等热量低碳高蛋白饮食结合有氧运动干预非酒精性脂肪性肝病的血清代谢组学分析

李美莺; 纪万里; 刘王振祖; 汪涛; 杜晟楠; 高静静; 蒋元烨; 胡诚

doi:10.3969/j.issn.1001-5256.2021.11.023

等热量低碳高蛋白饮食结合有氧运动干预非酒精性脂肪性肝病的血清代谢组学分析

DOI: 10.3969/j.issn.1001-5256.2021.11.023

李美莺^1a,
纪万里²,
刘王振祖^3a,
汪涛^1b,
杜晟楠^1b,
高静静^1b,
蒋元烨^1b, ,,
胡诚^3b, ,

1a.
上海市普陀区中心医院健康管理中心，上海 200062
1b.
上海市普陀区中心医院消化内科，上海 200062
2.
上海健康医学院药学院，上海 201318
3a.
上海中医药大学中药学院，上海 201203
3b.
上海中医药大学科技实验中心，上海 201203

基金项目:

国家自然科学基金青年项目 (81703879);

上海市卫计委临床研究专项面上项目 (201840377);

上海市卫计委临床研究专项面上项目 (201940449);

普陀区科委自主创新项目 (ptkwws201813);

上海中医药大学后备卓越中医人才项目 (20D-RC-02)

详细信息

通信作者:
蒋元烨，yuanye1014@126.com

胡诚，hucheng10200@163.com

中图分类号: R575.5
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出版历程
- 收稿日期: 2021-04-01
- 录用日期: 2021-05-06
- 出版日期: 2021-11-20

A serum metabolomics study on the intervention of nonalcoholic fatty liver disease by equicaloric low-carbohydrate high-protein diet combined with aerobic exercise

Meiying LI^1a,
Wanli JI²,
Wangzhenzu LIU^3a,
Tao WANG^1b,
Shengnan DU^1b,
Jingjing GAO^1b,
Yuanye JIANG^{1b
, ,},
Cheng HU^{3b
, ,}

1a.
Health Management Center, Putuo District Central Hospital, Shanghai 200062, China
1b.
Department of Gastroenrology, Putuo District Central Hospital, Shanghai 200062, China
2.
College of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
3a.
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
3b.
Science and Technology Experimental Center, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Research funding:

National Natural Science Foundation of China (81703879);

Shanghai Municipal Commission of Health and Family Planning General Project for Clinical Research of Health Industry (201840377);

Shanghai Municipal Commission of Health and Family Planning General Project for Clinical Research of Health Industry (201940449);

Putuo District of Shanghai Science And Technology Commission Research Project (ptkwws201813);

Reserve Excellent TCM Talents Project of Shanghai University of Traditional Chinese Medicine (20D-RC-02)

