离心15 min, 取上清300 μL, 使用旋转蒸发仪将其旋干, 加入100 μL 0.1%甲酸-乙腈复溶, 再次离心15 min, 取90 μL上清进样分析。

肝脏检测条件使用与Orbitrap Exploris 120质谱仪偶联的Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) 的UHPLC系统进行LC-MS/MS分析。流动相由25 mmol·L^-1乙酸铵和25 mmol·L^-1氨水(pH 9.75, A) 和乙腈(B) 组成。自动采样器温度为4 ℃, 注射体积为2 μL。在获取软件(Xcalabur) 的控制下, 使用轨道探测器120质谱仪能够获得信息依赖获取(IDA) 模式的质谱/质谱。在这种模式下, 采集软件连续评估全扫描质谱。ESI源条件设置为: 鞘层气体流量为50 arb, Aux气体流量为15 arb, 毛细管温度为320 ℃, 全MS分辨率为60 000, MS/MS分辨率为15 000, 碰撞能量: SNCE 20/30/40, 喷雾电压分别为3.8 kV (正) 或-3.4 kV (负)。

靶向代谢组学检测

肝脏样本制备解冻肝组织后称取20 mg, 加入200 μL裂解液, 使用研磨仪研磨, 离心取上清, 加入3倍含0.1%甲酸的乙腈(含内标D-虫荧光素游离酸, 0.1 mg·mL^-1) 沉淀蛋白2 h, 4 ℃、12 000 r·min^-1离心15 min, 取上清300 μL, 使用旋转蒸发仪将其旋干, 加入100 μL 0.1%甲酸/乙腈复溶, 再次离心15 min, 取90 μL上清进样分析。

色谱条件色谱柱为ACQUITY UPLC^TM BEH Amide (2.1 mm × 100 mm, 1.7 μm); 流动相: A (5 mmol·L^-1乙酸铵水溶液)-B (乙腈)。梯度洗脱, 前1.5 min为95%流动相A, 1.5~1.6 min梯度变化至75%流动相A, 1.6~3.2 min保持75%流动相A, 3.2~4.5 min变化至0%流动相A, 4.5~5.5 min保持0%流动相A, 5.5~5.6 min变化至95%流动相A并持续到7.5 min。流速为0.3 mL·min^-1; 柱温: 35 ℃; 进样体积为10 μL。

质谱条件采用Waters ACQUITY UPLC I-Class/Xevo TQ-XS三重四极杆质谱仪(配备ESI离子源), 正负离子扫描; 毛细管电压为: 1.0 KV; 离子源温度: 120 ℃, 去溶剂温度: 550 ℃; 锥孔气流速: 50 L·h^-1, 去溶剂气流速: 800 L·h^-1; 雾化气压: 6.0 bar; 电压为: 30, Ramp Trap碰撞能量设置为20~50, 离子质荷比范围设置为50~1 200 Da, 所有样品在全扫描模式(positive/negative) 下检测, 扫描时间设置为0.2 s, 其他参数默认仪器基础参数。

肝脏代谢组学数据处理与分析 将质谱所采集的小鼠肝组织原始图谱导入到XCMS 3.2.0软件进行峰识别、峰匹配、滤噪、标准化、归一化等预处理, 得到包含保留时间、m/z实测值、峰面积等信息的三维数据矩阵。将数据矩阵导入SIMCA-P (version 14.1) 软件进行主成分分析(PCA) 及正交偏最小二乘法-判别分析(OPLS-DA), 以变量重要性投影值(VIP) > 1预测数据贡献度, 差异倍数(FC) ≥ 1.2或FC ≤ 0.67及t检验所得P < 0.05为条件筛选差异代谢物, 结合质谱信息与HMDB等在线数据库及相关文献进一步匹配注释获得代谢物信息, 将其导入MetaboAnalyst (https://new.metaboanalyst.ca/) 进行通路富集分析, 以P < 0.05及通路影响值> 0.1为条件筛选关键代谢通路, 根据京都基因与基因组百科全书建立代谢通路网络图。

统计学分析 实验数据采用GraphPad Prism 9.5软件进行统计学分析, 多组间比较采用单因素方差分析(one-way ANOVA) 分析, 计量数据采用

