Muscone Protects Against Ferroptosis-Induced Injury in Models of Acute Ischemic Stroke by Modulating Snap25 Protein.

Background: Acute ischemic stroke (AIS) is one of the leading critical neurological conditions globally, resulting in significant adult mortality and disability. Previous studies have demonstrated a close relationship between AIS and the ferroptosis signaling pathway. Muscone, the primary active small-molecule component of musk, is a traditional Chinese medicine that exhibits significant pharmacological effects in reducing stroke injury. However, there is still only limited research on whether muscone can modulate ferroptosis-related injury in AIS, and on the underlying regulatory molecular mechanisms.

Methods: We utilized a transmission electron microscope and concurrently performed assays for glutathione peroxidase 4 (GPX4) activity, glutathione (GSH), reactive oxygen species (ROS), lipid peroxides, as well as cell viability and live/dead cell staining to investigate alterations in ferroptosis levels. RNA sequencing, bioinformatics analysis, and western blot (WB) assays were employed to evaluate the changes in synaptosome-associated protein 25 kDa (Snap25) expression levels. Furthermore, molecular docking, surface plasmon resonance (SPR) detection, and molecular dynamics (MD) simulation were implemented to examine the binding affinity and interaction between muscone and Snap25.

Results: RNA sequencing technology, bioinformatics analysis, and WB assays revealed that Snap25 was specifically downregulated under simulated AIS conditions. Snap25 knockdown and overexpression experiments were also conducted to elucidate the molecular mechanism by which muscone modulates Snap25 expression, thereby mitigating ferroptosis injury in AIS. Additionally, the results of molecular docking, SPR detection, and MD simulations indicate that muscone has multiple binding sites that allow it to bind directly to the Snap25 protein, thereby stabilizing the protein structure.

Conclusions: Our findings suggest that muscone produces an anti-AIS effect in the context of AIS injury by increasing Snap25 protein expression, thus reducing ferroptosis. This investigation offers insight into the anti-stroke mechanism of muscone and introduces a promising new treatment option for clinical AIS management.