Mechanism of Mahuang Xixin Fuzitang Against Migraine Based on Network Pharmacology and Experimental Validation

GE Fei; ZHANG Yao; HOU Jianchen; LUO Yamin; DONG Ruijuan; GE Dongyu; MENG Fengxian; TAO Xiaohua

doi:10.13422/j.cnki.syfjx.20220917

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Mechanism of Mahuang Xixin Fuzitang Against Migraine Based on Network Pharmacology and Experimental Validation

更新时间：2022-10-18

- Mechanism of Mahuang Xixin Fuzitang Against Migraine Based on Network Pharmacology and Experimental Validation
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 22, Pages: 106-115(2022)
- 作者机构：
  
  1.北京中医药大学中医学院，北京 100029
  2.北京中医药大学生命科学学院，北京 102488
  3.北京中医药大学东方医院，北京 100087
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220917
  CLC： R285;R289;R22;R2-031;R33
- Received：02 January 2022，
  
  Published Online：10 June 2022，
  
  Published：20 November 2022
- 稿件说明：
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葛飞,张瑶,侯鉴宸等.基于网络药理学及大鼠体内实验的方法探讨麻黄细辛附子汤干预偏头痛的作用机制[J].中国实验方剂学杂志,2022,28(22):106-115.

GE Fei,ZHANG Yao,HOU Jianchen,et al.Mechanism of Mahuang Xixin Fuzitang Against Migraine Based on Network Pharmacology and Experimental Validation[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(22):106-115.
葛飞,张瑶,侯鉴宸等.基于网络药理学及大鼠体内实验的方法探讨麻黄细辛附子汤干预偏头痛的作用机制[J].中国实验方剂学杂志,2022,28(22):106-115. DOI： 10.13422/j.cnki.syfjx.20220917.

GE Fei,ZHANG Yao,HOU Jianchen,et al.Mechanism of Mahuang Xixin Fuzitang Against Migraine Based on Network Pharmacology and Experimental Validation[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(22):106-115. DOI： 10.13422/j.cnki.syfjx.20220917.

摘要

目的

研究麻黄细辛附子汤（MXFT）干预偏头痛的作用机制。

方法

通过中药系统药理学数据库与分析平台（TCMSP）、SiwssTargetPrediction等数据库筛选MXFT活性成分及活性成分、偏头痛的相关靶点；运用STRING 11.5平台对药物和疾病的交集靶点建立潜在蛋白质-蛋白质相互作用（PPI）网络图；采用Metascape数据库对潜在交集靶点进行基因本体（GO）分析及京都基因与基因组百科全书（KEGG）富集分析；利用Cytoscape 3.7.1做MXFT成分-靶点-通路网络图，筛选度值较高的核心靶点；最后通过分子对接方法验证核心靶点与其映射成分的结合强度，将对接结果较好的核心靶点进行动物体内实验验证。选用SD大鼠48只，除空白组外，其余大鼠皮下注射硝酸甘油制备偏头痛大鼠模型，成模大鼠随机分为模型组、西药组、MXFT高、中、低剂量组。西药组给予佐米曲普坦片，治疗组给予MXFT高、中、低剂量干预。成模后隔日1次观测各组大鼠行为学及疼痛阈值变化。酶联免疫吸附测定试验（ELISA）检测血浆中降钙素基因相关肽（CGRP）、大鼠细胞外信号调节激酶2（ERK2）、c-fos原癌基因蛋白（FOS）水平。免疫组化技术、蛋白免疫印迹法（Western blot）检测SD大鼠三叉神经细胞外信号调节蛋白激酶1/2（ERK1/2，又称MAPK1/3）、蛋白激酶B1（Akt1）、蛋白激酶Cα（PRKCA）水平。

结果

网络药理学分析显示，MXFT治疗偏头痛的核心靶点为MAPK1、MAPK3、Akt1、PRKCA等；主要通路为神经活性配体/受体相互作用信号通路、钙离子信号通路、MAPK信号通路等；分子对接结果表明，MAPK1、MAPK3、Akt1、PRKCA、PRKCB、PRKCG与其映射成分具有较好的结合能力。动物实验结果显示，与空白组比较，模型组大鼠挠头次数显著增加（