$ \stackrel{-}{x} $

± s表示, P < 0.05表明具有统计学意义。

结果

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1 GPE对CCl₄诱导的小鼠肝组织病理学特征的影响

采用CCl₄诱导的方式构建肝纤维化小鼠模型, 造模的同时灌胃给药, 连续6周后取样(造模方法见图 1A)。与对照组相比, 模型组小鼠肝脏表面有较多小颗粒, 色泽苍白。经GPE干预后, 模型组小鼠肝脏表面较为光滑, 色泽鲜嫩接近空白对照组。H&E#38; E和Masson染色显示, 与对照组相比, 模型组小鼠肝脏内炎性细胞浸润明显, 胶原蓄积增多。与模型组相比, GPE组小鼠肝脏炎性细胞浸润与胶原蓄积明显减少, 肝纤维化有所改善(P < 0.05, P < 0.01, 图 1B、C)。

2 GPE对CCl₄诱导的小鼠血清生化指标的影响

采用ALT、AST和HYP试剂盒检测血清中GPE对小鼠肝损伤的影响, 结果显示, 与对照组相比, 模型组小鼠血清中ALT和AST酶活力及HYP的水平显著升高。与模型组相比, GPE组能显著降低血清中ALT和AST酶活力及HYP的水平(P < 0.05, P < 0.01, 图 1D)。采用RT-qPCR和Western blot检测GPE对小鼠肝纤维化的影响, 结果显示, 与对照组相比, 模型组小鼠肝内COL 1A1和α-SMA的基因和蛋白水平显著升高(P < 0.05, P < 0.01)。与模型组相比, GPE组能显著降低小鼠肝内COL 1A1和α-SMA的基因和蛋白表达水平(P < 0.05, P < 0.01)。提示GPE能够改善CCl₄诱导小鼠的肝纤维化(图 1E、F)。

3 GPE对CCl₄诱导的小鼠肝脏炎症的影响

采用RT-qPCR法检测GPE对小鼠肝脏炎症的影响, 结果显示, 与对照组相比, 模型组小鼠肝内Il-1β、Il-6和Tnf-α的基因水平显著升高(P < 0.05, P < 0.01)。与模型组相比, GPE组能显著降低小鼠肝内Il-1β、Il-6和Tnf-α的基因表达水平(P < 0.05, P < 0.01)。与H&E#38; E染色病理观察结果一致。提示CCl₄模型小鼠肝脏出现炎症, GPE能有效缓解炎症反应(图 1G)。

4 GPE对CCl₄诱导的小鼠肝脏代谢组学分析

4.1 代谢轮廓分析

正负离子模式下, 模型组和GPE组小鼠肝脏的总离子流图(TIC) 见图 2A~D, 结果显示, 各组肝组织谱图的峰形及峰面积有一定差异, 表明小鼠体内的代谢轮廓改变, 部分代谢物水平发生变化。采用正、负离子检查模式对模型组和GPE组小鼠肝组织中内源性代谢物进行PCA及OPLS-DA分析, PCA及OPLS-DA得分图中模型组与GPE组代谢轮廓分离显著, 聚类程度较好, 说明机体代谢状态发生改变, 内源性代谢产物在模型组与GPE组之间存在显著差异; 用置换检验(200次) 对模型的可靠性进行验证, 模型组与GPE组呈现较好的聚类效果, 在负离子模式下, 肝组织代谢模型组与GPE组截距为R² = 0.988, Q² = -0.208, 在正离子模式下, 模型组与GPE组R² = 0.992, Q² = -0.234; 说明模型验证有效, 具有良好的可靠性及预测能力, 见图 2。

4.2 相关代谢物筛选

在OPLS-DA分析基础上, 根据VIP > 1, P < 0.05及FC ≥ 1.2或FC ≤ 0.67筛选差异代谢物。提取差异代谢物的MS/MS数据, 与质谱数据库比对, 通过KEGG分析确定相关代谢物。最后, 筛选出47个代谢物, GPE干预显著上调39个代谢物, 下调8个代谢物。聚类分析表明, 模型组和GPE组体内代谢表现出显著差异(P < 0.05, P < 0.01), 见图 3A。

