Proteomics on the role of muscone in the “consciousness-restoring resuscitation” effect of musk on ischemic stroke,Journal of Ethnopharmacology

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Proteomics on the role of muscone in the “consciousness-restoring resuscitation” effect of musk on ischemic stroke
Journal of Ethnopharmacology ( IF 4.8 ) Pub Date : 2022-06-16 , DOI: 10.1016/j.jep.2022.115475
Bingbing Han ₁ , Yangang Zhao ₂ , Jing Yao ₃ , Na Li ₃ , Tianhe Fang ₁ , Yuan Wang ₁ , Zhaoqing Meng ₄ , Wei Liu ₃

Affiliation

Ethnopharmacological relevance

Musk is a representative drug of aroma-relieving traditional Chinese medicine, and it is a commonly used traditional Chinese medicine for the treatment of ischemic stroke. Muscone is the core medicinal component of musk.

Aim of the study

We sought to identify the target of muscone in the treatment of ischemic stroke using network pharmacology, an animal model of ischemic stroke, and differential proteomics.

Materials and methods

The drug targets of muscone in the treatment of ischemic stroke were predicted and analyzed using information derived from sources such as the Traditional Chinese Medicine Systems Pharmacology database and Swiss Target Prediction tool. The animal model of focal cerebral ischemia was established by suture-based occlusion of the middle cerebral artery of rats. The rats were divided into six groups: sham-operated control, model, musk, muscone1, muscone2, and muscone3. Neurological deficit scores were calculated after intragastric administration of musk or muscone. The microcirculation blood flow of the pia mater was detected using a laser speckle blood flow meter. The cerebral infarction rate was detected by 2,3,5-triphenyltetrazolium chloride staining. The necrosis rate of the cerebral cortex and the hippocampal neurons was detected by hematoxylin and eosin staining. Blood-brain barrier damage was detected by the Evans blue method. Quantitative proteomics analysis in the sham-operated control, model, and muscone groups was performed using tandem-mass-tags. Considering fold changes exceeding 1.2 as differential protein expression, the quantitative values were compared among groups by analysis of variance. Furthermore, a protein-protein interaction network was constructed, and differentially expressed proteins were analyzed by gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Results

Network pharmacology identified 339 targets for the intersection of 17 components of musk and cerebral ischemia-reperfusion injury. The GO and KEGG enrichment items mainly identified regulation of neuronal synaptic structure and transfer function, synaptic neurotransmitters, and receptor activity. Zoopery showed that the model group had a higher behavioral score, cerebral infarction rate, cortical and hippocampal neuron death rate, Evans blue exudation in the brain, and bilateral pia mater microcirculation blood flow differences than the sham-operated control group (P <0.01). Compared with the model group, the behavioral score, infarction rate, hippocampal neuronal mortality, and Evans blue content decreased significantly in the musk, muscone2, and muscone3 groups (P <0.05). Proteomic analysis showed that 160 genes were differentially expressed among the sham-operated control, model, and muscone groups. GO items with high enrichment included neuronal synapses, postsynaptic signal transduction, etc. KEGG items with high enrichment included cholinergic synapses, calcium signaling pathway, dopaminergic synapses, etc. Protein interaction analysis revealed that the top three protein pairs were Ndufa10/Ndufa6, Kcna2/Kcnab2, and Gsk3b/Traf6.

Conclusions

Muscone can reduce neuronal necrosis, protect the blood-brain barrier, and improve the neurological damage caused by cerebral ischemia via molecular mechanisms mainly involving the regulation of neuronal synaptic connections. Muscone is an important active component responsible for the “consciousness-restoring resuscitation” effect of musk on ischemic stroke.

中文翻译：

蛋白质组学研究麝香酮在麝香对缺血性脑卒中“意识恢复复苏”作用中的作用

民族药理学相关性

麝香是中药解香的代表药，是治疗缺血性中风的常用中药。麝香酮是麝香的核心药用成分。

研究目的

我们试图使用网络药理学、缺血性中风动物模型和差异蛋白质组学来确定麝香酮治疗缺血性中风的靶点。

材料和方法

使用来自传统中药系统药理学数据库和瑞士靶点预测工具等来源的信息预测和分析麝香酮治疗缺血性卒中的药物靶点。采用缝合法封闭大鼠大脑中动脉，建立局灶性脑缺血动物模型。将大鼠分为6组：假手术对照组、模型组、麝香组、muscone1组、muscone2组和muscone3组。在麝香或麝香酮灌胃后计算神经功能缺损评分。使用激光散斑血流仪检测软脑膜微循环血流。脑梗塞2,3,5-三苯基四唑氯化物染色检测速率。苏木精伊红染色检测大脑皮层和海马神经元的坏死率。埃文斯蓝法检测血脑屏障损伤。使用串联质量标签对假手术对照组、模型组和麝香组进行定量蛋白质组学分析。考虑倍数变化超过1.2作为差异蛋白表达，通过方差分析比较组间的定量值。此外，构建了蛋白质-蛋白质相互作用网络，并通过基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析对差异表达的蛋白质进行了分析。

结果

网络药理学确定了麝香 17 种成分与脑缺血再灌注损伤相交的 339 个靶点。GO和KEGG富集项目主要识别神经元突触结构和传递功能、突触神经递质和受体活性的调节。动物实验显示，模型组的行为评分、脑梗死率、皮质和海马神经元死亡率、脑内埃文斯蓝渗出、双侧软脑膜微循环血流差异均高于假手术对照组（P<0.01） . 与模型组相比，麝香、麝香2、麝香3组的行为评分、脑梗死率、海马神经元死亡率、埃文斯蓝含量均显着降低（P<0.05）。蛋白质组学分析表明，160 个基因在假手术对照组、模型组和麝香组之间有差异表达。高富集的GO项目包括神经元突触、突触后信号转导等。高富集的KEGG项目包括胆碱能突触、钙信号通路、多巴胺能突触等。蛋白质相互作用分析显示，前三位的蛋白质对分别是Ndufa10/Ndufa6、Kcna2/Kcnab2和Gsk3b/Traf6。