Network pharmacology study of Cassiae Semen in treatment of hyperlipidemia and experimental validation in zebrafish

ZHOU Chang; PENG Longping; DONG Yidan; MA Mengjiao; ZHOU Maolin; LI Qiang; WANG Youhua

doi:10.16306/j.1008-861x.2024.04.010

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Network pharmacology study of Cassiae Semen in treatment of hyperlipidemia and experimental validation in zebrafish

Experiment Research | 更新时间：2024-08-13

- Network pharmacology study of Cassiae Semen in treatment of hyperlipidemia and experimental validation in zebrafish
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 38, Issue 4, Pages: 71-80(2024)
- 作者机构：
  
  1.上海中医药大学附属龙华医院（上海　200032）
  2.复旦大学附属儿科医院（上海　201102）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2024.04.010
  CLC：
- Published：25 July 2024，
  
  Received：15 March 2023，
  
  Revised：22 May 2023，
- 稿件说明：
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周嫦,彭珑萍,董艺丹等.决明子治疗高脂血症的网络药理学研究及斑马鱼实验验证[J].上海中医药大学学报,2024,38(04):71-80.

ZHOU Chang,PENG Longping,DONG Yidan,et al.Network pharmacology study of Cassiae Semen in treatment of hyperlipidemia and experimental validation in zebrafish[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(04):71-80.
周嫦,彭珑萍,董艺丹等.决明子治疗高脂血症的网络药理学研究及斑马鱼实验验证[J].上海中医药大学学报,2024,38(04):71-80. DOI： 10.16306/j.1008-861x.2024.04.010.

ZHOU Chang,PENG Longping,DONG Yidan,et al.Network pharmacology study of Cassiae Semen in treatment of hyperlipidemia and experimental validation in zebrafish[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(04):71-80. DOI： 10.16306/j.1008-861x.2024.04.010.

摘要

目的

基于网络药理学探讨决明子治疗高脂血症的作用机制，并通过斑马鱼实验进行验证。

方法

在中药分子机制生物信息学注释数据库（BATMAN-TCM）和中药系统药理学分析平台（TCMSP）检索并筛选决明子活性成分及相关靶点，通过GeneCards、DisGeNET、在线人类孟德尔遗传数据系统（OMIM）检索得到与高脂血症有关的靶点，利用Venny 2.1.0平台获得药物与疾病的共同靶点。使用Cytoscape3.8.2绘制决明子-活性成分-作用靶点网络图，采用String数据库构建蛋白质-蛋白质相互作用（PPI）网络图，将决明子-高脂血症共同靶点通过注释、可视化和集成发现数据库（DAVID）进行基因本体（GO）功能及京都基因与基因组百科全书（KEGG）通路富集分析。根据网络药理学研究结果，推测橙黄决明素（AO）为决明子降血脂的有效作用成分，在此基础上进行斑马鱼实验验证。以蛋黄液高脂饮食诱导斑马鱼高脂血症模型，造模后给予AO干预，验证AO治疗高脂血症的效果及作用机制。首先进行AO对高脂血症斑马鱼的毒性实验，确定AO最大给药浓度；后续观察AO对高脂血症斑马鱼总胆固醇（TC）、三酰甘油（TG）含量及肝脏组织形态学的影响；采用实时荧光定量PCR检测核心靶点mRNA在高脂血症斑马鱼中的表达。

结果

共获得包括AO在内的13个决明子活性成分，决明子作用于高脂血症的潜在靶点109个，包括核心靶点肿瘤坏死因子（TNF）、白介素（IL）-1β、前列腺素内过氧化物合酶2（PTGS2）、半胱氨酸天冬氨酸蛋白酶3（CASP3）、过氧化物酶体增殖物激活受体-γ（PPAR-γ）等，主要涉及IL-17信号通路、TNF信号通路、脂质和动脉粥样硬化相关作用通路等。斑马鱼验证实验结果显示：造模后斑马鱼出现明显的脂质聚积，AO可显著降低高脂血症斑马鱼组织中的TG、TC含量（

0.05，

0.01），减少肝脏组织内脂肪空泡和脂滴形成，并可显著下调核心靶点

TNF

、

1β

mRNA表达（

0.01）。

结论

基于网络药理学获得了决明子治疗高脂血症的核心通路与靶点，并通过斑马鱼实验初步揭示AO对高脂血症的改善效果，其机制可能是通过TNF-α、IL-1β

等核心靶点发挥治疗作用，旨在为后期研究AO在高脂血症中的临床应用提供参考依据。

Abstract

Objective： To investigate the mechanisms of Cassiae Semen in treating hyperlipidemia based on network pharmacology and be validated through zebrafish experiments.

Methods

The active ingredients and related targets of Cassiae Semen were searched and screened in the Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine （BATMAN-TCM） and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Hyperlipidemia-related target genes were obtained through searching GeneCards， DisGeNET and Online Mendelian Inheritance in man （OMIM）. The common targets of drugs and diseases were obtained by Venny 2.1.0 platform. The Cassiae Semen-active ingredients-potential target network diagram was drawn by Cytoscape3.8.2， the protein-protein interaction （PPI） network diagram was constructed by String database， and the Cassiae Semen-hyperlipidemia common targets were subjected to Gene Ontology （GO） enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis through

the Database for Annotation， Visualization and Integrated Discovery （DAVID）. According to the results of the network pharmacology study， aurantio-obtusin

（AO） was hypothesized to be the effective acting component of Cassiae Semen in lowering blood lipids， and then the zebrafish experiments were carried out to verify based on above. The zebrafish hyperlipidemia model was induced by high-fat diet with egg yolk liquid， and was treated by AO after modeling， in order to verify the effect and mechanisms of AO in treating hyperlipidemia. Firstly， the toxicity experiment of AO on hyperlipidemic zebrafish was performed to determine the maximum administration concentration of AO. Subsequently， the effects of AO on total cholesterol （TC） and triglyceride （TG） contents and liver histomorphometry of hyperlipidemic zebrafish were observed. Quantitative real-time PCR was used to detect the expressions of core target mRNAs in hyperlipidemic zebrafish.

Results

Thirteen active components of Cassiae Semen including AO were obtained. A total of 109 potential targets of Cassiae Semen acting on hyperlipidemia were obtained， including the core targets tumor necrosis factor （TNF）， interleukin （IL）-1β， prostaglandin-endoperoxide synthase 2 （PTGS2）， Caspase 3 （CASP3）， peroxisome proliferator activated receptor-γ （PPAR-γ）， etc.， which were mainly involved in IL-17 signaling pathway， TNF signaling pathway， as well as lipid and atherosclerosis-related pathways. The results of zebrafish validation experiments showed that， the significant lipid accumulation in zebrafish after modeling occurred， and AO could significantly reduce the TG and TC contents in hyperlipidemic zebrafish tissue （

0.05，

0.01）， reduce the fat vacuole and lipid droplet in liver， and significantly down-regulate the expressions of core targets

TNF

and

1β

mRNA （

0.01）.

Conclusion

Based on network pharmacology， the core pathways and targets of Cassiae Semen in the treatment of hyperlipidemia are obtained， and the improvement effect of AO on hyperlipidemia is preliminarily revealed by zebrafish experiments， it may play the therapeutic effect through the core targets such as TNF-α， IL-1β， etc.， in order to provide a reference for the later study of the clinical application of AO in hyperlipidemia.

关键词

高脂血症决明子橙黄决明素网络药理学斑马鱼

Keywords

hyperlipidemiaCassiae Semenaurantio-obtusinnetwork pharmacologyzebrafish

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