4.3 代谢通路分析

将筛选出的代谢物导入MetaboAnalyst进行代谢通路富集分析, 探究代谢物之间的潜在联系。结果发现, GPE干预CCl₄小鼠主要通过影响果糖与甘露糖代谢, 氨基糖与核苷酸代谢, 淀粉和蔗糖代谢, 苯丙氨酸、酪氨酸和色氨酸的生物合成, 鞘脂代谢和半乳糖代谢等6条关键代谢通路发挥治疗作用, GPE干预CCl₄小鼠的相关代谢通路见图 3B, GPE干预CCl₄小鼠的代谢物信息见表 2。

4.4 靶向代谢组学检测分析

使用TQXS三重串联四级杆液质联用仪检测小鼠肝组织中关键代谢通路中的代谢物含量, 结果显示, 与对照组相比, 模型和GPE组主要影响了D-甘露糖、6-磷酸甘露糖、D-果糖、2-磷酸果糖、D-蔗糖、海藻糖、谷氨酸、苯丙氨酸、酪氨酸、L-2-氨基-3-氧代丁酸、乳酸、2-羟基丁酸和牛磺酸共13个差异代谢物, 与对照组相比, 模型组小鼠体内的海藻糖、谷氨酸、苯丙氨酸、酪氨酸、L-2-氨基-3-氧代丁酸、乳酸、2-羟基丁酸和牛磺酸含量显著升高; 与模型组相比, GPE小鼠体内的海藻糖、谷氨酸、苯丙氨酸、酪氨酸、L-2-氨基-3-氧代丁酸、乳酸、2-羟基丁酸和牛磺酸水平显著被逆转; 与对照组相比, 模型组小鼠体内的D-甘露糖、6-磷酸甘露糖、D-果糖、2-磷酸果糖和D-蔗糖的含量显著下降; 与模型组相比, GPE小鼠体内D-甘露糖、6-磷酸甘露糖、D-果糖、2-磷酸果糖和D-蔗糖的水平有显著回升(P < 0.05, P < 0.01), 见图 4。这些代谢物主要涉及果糖与甘露糖代谢和芳香氨基酸代谢两条关键代谢通路, 见图 5。表明GPE的抗肝纤维化作用可能与调控果糖与甘露糖代谢途径和芳香氨基酸代谢途径中的此13种代谢物密切相关。

讨论

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慢性肝病死亡率不断上升, 已成为一个重大的全球健康问题^[24]。肝纤维化是各种慢性肝病在进展过程中均会出现的病理状态, 减轻肝纤维化是目前已被广泛认可的治疗慢性肝病方法。绞股蓝皂苷是扶正化瘀胶囊抗炎、抗肝纤维化的主要活性成分群之一, 能够通过抑制肝星状细胞激活、肝祖细胞向成纤维细胞转化和抗肝细胞凋亡等多种机制发挥作用^[25-27]。其通常采用溶剂法进行提取, 其中乙醇提取皂苷含量最佳^{[28, 29]}。课题组前期研究表明, 绞股蓝醇提物能够通过抑制PDK1/Bcl-2通路发挥抗肝细胞凋亡、保护肝脏的作用^[22], 但是绞股蓝的保肝、抗肝纤维化作用及机制还需要更深入的探索。代谢组学主要研究机体整体水平下内源性小分子代谢物的代谢轮廓^[30], 来阐明疾病潜在的发生机制及治疗靶点。本研究采用CCl₄诱导的肝纤维化小鼠模型, 探讨了GPE对肝纤维化小鼠的改善作用, 从肝脏代谢组学的角度阐述GPE抗炎、抗肝纤维化的潜在作用机制。

本研究表明, GPE能够改善肝组织形态、减少胶原蓄积, 抑制肝损伤和肝纤维化的进展。肝脏是机体主要的代谢场所, 通过对小鼠肝脏的代谢组学分析, 筛选发现GPE主要调控了47个关键代谢物, 通路富集分析发现GPE主要调节果糖与甘露糖代谢和氨基酸代谢途径, 这可能是GPE改善CCl₄诱导的小鼠模型肝纤维化的潜在机制之一。