摘要

摘要: 目的收集非酒精性脂肪性肝病(NAFLD)患者的血清并对生活方式干预前后的血清代谢生物标志物变化情况进行分析。方法收集2019年1月—2020年1月于上海市普陀区中心医院消化科及住院部诊断为NAFLD的患者23例，患者采取有氧运动及等热量低碳水化合物高蛋白饮食进行干预，从体检中心选择13例健康志愿者作为对照组。NAFLD患者于干预前后进行常规的基本信息采集，并分别采血2次检测肝功能、血糖、血脂等指标，另分装部分血清用于血清代谢组学分析。将血清样本通过处理后使用超高效液相色谱串联高分辨质谱(UPLC-Q-Orbitrap/MS)进行分析。采集的数据经Compound Discover处理后运用主成分分析(PCA) 和正交偏最小二乘判别法分析构建患者与正常健康人的差异血清代谢物谱，并对差异代谢通路进行富集分析。正态分布的计量资料两组比较采用独立样本t检验；非分正态分布的计量资料两组比较采用Wilcoxon非参数检验。结果经生活方式干预后，患者的BMI及体质量均明显降低(P值均＜0.01)；血清生化指标ALP、Alb、GGT、ALT表达水平均降低(P值均＜0.05)，总蛋白(TP)显著降低(P＜0.01)，胆碱酯酶(ChE)、AST、血糖(GLU)改善不明显；血脂四项中TG的水平显著减低(P＜0.01)，HDL-C、LDL-C、TC改善不显著。代谢组学研究发现NAFLD患者生活方式干预前后，血清中的代谢产物有33种代谢物发生了明显改变。此外，PCA结果显示NAFLD患者经干预后，体内的代谢产物水平趋于健康人。通过信号通路分析，发现运动饮食主要影响了胆汁酸、不饱和脂肪酸合成及苯丙氨酸代谢等通路。结论生活方式干预可不同程度的减轻NAFLD患者的体质量，改善血清生化指标，改善NAFLD患者的异常代谢途径，这对指导临床医生为NAFLD患者制订合理的饮食及运动策略，预防NAFLD进展具有一定的临床价值和意义。
- 非酒精性脂肪性肝病 /
- 代谢组学 /
- 生物标记 /
- 生活方式
Abstract: Objective To collect the serum samples of patients with nonalcoholic fatty liver disease (NAFLD), and to investigate the changes in serum metabolic biomarkers before and after lifestyle intervention. Methods A total of 23 patients who were diagnosed with NAFLD in Department of Gastroenterology and Inpatient Department, Putuo District Central Hospital of Shanghai, from January 2019 to January 2020 were enrolled, and all patients received the intervention with aerobic exercise and equicaloric low-carbohydrate high-protein diet. A total of 13 healthy volunteers who underwent physical examination in Physical Examination Center were enrolled as control group. For the patients with NAFLD, basic information was collected before and after intervention, blood samples were collected twice to measure liver function, blood glucose, and blood lipids, and part of serum was used for serum metabolomics analysis. The serum samples were analyzed by ultra-performance liquid chromatography/tandem high-resolution mass spectrometry. The data collected were processed in Compound Discover, and then principal component analysis (PCA) and orthogonal partial least squares discriminant analysis were used to establish the profile of differentially expressed blood metabolites between patients and healthy people and perform the enrichment analysis of differentially expressed metabolic pathways. The independent samples t-test was used for comparison of normally distributed continuous data between two groups, and the Wilcoxon non-parametric test was used for comparison of non-normally distributed continuous data between two groups. Results After lifestyle intervention, the patients had significant reductions in body mass index (P < 0.01), body weight (P < 0.01), and serum biochemical parameters alkaline phosphatase, albumin, gamma-glutamyl transpeptidase, and alanine aminotransferase (all P < 0.05), as well as a significant reduction in total protein (P < 0.01), while there were no significant improvements in cholinesterase, aspartate aminotransferase, and glucose. As for the four items for blood lipids, there was a significant reduction in triglyceride (P < 0.01), while there were no significant improvements in high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total cholesterol. The metabolomics analysis showed that 33 serum metabolites changed significantly after lifestyle intervention. In addition, PCA results showed that after intervention, the level of metabolites in patients tended to be normal. The signaling pathway analysis showed that exercise and diet mainly affected the pathways of bile acid, unsaturated fatty acid synthesis, and phenylalanine metabolism. Conclusion Lifestyle intervention can achieve varying degrees of reduction in the body weight of patients with NAFLD, improve serum biochemical parameters, and regulate the abnormal metabolic pathway in patients with NAFLD, which has important clinical value and significance for guiding clinicians to formulate reasonable diet and exercise strategies for patients with NAFLD and prevent the progression of NAFLD.

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图 1 健康对照组及NAFLD患者运动饮食干预前后的PCA

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图 2 NAFLD患者运动饮食干预前后的OPLS-DA

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图 3 NAFLD患者经运动饮食干预前后的差异代谢物热图

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图 4 差异代谢产物通路富集分析

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表 1 梯度洗脱程序

时间(min)	流速(mL/min)	A(%)	B(%)
0	0.3	95	5
2	0.3	95	5
12	0.3	5	95
15	0.3	5	95
17	0.3	95	5

