Proteomics analysis of brain tissues of cerebral ischemic rat treated with Xingnaojing injection and its brain component muscone

Cerebral ischemia-reperfusion injury is categorized as "stroke" in traditional Chinese medicine. For thousands of years, traditional Chinese medicine has accumulated rich experience in the treatment of stroke and Other Diseases, and with remarkable curative effects. Currently, Xingnaojing injection and its component musk are commonly used in the treatment of acute stroke, and muscone is the main active ingredient of musk. In this study, a rat model of transient middle cerebral artery occlusion was established, and the neuroprotective effects of Xingnaojing and muscone on transient middle cerebral artery occlusion rats were validated by Zea-Longa neurological function score, behavioral test and 2,3,5-triphenyltetrazolium chloride staining. Quantitative proteomics analysis was then performed on the brain tissues from different groups to investigate the mechanisms by which Xingnaojing and muscone act on cerebral ischemia-reperfusion injury. Our data indicate that Xingnaojing and muscone significantly affect proteins related to Oxidative Phosphorylation in CIRI rats, highlighting mitochondrial energy metabolism as a potentially important pathway contributing to their neuroprotective effects. Furthermore, the limited proteolysis-coupled mass spectrometry, target-responsive accessibility profiling, and lysine reactivity profiling methods were used to identify the direct protein targets of muscone in rat brain tissue lysate. A total of 36 potential target proteins were commonly identified by all the three methods. Bioinformatics analysis suggested that muscone was more significantly enriched in glycolysis/gluconeogenesis related pathways and closely associated with Oxidative Phosphorylation. Finally, the glycolytic key enzyme phosphoglycerate kinase 1, one of the binding proteins with muscone, was selected and verified by drug affinity responsive target stability. The molecular docking and dynamics simulation analysis further confirmed the interaction of glycolytic key enzyme phosphoglycerate kinase 1 and muscone. This study provides evidences for the clinical application and mechanisms of Xingnaojing and muscone in treating cerebral ischemia-reperfusion injury, and identifies candidate protein targets of muscone.

Cerebral ischemia-reperfusion injury; Muscone; Proteomics; Target protein; Xingnaojing injection.