甘露糖是一种葡萄糖的2-外聚体^[31], 在人体内参与多种代谢, 既往认为甘露糖只是一种常见的单糖, 对其生物学功能重视不足。然而, 最新研究发现其与免疫细胞的功能高度相关, 是当前免疫代谢的研究热点之一^{[32, 33]}。大量研究表明, 补充甘露糖具有减轻肝纤维化^{[34, 35]}、抗肿瘤^{[36, 37]}、抑制肝癌细胞生长^{[38, 39]}的作用。研究表明, 超生理水平的D-甘露糖能够抑制自身免疫性糖尿病和气道炎症小鼠模型的免疫病理^[37], 弥补甘露糖磷酸异构酶的缺失, 减弱肝星状细胞活化改善肝纤维化^[34]。本研究结果显示, CCl₄小鼠模型体内D-甘露糖及其下游代谢物6-磷酸甘露糖的含量显著降低, 表明在肝纤维化疾病状态下, 小鼠肝脏中的甘露糖代谢受到影响, 而GPE治疗后D-甘露糖和6-磷酸甘露糖的水平显著回升。果糖是甘露糖的上游代谢物, 对肝脏代谢具有直接作用^[40]。在本研究中, CCl₄小鼠模型肝脏内D-果糖及其下游代谢物2-磷酸果糖的含量显著降低, GPE治疗后D-果糖与2-磷酸果糖的水平明显回调。本研究表明, GPE可显著改善CCl₄小鼠模型的肝功能和肝内胶原蓄积, 并首次发现该作用与甘露糖代谢通路密切相关, 鉴于甘露糖及其代谢通路上的代谢物具有极为重要的免疫调控作用, 因此, GPE具有较高的临床应用价值, 是否能够通过调控甘露糖代谢途径发挥抗纤维化作用, 值得深入研究。

苯丙氨酸属芳香族氨基酸, 能够在体内经其羟化酶氧化生成酪氨酸, 与酪氨酸一起合成神经递质和激素参与机体糖代谢^[41]。据报道, 苯丙氨酸/酪氨酸代谢与非酒精性脂肪肝病的发生发展密切相关^{[42, 43]}。非酒精性脂肪性肝病患者体内谷氨酸、苯丙氨酸和酪氨酸等氨基酸的浓度显著升高^[44]。本研究中肝纤维化小鼠模型肝脏中谷氨酸、苯丙氨酸和酪氨酸等代谢物水平显著升高, 表明肝纤维化小鼠体内氨基酸代谢紊乱。GPE治疗能够降低模型组小鼠肝内谷氨酸、苯丙氨酸、酪氨酸等氨基酸的水平。本文研究发现GPE抗肝纤维化作用与芳香族氨基酸中的苯丙氨酸/酪氨酸代谢密切相关。此外, 芳香族氨基酸能够通过PI3K/AKT信号通路调节糖代谢紊乱^[45-47]。综上, 本研究首次发现, GPE改善肝脏糖代谢紊乱发挥抗炎、抗肝纤维化作用可能与芳香氨基酸的苯丙氨酸/酪氨酸代谢密切相关。

综上所述, 本研究通过非靶向代谢组学和靶向代谢组学序贯分析, 发现并验证了GPE抗肝纤维化作用可能与果糖与甘露糖代谢和芳香氨基酸代谢等途径密切相关。后续本课题组将通过同位素示踪、光亲合探针钓靶等方式对GPE抗肝纤维化机制进行更深入的研究, 以期为GPE抗肝纤维化治疗的临床应用和产品开发提供参考依据。

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国家自然科学基金项目(82074112)
名贵中药资源可持续利用能力建设项目(2060302)

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2025年第60卷第4期

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doi: 10.16438/j.0513-4870.2024-1041

接收时间：2024-10-24
首发时间：2025-10-29
出版时间：2025-04-12

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收稿日期：2024-10-24
修回日期：2025-02-09

基金

国家自然科学基金项目(82074112)

名贵中药资源可持续利用能力建设项目(2060302)

作者信息

¹ 山西中医药大学, 中药与食品工程学院, 山西晋中 030619

² 首都医科大学中医药学院, 中医药防治传染性重症肝病研究与转化实验室, 北京 100069

³ 首都医科大学附属北京中医医院, 北京 100010

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鲁雅雯，Tel: 86-351-3179903, E-mail: ywlu0411@163.com

