Dl-3-n-butylphthalide reduces urokinase-induced hemorrhagic transformation after stroke by targeting NET-mediated microglial activation.

Urokinase (UK) administration is recommended for patients with acute ischemic stroke (AIS) within 6 h of onset; but its use beyond this time window may increase the risk of hemorrhagic transformation (HT). Clinical studies have shown that the administration of dl-3-n-butylphthalide (NBP) combined with UK improves AIS neurological deficits, but the potential mechanisms remain unclear. In the present study, in vivo experiments revealed that a combination of NBP (2.5 mg/kg) and UK (100,000 U/kg) extended the UK therapeutic time window to 9 h in embolic middle cerebral artery occlusion (eMCAO)-operated rats. This effect may be attributed to NBP-mediated inhibition of blood-brain barrier damage, microglial polarization and neutrophil extracellular trap (NET) formation, which suppresses UK-induced HT. In vitro, NBP (10, 100 μM) inhibited UK-induced (2000 U/mL) NET formation by neutrophils derived from eMCAO-treated rats. In addition, the effect UK-treated conditioned medium of neutrophils on promoting the microglia to transform into M1 phenotype was alleviated by NBP after oxygen glucose deprivation (OGD). In summary, our research demonstrates that NBP administration prolongs the therapeutic time window for UK and mitigates the UK-induced HT after stroke by targeting NET-mediated microglial activation. These findings provide strong pharmacological evidence for their therapeutic combination.