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表 2 2组基线资料及血清生化参数比较

参数	健康对照组(n=13)	NAFLD组(n=23)	统计值	P值
年龄(岁)	42±14	48±14	t=1.208	0.235
体质量(kg)	82.00(74.00~90.00)	71.00(68.00~77.85)	Z=2.408	0.015
BMI(kg/m²)	27.08(25.86~27.85)	24.98(24.22~26.61)	Z=-2.289	0.022
腰围(cm)	102.38±10.02	94.50±14.17	t=1.721	0.094
TBil(μmol/L)	11.00(10.00~14.00)	13.00(10.00~14.20)	Z=-0.564	0.580
DBil(μmol/L)	2.21±0.75	2.38±0.75	t=-0.625	0.536
ALP(U/L)	70.00(61.00~87.00)	86.00(69.00~100.50)	Z=-1.648	0.100
TP(g/L)	72.00(67.00~75.00)	76.00(72.50~79.15)	Z=-1.404	0.169
Alb(μmol/L)	30.00(27.00~31.00)	39.00(30.00~45.50)	Z=-1.584	0.015
GGT(U/L)	30.00(25.00~46.00)	55.00(32.50~75.00)	Z=-2.208	0.026
ChE(U/L)	9732.85±1244.79	10 047.35±1488.81	t=-0.627	0.535
ALT(U/L)	43.00(28.00~47.00)	61.00(28.50~102.50)	Z=-1.763	0.031
AST(U/L)	26.00(25.00~35.00)	32.00(23.00~52.50)	Z=-1.172	0.253
GLU(mmol/L)	4.80(4.50~5.10)	5.50(5.10~5.65)	Z=-2.295	0.022
HDL-C(mmol/L)	1.26±0.33	1.15±0.25	t=1.168	0.410
LDL-C(mmol/L)	3.73±0.68	3.44±1.10	t=0.835	0.282
TC(mmol/L)	4.36±1.79	5.05±1.76	t=-1.093	0.018
TG(mmol/L)	1.72(1.43~2.89)	2.63(1.82~3.69)	Z=-1.268	0.021
注：TP，总蛋白；ChE，胆碱酯酶；GLU，血糖。

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表 3 NAFLD组干预前后临床资料及血清生化参数比较

参数	干预前	干预后	统计值	P值
体质量(kg)	71.00(68.00~77.85)	68.00(63.00~72.75)	Z=-1.442	0.149
BMI(kg/m²)	24.98(24.22~26.61)	24.17(23.07~25.53)	Z=-1.736	0.083
腰围(cm)	94.50±14.17	90.12±13.91	t=1.035	0.306
TBil(μmol/L)	13.00(10.00~14.20)	13.00(10.50~14.50)	Z=-0.343	0.731
DBil(μmol/L)	2.38±0.75	2.28±0.74	t=0.434	0.666
ALP(U/L)	86.00(69.00~100.50)	85.00(68.00~99.50)	Z=-0.066	0.947
TP(g/L)	76.00(72.50~79.15)	74.00(72.00~79.50)	Z=-0.264	0.792
Alb(μmol/L)	39.00(30.00~45.50)	36.00(29.90~45.50)	Z=-1.004	0.035
GGT(U/L)	55.00(32.50~75.00)	41.00(22.50~46.00)	Z=-2.089	0.037
ChE(U/L)	10 047.35±1488.81	9575.26±1938.16	t=0.906	0.370
ALT(U/L)	61.00(28.50~102.50)	32.00(24.50~55.00)	Z=-1.726	0.044
AST(U/L)	32.00(23.00~52.50)	34.00(21.00~45.00)	Z=-1.431	0.152
GLU(mmol/L)	5.50(5.10~5.65)	5.30(4.45~5.50)	Z=-1.389	0.165
HDL-C(mmol/L)	1.15±0.25	1.15±0.21	t=-0.111	0.765
LDL-C(mmol/L)	3.44±1.10	3.35±0.81	t=0.301	0.109
TC(mmol/L)	5.05±1.76	4.27±1.38	t=1.635	0.013
TG(mmol/L)	2.63(1.82~3.69)	1.59(1.22~2.30)	Z=-2.702	0.007

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