王伽伯，E-mail: jiabo_wang@ccmu.edu.cn

李会芳，E-mail: lihuifangzy@sxtcm.edu.cn

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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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TxT

No.	Gene name	Sequence (5′ to 3′)	T_m/℃
1	M-α-sma-F	GGCACCACTGAACCCTAAGG	20
2	M-α-sma-R	ACAATACCAGTTGTACGTCCAGA	23
3	M-Col1-F	TAAGGGTCCCCAATGGTGAGA	21
4	M-Col1-R	GGGTCCCTCGACTCCTACAT	20
5	M-Il-1β-F	GCAACTGTTCCTGAACTCAACT	22
6	M-Il-1β-R	ATCTTTTGGGGTCCGTCAACT	21
7	M-Il-6-F	TAGTCCTTCCTACCCCAATTTCC	23
8	M-Il-6-R	TTGGTCCTTAGCCACTCCTTC	21
9	M-Tnf-α-F	CCCTCACACTCAGATCATCTTCT	23
10	M-Tnf-α-R	GCTACGACGTGGGCTACAG	19
11	M-β-actin-F	CCACAGCTGAGAGGGAAATC	20
12	M-β-actin-R	AAGGAAGGCTGGAAAAGAGC	20

No.

Gene name

Sequence (5′ to 3′)

