Adjuvant therapy with total salvianolic acid injection enhances the safety and efficacy of recombinant prourokinase in ischemic stroke by mitigating blood–brain barrier disruption

Background: Late-phase thrombolysis within the 4.5-hour window for acute ischemic stroke exacerbates blood-brain barrier (BBB) disruption, increasing the risk of hemorrhagic transformation. Recombinant human prourokinase (rhPro-UK) is an emerging thrombolytic agent, but its impact on BBB integrity compared to alteplase (recombinant tissue plasminogen activator, rt-PA) and its potential additive effect with adjuvant neuroprotectants like Total Salvianolic Acid Injection (TSI) remain unclear. Methods: A mouse thromboembolic stroke model was established, and treatments were administered intravenously 4.5 h post-occlusion. Mice were randomized into groups including sham, thrombus, rt-PA, rhPro-UK, TSI, and rhPro-UK + TSI. Cerebral blood flow, thrombus area, infarct volume, neurological scores, and BBB integrity (Evans blue leakage, cerebral edema, hemorrhage) were assessed. In vitro, human brain microvascular endothelial cells (HBMECs) underwent oxygen-glucose deprivation/reoxygenation (OGD/R) with or without rhPro-UK and TSI. Transendothelial electrical resistance, permeability, junctional proteins, matrix metalloproteinase (MMP)-2/9 activity, and oxidative stress markers were evaluated. Data were analyzed by ANOVA (Analysis of Variance) with Bonferroni post-hoc tests. Results: rhPro-UK induced less Evans blue leakage and hemorrhagic transformation than rt-PA. Combined rhPro-UK and TSI treatment, but not monotherapies, significantly reduced cerebral infarction and improved neurological function. This combination preserved BBB integrity by preventing the degradation of junctional proteins (occludin, VE-cadherin) and basement membrane components (collagen IV). In HBMECs, TSI co-treatment attenuated OGD/R+rhPro-UK-induced barrier dysfunction, suppressed reactive oxygen species (ROS) production, and reduced MMP‑2‑related gelatinolytic activity without altering MMP-2/9 expression. TSI also mitigated membrane translocation of the NADPH oxidase (NOX) subunits gp91phox and p47phox. Conclusions: rhPro-UK exhibits a superior safety profile over rt-PA regarding BBB disruption. TSI potentiates rhPro-UK-mediated protection to improve BBB integrity during late-phase thrombolysis by suppressing NOX-2-derived oxidative stress and subsequent MMP-2 activation. This combinatory strategy represents a promising therapeutic approach to enhance the safety and efficacy of delayed thrombolysis within the standard 4.5‑hour window for ischemic stroke.