基于液相色谱-质谱代谢组学方法研究中药定喘汤对呼吸道合胞病毒感染的疗效

doi:10.3724/SP.J.1123.2020.06013

色谱 ›› 2021, Vol. 39 ›› Issue (3): 281-290.DOI: 10.3724/SP.J.1123.2020.06013

基于液相色谱-质谱代谢组学方法研究中药定喘汤对呼吸道合胞病毒感染的疗效

欧阳瑒¹^,², 迟磊³, 徐超³, 赵欣捷¹^,^*(), 崔振泽³^,^*()

1.中国科学院分离分析化学重点实验室, 中国科学院大连化学物理研究所, 辽宁大连 116023
2.中国科学院大学, 北京 100049
3.大连医科大学附属大连市儿童医院, 辽宁大连 116012

收稿日期:2020-06-09 出版日期:2021-03-08 发布日期:2021-02-03
通讯作者: 赵欣捷,崔振泽
作者简介:Tel:(0411)62926901,E-mail: cuizhenze@126.com(崔振泽).
*Tel:(0411)84379532,E-mail: xj_zhao1@126.com(赵欣捷);
第一联系人：
# 共同第一作者.
基金资助:
国家自然科学基金(81473726)

Liquid chromatography-mass spectrometry-based metabolomics study of the efficacy of Chinese medicine asthma-relieving decoction on respiratory syncytial virus infection

OUYANG Yang¹^,², CHI Lei³, XU Chao³, ZHAO Xinjie¹^,^*(), CUI Zhenze³^,^*()

1. Chinese Academy of Sciences Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. Dalian Children’ s Hospital of Dalian Medical University, Dalian 116012, China;

Received:2020-06-09 Online:2021-03-08 Published:2021-02-03
Contact: ZHAO Xinjie,CUI Zhenze
Supported by:
National Natural Science Foundation of China(81473726)

摘要/Abstract

摘要：

呼吸道合胞病毒(RSV)容易引发下呼吸道感染,尤其是小儿毛细支气管炎。中药定喘汤用于RSV感染的治疗在中国有着广泛的临床实践基础。由于中药体系的复杂性,其治疗机制和主要药效成分还不够明确。采用代谢组学方法分析中药药效,可以为传统中药治疗提供现代科学论证。该研究对大鼠各干预组分别采用中药定喘汤全方,宣、降、清分解剂以及利巴韦林灌胃治疗,正常组大鼠和RSV模型组大鼠均采用生理盐水灌胃。分别取大鼠在第0、3、7天的眼底血浆进行基于液相色谱-串联质谱法的非靶向代谢组学分析,以研究RSV感染引起的代谢组改变、定喘汤对RSV感染相关代谢物的调控及定喘汤的主要药效来源。对检测的代谢物离子进行多变量分析,建立主成分分析模型,结果显示在RSV感染及治疗进程中,中药定喘汤组与利巴韦林阳性对照组对RSV感染大鼠产生的总体代谢调控效果类似。在3种定喘汤分解剂中,组成成分为麻黄和白果的宣法分解剂对代谢组的调控作用与定喘汤更为接近,可能为定喘汤的主要药效来源。基于配对t检验发现大鼠在感染RSV后有83种代谢物发生了显著性改变,表明RSV感染可造成大鼠多条代谢通路紊乱。第3天时中药定喘汤对包括胆汁酸、氨基酸、有机酸、脂质等在内的代谢物有治疗效果。3种分解剂中,宣法分解剂对代谢物的调节作用与定喘汤类似,而降法分解剂和清法分解剂对大鼠因感染RSV引起的酰基肉碱的异常升高有明显下调作用。各组都有维持肠道菌群和免疫系统稳定的药效。该研究采用代谢组学方法评价定喘汤的药效,发现了定喘汤治疗后改变的代谢物的种类及变化规律,为定喘汤的治疗机理和活性成分的研究提供了理论支持。

关键词: 液相色谱-串联质谱法, 呼吸道合胞病毒, 代谢组学, 中药, 定喘汤

Abstract:

Respiratory syncytial virus (RSV) can cause lower respiratory tract infections, such as bronchiolitis in infants. In China, traditional asthma-relieving medicine has numerous clinical applications in the treatment of RSV infections. However, due to the complexity of the traditional Chinese medicine system, its therapeutic mechanism and main pharmacological components remain unclear. Metabolomics can be used to analyze the efficacy of traditional Chinese medicine to provide modern scientific evidence for such treatments. In this study, an animal model experiment was performed with seven groups of three-week-old rats. The model group and five intervention groups were inoculated nasally with RSV for three consecutive days, and the normal group was treated with the same amount of saline for three consecutive days under the same conditions. In parallel, the five intervention groups were treated separately with the following via intragastric administration for seven consecutive days: asthma-relieving traditional Chinese medicine decoction, its three constituent agents (ascending (xuan) therapy, descending (jiang) therapy, pyretic clearing (qing) therapy), and ribavirin. Both normal group and RSV model group were administered with normal saline via intragastric administration as controls for seven consecutive days. The fundus plasma of rats in each group was collected on day 0, day 3, and day 7. Liquid chromatography-mass spectrometry-based untargeted metabolomics analysis was performed to investigate the changes in the metabolome after RSV infection, the effects of the asthma-relieving decoction on the regulation of metabolites related to RSV infection, and the primary source of efficacy. The detected metabolite ions were corrected using internal standards. Multivariate analysis of ions with an RSD value of less than 30% in quality control (QC) samples was used to construct principal component analysis models to monitor the overall metabolic changes of each group. The results showed that, during RSV infection and treatment, the asthma-relieving decoction and the positive control ribavirin had similar effects on the overall metabolic regulation of RSV-infected rats. Among the three asthma-relieving decoction constituent agents, the ascending (xuan) therapy agents which was composed of ephedra and ginkgo had a closer metabolic regulation effect with asthma-relieving decoction, and might be the main source of pharmacological efficacy. Based on the retention time, m/z value and tandem mass spectra in the database established by our laboratory, a total of 150 metabolites were identified. Paired t-tests were performed using data of the identified metabolites before and after RSV infection in each group, and it was found that 83 metabolite levels significantly changed after RSV infection, indicating that RSV infection could lead to disorders of multiple metabolic pathways in rats. The altered pathways included aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, primary bile acid biosynthesis, phenylalanine metabolism and sphingomyelin metabolism. On the third day, the asthma-relieving decoction had regulatory effects on several metabolites such as bile acids, amino acids, organic acids, lipids, etc. Among the three asthma-relieving decoction constituent agents, the ascending (xuan) therapy agents had more similar effects on the regulation of metabolites with the asthma-relieving decoction. On the other hand, the descending (jiang) therapy agents and pyretic clearing (qing) therapy agents down-regulated the abnormal increase in acylcarnitine caused by the RSV infection. Additionally, both asthma-relieving decoction and its constituent agents could maintain the stability of the immune system and metabolism of the intestinal flora in rats. This study used metabolomics to evaluate the efficacy of an asthma-relieving decoction and demonstrate the metabolites and the corresponding changes after asthma-relieving decoction-based treatment. It provides theoretical support for research on the therapeutic mechanism and active ingredients of asthma-relieving decoction.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), respiratory syncytial virus (RSV), metabolomics, Chinese medicine, asthma-relieving decoction

中图分类号:

O658

欧阳瑒, 迟磊, 徐超, 赵欣捷, 崔振泽. 基于液相色谱-质谱代谢组学方法研究中药定喘汤对呼吸道合胞病毒感染的疗效[J]. 色谱, 2021, 39(3): 281-290.

OUYANG Yang, CHI Lei, XU Chao, ZHAO Xinjie, CUI Zhenze. Liquid chromatography-mass spectrometry-based metabolomics study of the efficacy of Chinese medicine asthma-relieving decoction on respiratory syncytial virus infection[J]. Chinese Journal of Chromatography, 2021, 39(3): 281-290.

图/表 7

图1 大鼠喂养、感染和给药过程图示

Fig. 1 Schematic representation of rat feeding, infection, and dosage method RSV: respiratory syncytial virus.

图2 各组大鼠的平均病毒载量和平均肺指数柱状图

Fig. 2 Histogram of mean viral load and mean lung index of rats in each group a. The mean viral load on day 3; b. The mean viral load on day 7; c. The mean lung index on day 7; The differences between model group and other groups were assessed by independent samples t-test, and metabolites with p<0.05 were marked by stars; Error bars indicate SD values. The letters Z, M, L, D, D-X, D-J, and D-Q represent normal group, RSV model group, ribavirin group, asthma-relieving decoction group, ascending (xuan) therapy group, descending (jiang) therapy group and pyretic clearing (qing) therapy group, respectively.

图3 (a)正离子和(b)负离子检测模式下质量控制(QC)样品的RSD值

Fig. 3 RSDs of quality control (QC) samples in (a) positive and (b) negative ion modes

图4 (a)第0天、(b)第3天和(c)第7天的主成分分析(PCA)得分图

Fig. 4 Score plots of principal component analysis (PCA) of (a) day 0, (b) day 3, and (c) day 7

图5 RSV感染对大鼠代谢通路的影响

Fig. 5 Effects of RSV infections on the metabolic pathways in rats

图6 (a)第3天和(b)第7天时各组中与RSV感染有相同变化趋势的显著性代谢物的个数

Fig. 6 Numbers of significant metabolites in each group that had similar changes with RSV infection on (a) day 3 and (b) day 7

图7 与RSV感染相关的56个代谢物在各组第3天含量的热图

Fig. 7 Heat map of 56 metabolites related to RSV infection in each group on day 3 Numbers “1” and “2” represent metabolites whose levels were significantly up-regulated or down-regulated on day 3 after RSV infection, respectively; The letter “a” indicates abnormal metabolites that were corrected by using both ribavirin and asthma-relieving decoction; “b” indicates abnormal metabolites that were only corrected by using ribavirin; “c” indicates abnormal metabolites that were only corrected by using asthma-relieving decoction; “d” indicates abnormal metabolites that were regulated by using both ribavirin and asthma-relieving decoction. Abbreviation: CA: cholic acid; TCA: taurocholic acid; TUDCA: tauroursodesoxycholic acid; LPC: lysophosphatidylcholine; SM: sphingomyelin; PC: phosphatidylcholine; TCDCA: taurochenodexycholic acid; DCA: deoxycholic acid; UDCA: ursodeoxycholic acid; GUDCA: glycoursodeoxycholic acid; LPE: lysophosphatidylethanolamine; FFA: free fatty acid.

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基于液相色谱-质谱代谢组学方法研究中药定喘汤对呼吸道合胞病毒感染的疗效

Liquid chromatography-mass spectrometry-based metabolomics study of the efficacy of Chinese medicine asthma-relieving decoction on respiratory syncytial virus infection

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