T_m/℃

M-α-sma-F

GGCACCACTGAACCCTAAGG

M-α-sma-R

ACAATACCAGTTGTACGTCCAGA

M-Col1-F

TAAGGGTCCCCAATGGTGAGA

M-Col1-R

GGGTCCCTCGACTCCTACAT

M-Il-1β-F

GCAACTGTTCCTGAACTCAACT

M-Il-1β-R

ATCTTTTGGGGTCCGTCAACT

M-Il-6-F

TAGTCCTTCCTACCCCAATTTCC

M-Il-6-R

TTGGTCCTTAGCCACTCCTTC

M-Tnf-α-F

CCCTCACACTCAGATCATCTTCT

M-Tnf-α-R

GCTACGACGTGGGCTACAG

M-β-actin-F

CCACAGCTGAGAGGGAAATC

M-β-actin-R

AAGGAAGGCTGGAAAAGAGC

No.	Metabolite	HMDB ID	Ion mode	m/z	VIP	FC (GPE group vs CCl₄ group)
1	Montecristin	HMDB0029795	POS	575.503 7	1.348 98	5.733 3
2	PC(35∶3)	HMDB0007947	POS	770.567 6	1.770 6	2.669 1
3	PC(18∶3(6Z, 9Z, 12Z)/15∶0)	HMDB0008165	POS	742.536 1	1.877 32	3.242 5
4	PE(18∶1(11Z)/18∶2(9Z, 12Z))	HMDB0009027	POS	742.536 1	1.877 32	3.242 5
5	PC(18∶1(9Z)/P-16∶0)	HMDB0008126	POS	744.592 7	1.151 39	6.334 7
6	Ethyl glucuronide	HMDB0010325	NEG	221.066 5	19.714 5	158.64
7	Nobiletin	HMDB0029540	NEG	401.128 2	7.564 67	281.49
8	Ascorbic acid	HMDB0000044	NEG	175.024 8	2.733 84	9.929 2
9	N-Acetylmethionine	HMDB0011745	NEG	190.053 9	2.787 97	2.147 7
10	2, 2-Dimethylsuccinic acid	HMDB0002074	NEG	145.050 6	1.509 26	29.53
11	Pentadecanoic acid	HMDB0000826	NEG	241.216 7	1.293 2	1.742 6
12	4-Nitroaniline	HMDB0246529	NEG	137.0354	1.116 37	0.167 88
13	N-Acetylphenylalanine	HMDB0000512	NEG	206.082	1.647 33	2.715 6
14	PC(18∶0/18∶1(9Z))	HMDB0008038	POS	788.609 7	3.825 21	0.514 82
15	Acetylleucine	HMDB0011756	NEG	172.097 8	1.465 7	1.949 9
16	Cer(d18∶1/16∶0)	HMDB0004949	POS	520.507 2	1.187 38	0.591 63
17	Bathophenanthroline	HMDB0246617	POS	333.132 8	1.107 16	2.571 1
18	Doxycycline	HMDB0014399	NEG	443.140 3	2.578 83	178.88
19	Buprenorphine	HMDB0015057	POS	468.307 3	1.883 29	1.527 2
20	Glucose 6-phosphate	HMDB0001401	NEG	259.021 9	1.781 33	1.985 1
21	Mannose 6-phosphate	HMDB0001078	NEG	259.021 9	1.781 33	1.985 1
22	Fructose 1-phosphate	HMDB0001076	NEG	259.021 9	1.781 33	1.985 1
23	Mannose 1-phosphate	HMDB0006330	NEG	259.021 9	1.781 33	1.985 1
24	Sorbose	HMDB0246950	NEG	179.056 1	1.918 27	2.200 1
25	Mannose	HMDB0000169	NEG	179.056 1	1.918 27	2.200 1
26	Fructose	HMDB0000660	NEG	179.056 1	1.918 27	2.200 1
27	Castamollissin	HMDB0038888	POS	469.096 1	1.650 57	2.170 1
28	Trigoforin	HMDB0029495	POS	189.086 9	1.510 57	1.827 7
29	N-[(4E, 8E)-1, 3-dihydroxyoctadeca-4, 8-dien-2-yl]hexadecanamide	HMDB0035480	POS	536.504	1.134 28	0.503 58
30	N-Acetylglutamine	HMDB0006029	NEG	187.071 9	2.388 84	1.787 9
31	N-Acetylhistidine	HMDB0032055	NEG	196.072 6	1.082	1.694 4
32	Dehydroascorbic acid (oxidized vitamin C)	HMDB0001264	NEG	173.008 7	1.535 82	1.602 5
33	Ribitol	HMDB0000508	NEG	151.061	1.152 99	1.79
34	Tyrosine	HMDB0000158	POS	182.081 1	6.640 24	1.684 9
35	O-Tyrosine	HMDB0006050	POS	182.081 1	6.640 24	1.684 9
36	Heptadecanoic acid	HMDB0002259	NEG	269.247 8	1.259 36	1.516 6
37	Methylimidazoleacetic acid	HMDB0002820	POS	141.065 6	1.402 39	3.025 8
38	PC(14∶1(9Z)/16∶0)	HMDB0007902	POS	704.522 7	1.451 5	2.455 9
39	Mangiferdesmethylursanone	HMDB0036018	POS	429.372 5	1.182 26	0.591 78
40	Allantoin	HMDB0000462	NEG	157.036 2	3.727 13	0.607 9
41	Chromone	HMDB0032938	POS	147.044 1	1.069 52	2.184 2
42	Isovalerylglycine	HMDB0000678	NEG	158.082 1	1.133 26	0.518 85
43	2-Hydroxyethanesulfonic acid	HMDB0003903	NEG	124.991 3	1.966 18	0.297 07
44	Cytidine 5′-diphosphocholine (CDP-choline)	HMDB0001413	POS	489.113 5	1.082 65	1.648 2
45	Glutathione	HMDB0000125	NEG	306.076	4.332 64	1.860 4
46	Fagomine	HMDB0033453	POS	148.096 6	1.714 51	1.918 5
47	PC(20∶1(11Z)/15∶0)	HMDB0008297	POS	774.597 6	1.706 96	2.003 6

No.

Metabolite

HMDB ID

Ion mode

m/z

VIP

FC (GPE group vs CCl₄ group)

Montecristin

HMDB0029795

POS

575.503 7

1.348 98

5.733 3

PC(35∶3)

HMDB0007947

POS

770.567 6

1.770 6

2.669 1

PC(18∶3(6Z, 9Z, 12Z)/15∶0)

HMDB0008165

POS

742.536 1

1.877 32

3.242 5

PE(18∶1(11Z)/18∶2(9Z, 12Z))

HMDB0009027

POS

742.536 1

1.877 32

3.242 5

PC(18∶1(9Z)/P-16∶0)

