Muscone ameliorates cerebral ischemia-reperfusion-induced BBB injury through PKA/RHOA/MLC pathway

Cerebral ischemia-reperfusion (CIR) injury disrupts the blood-brain barrier (BBB), leading to exacerbated brain damage. Muscone, the main active component of musk, has been reported to exert neuroprotective effects, but its mechanism in protecting BBB integrity remains unclear. In a mouse model of middle cerebral artery occlusion and reperfusion, muscone treatment significantly reduced Zea Longa scores, cerebral infarct volume, and increased the proportion of normal neurons. Laser speckle contrast imaging and small animal super-resolution ultrasound imaging showed that muscone promoted blood flow restoration in the ischemic hemisphere. Muscone also inhibited apoptosis of brain microvascular endothelial cells (BMECs), as evidenced by a decreased proportion of TUNEL⁺/CD31⁺ cells, reduced expression of pro-apoptotic proteins BAX and Cleaved-Caspase-3, and increased expression of anti-apoptotic protein Bcl-2. Furthermore, muscone attenuated the degradation of tight junction proteins (ZO-1, Occludin, Claudin-5) and reduced Evans blue leakage, indicating preserved BBB integrity. Mechanistically, muscone increased the phosphorylation of PKA and RHOA, while decreasing p-MLC expression in the ischemic hemisphere. And the PKA inhibitor H-89 reduced the protective effects of muscone on BMEC apoptosis, tight junction degradation, and Evans blue leakage, ultimately leading to increased Zea Longa scores, infarct volume, and neuronal damage. These findings demonstrate that muscone may exert a protective effect against CIR-induced BBB injury by inhibiting BMEC apoptosis and tight junction degradation through activation of the PKA/RHOA/MLC pathway. This study provides new insights into the mechanism of muscone and supports its potential application in ischemic stroke therapy.