0.01），疼痛阈值显著降低（

0.01）；与模型组比较，各给药组大鼠挠头次数显著减少（

0.01），疼痛阈值显著提高（

0.01）。与空白组比较，模型组大鼠血浆中CGRP、ERK2、FOS蛋白水平明显增高（

0.05，

0.01），三叉神经节中Akt1、ERK1/2、PRKCA蛋白水平明显增高（

0.05，

0.01）。与模型组比较，各给药组血浆中CGRP、ERK2、FOS蛋白水平，三叉神经节中Akt1、ERK1/2、PRKCA蛋白水平明显降低（

0.05，

0.01）。

结论

MXFT抗偏头痛具有多成分-多靶点-多通路的特点，其作用机制可能与抑制血管扩张、减少炎症因子释放、抑制神经元过度活跃等多途径发挥作用。

Abstract

Objective

To study the mechanism of Mahuang Xixin Fuzitang （MXFT） against migraine.

Method

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， SiwssTargetPrediction and other databases were used to screen the active components and action targets of MXFT as well as migraine-related targets. The potential protein-protein interaction （PPI） network diagram was plotted for the intersection targets of MXFT and migraine using STRING 11.5. Metascape was used for Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis of potential intersection targets. The component-target-pathway network of MXFT was constructed by Cytoscape 3.7.1 to screen core targets with high degree value. Finally， the binding strength between core target and its mapping components was verified by molecular docking， and the core targets with desirable docking results were verified by animal experiments

in vivo

. Forty eight SD rats were selected， and except the blank group， the other rats were subcutaneously injected with nitroglycerin to prepare the migraine rat model. The modeled rats were randomly divided into model group， positive drug group and MXFT high-， medium- and low-dose groups. The positive drug group was given zolmitriptan tablets， and the MXFT high-， medium- and low-dose groups were given high， medium and low doses of MXFT， respectively. The changes of behavior and pain threshold of rats in each group were observed every other day after modeling. The levels of calcitonin gene-related peptide （CGRP）， extracellular signal-regulated kinase 2 （ERK2） and c-fos proto-oncogene （FOS） protein in plasma were detected by enzyme-linked immunosorbent assay （ELISA）. Immunohistochemical technique and Western blot were employed to determine the levels of extracellular signal-regulated kinase 1/2 （ERK1/2， also known as MAPK1/3） and protein kinase B 1 （Akt1）， protein kinase C

（PRKCA） in trigeminal nerve of SD rats.

Result

The network pharmacology showed that the core targets of MXFT in the treatment of migraine were MAPK1， MAPK3， Akt1， PRKCA， etc.， mainly involving neuroactive ligand-receptor interaction signaling pathway， calcium signaling pathway， MAPK signaling pathway， etc. The molecular docking demonstrated that MAPK1， MAPK3， Akt1， PRKCA， PRKCB and PRKCG had good binding ability with their mapping components. The animal experiments indicated that compared with the conditions in the blank group， the number of head scratching in the model group was increased （

0.01）， and the pain threshold was decreased （

0.01）. Compared with the conditions in the model group， the number of head scratching in each administration group was reduced （

0.01）， and the pain threshold was increased （

0.01）. In addition， the levels of CGRP， ERK2 and FOS proteins in plasma， and Akt1， ERK1/2 and PRKCA proteins in trigeminal ganglion of the model group were higher than those of the blank group （

0.05，

0.01）. The levels of CGRP， ERK2 and FOS proteins in plasma and Akt1， ERK1/2 and PRKCA proteins in trigeminal ganglion of each administration group were lower than those of the model group （

0.05，

0.01）.

Conclusion

MXFT had multi-component， multi-target and multi-pathway characteristics in the treatment of migraine， and the mechanism might be related to inhibiting vasodilation， reducing the release of inflammatory factors and inhibiting neuronal hyperactivity.

关键词

Keywords

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