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Systematic Study on Molecular Mechanism of Aconitum Alkaloid in Treatment of Rheumatoid Arthritis
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 5, Pages: 171-181(2021)
- 作者机构：
内蒙古医科大学 药学院，内蒙古医科大学 新药安全评价研究中心，内蒙古自治区 新药筛选工程研究中心，呼和浩特 010110
- 作者简介：
- 基金信息：
DOI：10.13422/j.cnki.syfjx.20210101
CLC： R2-0;R22;R285.5;R289Published：05 March 2021，
Published Online：06 January 2021，
Received：13 October 2020，
- 稿件说明：
扫 描 看 全 文
刘婷婷,肖志彬,陈琪等.基于系统药理学探讨乌头生物碱治疗类风湿性关节炎的分子机制[J].中国实验方剂学杂志,2021,27(05):171-181.
LIU Ting-ting,XIAO Zhi-bin,CHEN Qi,et al.Systematic Study on Molecular Mechanism of Aconitum Alkaloid in Treatment of Rheumatoid Arthritis[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(05):171-181.
刘婷婷,肖志彬,陈琪等.基于系统药理学探讨乌头生物碱治疗类风湿性关节炎的分子机制[J].中国实验方剂学杂志,2021,27(05):171-181. DOI： 10.13422/j.cnki.syfjx.20210101.
LIU Ting-ting,XIAO Zhi-bin,CHEN Qi,et al.Systematic Study on Molecular Mechanism of Aconitum Alkaloid in Treatment of Rheumatoid Arthritis[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(05):171-181. DOI： 10.13422/j.cnki.syfjx.20210101.
摘要
目的
2
基于系统药理学探讨乌头生物碱中化学成分对脂多糖（LPS）诱导的RAW264.7巨噬细胞炎症反应的影响及其机制。
方法
2
TCMSP数据库结合口服生物利用度（OB）≥30%，类药性（DL）≥0.18搜集草乌化学成分，通过Pubchem数据库以及Swiss Target Prediction数据库预测各化学成分的潜在作用靶点；从GeneCards数据库搜集类风湿性关节炎（RA）靶点，两者取交集，DAVID数据库进行基因本体（GO）分类富集分析和Pathway富集分析。Cytoscape构建“化学成分-潜在作用靶点-信号通路-疾病”网络拓扑图；结合STRING数据库和Cytoscape软件构建蛋白质-蛋白质相互作用（PPI）网络。体外实验通过LPS刺激RAW264.7巨噬细胞，建立体外巨噬细胞炎症模型，采用蛋白免疫印迹法检测乌头生物碱中化学成分对LPS诱导的RAW264.7细胞肿瘤坏死因子-
α
（TNF-
α
），环氧化酶（COX-2）蛋白表达的影响以及对JAK激酶（Janus kinase），核转录因子-
κ
B（NF-
κ
B）信号通路主要蛋白表达的影响。
结果
2
通过TCMSP数据库检索以及查阅文献共得到27个化学成分，经筛选后（OB≥30%，DL≥0.18）得到12个化学成分，通过数据库预测并筛选后得到177个潜在作用靶点，与4 329个RA靶点取交集后得到97个靶点作为治疗RA的潜在作用靶点。通过DAVID数据库富集到生物过程（BP）经筛选后共32条（
P
<
0.01，FDR
<
0.01）；所处细胞中的位置（CC）经筛选后共有5个位置（
P
<
0.01，FDR
<
0.01）；分子功能（MF）经筛选后得到12种分子功能（
P
<
0.01，FDR
<
0.01）。通过构建网络拓扑图说明乌头生物碱在治疗RA时不同的化学成分可作用于相同靶点，同一化学成分也作用于不同的靶点；乌头生物碱既可通过不同靶点与同一通路相连接，也可通过同一靶点与不同通路相连接，说明不同的靶点具有协同作用，充分体现了乌头生物碱多成分、多靶点、多通路的复杂机制。加入不同浓度的LPS刺激（质量浓度为0~200 μg·L
-1
）能够显著上调RAW264.7巨噬细胞COX-2蛋白表达水平（
P
<
0.05），说明炎症模型成功。与正常组比较，RAW264.7细胞炎症模型中TNF-
α
，COX-2蛋白表达显著增加（
P
<
0.05）；与模型组比较，BLA，songorine，yunaconitine，karacoline各组TNF-
α
，COX-2蛋白表达水平不同程度降低（
P
<
0.05）。与正常组比较，RAW264.7细胞炎症模型中白细胞介素-1受体相关激酶4（IRAK-4），NF-
κ
B，JAK1，信号转导与转录激活因子3（STAT3）水平显著增加（
P
<
0.05）；与模型组比较，BLA，songorine，yunaconitine，karacoline各组IRAK4，NF-
κ
B，JAK1，STAT3水平显著降低（
P
<
0.05）。
结论
2
基于系统药理学和体外实验对相关靶点和信号通路进行分析，为了解RA的发病机制，揭示乌头生物碱治疗RA的分子机制提供新见解，也为蒙药在现代医学中的应用提供新思路。
Abstract
Objective
2
To explore the effect of chemical compound of aconitum alkaloid on the lipopolysaccharide （LPS）-induced inflammatory response of RAW264.7 macrophages and investigate its mechanism.