HMDB0008126

POS

744.592 7

1.151 39

6.334 7

Ethyl glucuronide

HMDB0010325

NEG

221.066 5

19.714 5

158.64

Nobiletin

HMDB0029540

NEG

401.128 2

7.564 67

281.49

Ascorbic acid

HMDB0000044

NEG

175.024 8

2.733 84

9.929 2

N-Acetylmethionine

HMDB0011745

NEG

190.053 9

2.787 97

2.147 7

2, 2-Dimethylsuccinic acid

HMDB0002074

NEG

145.050 6

1.509 26

29.53

Pentadecanoic acid

HMDB0000826

NEG

241.216 7

1.293 2

1.742 6

4-Nitroaniline

HMDB0246529

NEG

137.0354

1.116 37

0.167 88

N-Acetylphenylalanine

HMDB0000512

NEG

206.082

1.647 33

2.715 6

PC(18∶0/18∶1(9Z))

HMDB0008038

POS

788.609 7

3.825 21

0.514 82

Acetylleucine

HMDB0011756

NEG

172.097 8

1.465 7

1.949 9

Cer(d18∶1/16∶0)

HMDB0004949

POS

520.507 2

1.187 38

0.591 63

Bathophenanthroline

HMDB0246617

POS

333.132 8

1.107 16

2.571 1

Doxycycline

HMDB0014399

NEG

443.140 3

2.578 83

178.88

Buprenorphine

HMDB0015057

POS

468.307 3

1.883 29

1.527 2

Glucose 6-phosphate

HMDB0001401

NEG

259.021 9

1.781 33

1.985 1

Mannose 6-phosphate

HMDB0001078

NEG

259.021 9

1.781 33

1.985 1

Fructose 1-phosphate

HMDB0001076

NEG

259.021 9

1.781 33

1.985 1

Mannose 1-phosphate

HMDB0006330

NEG

259.021 9

1.781 33

1.985 1

Sorbose

HMDB0246950

NEG

179.056 1

1.918 27

2.200 1

Mannose

HMDB0000169

NEG

179.056 1

1.918 27

2.200 1

Fructose

HMDB0000660

NEG

179.056 1

1.918 27

2.200 1

Castamollissin

HMDB0038888

POS

469.096 1

1.650 57

2.170 1

Trigoforin

HMDB0029495

POS

189.086 9

1.510 57

1.827 7

N-[(4E, 8E)-1, 3-dihydroxyoctadeca-4, 8-dien-2-yl]hexadecanamide

HMDB0035480

POS

536.504

1.134 28

0.503 58

N-Acetylglutamine

HMDB0006029

NEG

187.071 9

2.388 84

1.787 9

N-Acetylhistidine

HMDB0032055

NEG

196.072 6

1.082

1.694 4

Dehydroascorbic acid (oxidized vitamin C)

HMDB0001264

NEG

173.008 7

1.535 82

1.602 5

Ribitol

HMDB0000508

NEG

151.061

1.152 99

1.79

Tyrosine

HMDB0000158

POS

182.081 1

6.640 24

1.684 9

O-Tyrosine

HMDB0006050

POS

182.081 1

6.640 24

1.684 9

Heptadecanoic acid

HMDB0002259

NEG

269.247 8

1.259 36

1.516 6

Methylimidazoleacetic acid

HMDB0002820

POS

141.065 6

1.402 39

3.025 8

PC(14∶1(9Z)/16∶0)

HMDB0007902

POS

704.522 7

1.451 5

2.455 9

Mangiferdesmethylursanone

HMDB0036018

POS

429.372 5

1.182 26

0.591 78

Allantoin

HMDB0000462

NEG

157.036 2

3.727 13

0.607 9

Chromone

HMDB0032938

POS

147.044 1

1.069 52

2.184 2

Isovalerylglycine

HMDB0000678

NEG

158.082 1

1.133 26

0.518 85

2-Hydroxyethanesulfonic acid

HMDB0003903

NEG

124.991 3

1.966 18

0.297 07

Cytidine 5′-diphosphocholine (CDP-choline)

HMDB0001413

POS

489.113 5

1.082 65

1.648 2

Glutathione

HMDB0000125

NEG

306.076

4.332 64

1.860 4

Fagomine

HMDB0033453

POS

148.096 6

1.714 51

1.918 5

PC(20∶1(11Z)/15∶0)

HMDB0008297

POS

774.597 6

1.706 96

2.003 6