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

Abstract

Ethnopharmacological relevanceMusk 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 studyWe 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 methodsThe 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. ResultsNetwork 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. ConclusionsMuscone 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.