Method
2
The chemical compounds of Aconitum Kusnezoffii Reichb were collected from TCMSP database with consideration of oral bioavailability （OB）≥30% and drug-likeness （DL）≥0.18. The potential targets of each chemical component were predicted with use of Pubchem database and Swiss Target Prediction database. Rheumatoid arthritis （RA） targets were collected from GeneCards database and selected by intersection screening. Gene ontology （GO） classification enrichment and Pathway enrichment analysis were carried out with use of DAVID database. Cytoscape was used to construct "Chemical Compound-Potential Targets-Pathway-Disease" network. Protein-protein interaction （PPI） network was constructed by using STRING database and Cytoscape software. RAW264.7 macrophages were stimulated by LPS to establish macrophage inflammation model
in vitro
. Western blot was used to detect the effects of chemical compounds on the expression of tumor necrosis factor-
α
（TNF-
α
） and cyclooxygenase-2 （COX-2） in RAW264.7 cells induced by LPS， as well as on the expression of JAK kinase and nuclear transcription factor- kappa B （NF-
κ
B） signal pathway.
Result
2
A total of 27 chemical compounds were obtained by searching TCMSP database and consulting literature （OB≥30%， DL≥0.18）. 12 chemical compounds were obtained after screening. 177 potential targets were obtained after database prediction and screening， and 97 targets were obtained as potential targets for the treatment of RA after intersection between 177 potential targets and 4 329 RA targets. A total of 32 biological processes （BP）， 5 cellular components （CC）， and 12 molecular functions （MF） were enriched by DAVID database. The construction of network topology map showed that different chemical compounds can act on the same target and the same chemical compound can also act on different targets in the treatment of RA. Aconitum alkaloid can be connected with the same pathway through different targets or with different pathways through the same target， indicating that different targets may have synergistic effect， which fully reflected the complex multi-compound， multi-targets and multi-pathways mechanism. Different concentrations of LPS in stimulation （0-200 μg·L
-1
） can significantly up-regulate the expression of COX-2 protein in RAW264.7 macrophages （
P
<
0.05）， indicating that the inflammatory model was successful. Compared with the normal group， the expression of TNF-
α
and COX-2 protein in the inflammatory model of RAW264.7 cells increased significantly（
P
<
0.05）， while the expression of TNF-
α
and COX-2 protein in bulleyaconitine A（BLA）， songorine， yunaconitine and karacoline groups decreased in varying degrees compared with the model group （
P
<
0.05）. Compared with the normal group， the expression of IRAK4， NF-
κ
B， JAK1 and STAT3 in the inflammatory model of RAW264.7 cells were significantly increased （
P
<
0.05）， while such levels in BulleyaconitineA， songorine， yunaconitine and Karacoline groups were significantly lower than those in the model group（
P
<
0.05）.
Conclusion
2
Based on systematic pharmacology and
in vitro
experiments， the related targets and signal pathways were analyzed to provide new insights into the pathogenesis of RA， reveal the molecular mechanism of aconitum alkaloid in the treatment of RA， and provide new ideas for the application of Mongolian medicine in modern medicine.
关键词
乌头生物碱脂多糖类风湿性关节炎炎症因子系统药理学分子机制
Keywords
aconitum alkaloidlipopolysacchariderheumatoid arthritisinflammatory factorssystematic pharmacologymolecular mechanism
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