Baicalin Attenuates Blood-Brain Barrier Disruption and Hemorrhagic transformation and Improves Neurological Outcome in Ischemic Stroke Rats with Delayed t-PA Treatment: Involvement of ONOO-MMP9 pathway

Abstract

Stroke is the main cause of death and disability all over the world and tissue plasminogen activator (t-PA) remains the only FDA-approved thrombolytic drug for ischemic stroke treatment. However, t-PA has a limited time window of 4.5 hours and treatment beyond this time window increases the rate of hemorrhagic transformation (HT), which in turn limits its clinical application. We previously reported that peroxynirite, a representative reactive nitrogen species produced during brain ischemia-reperfusion, plays an important role in mediating HT via the activation of MMPs. In the present study, we firstly evaluated the role of peroxynitrite in mediating HT in ischemic stroke patients. Increased plasma nitrotyrosine (NT) level, a footprint marker of peroxyntrite was associated with HT and NIHSS neurological scores in ischemic stroke patients. We then tested the hypothesis that baicalin, an active compound of medicinal plant, could attenuate HT in cerebral ischemia stroke with delayed t-PA treatment. Male Sprague-Dawley rats were subject to middle cerebral artery occlusion (MCAO) for 4.5 hours and then continuously received t-PA infusion (10mg/kg) for 0.5 hour followed by 19 hours’ reperfusion. Baicalin (50, 100, 150mg/kg) was administrated via femoral vein 4.5 hours after MCAO cerebral ischemia. Delayed t-PA infusion significantly increased the mortality rate, induced blood-brain barrier (BBB) damage, brain edema, HT, and apoptotic cell death in the ischemic brains and exacerbated neurological outcomes in rats 24 hours after MCAO cerebral ischemia. Co-treatment of baicalin significantly reduced the mortality rates, ameliorated the t-PA-mediated BBB disruption, brain edema, cell apoptosis and HT, and improved the neurological outcomes. Similar effects were achieved by FeTMPyP, a peroxynitrite decomposition catalyst, which served as a positive control. Furthermore, it was shown baicalin directly scavenged peroxynitrite in vitro and in vivo as evident by the UPLC analysis, HK-Yellow AM probe staining in cell culture and rat brains. Baicalin inhibited MMP-9 expression and activity, reduced Aquporin-4 (AQP4) expression and preserved tight junction ZO-1 in the ischemic brains with the delayed t-PA treatment. We observed the 3-NT and MMP-9 expressions in brain microvessels where showed Evans blue leakage in ischemic brains, indicating their involvement in BBB damage. In addition, baicalin also protected brain microvascular endothelial b.End3 cells from oxygen and glucose deprivation (OGD) and t-PA treatment in vitro. Baicalin had no effect on the fibrinolytic function of t-PA as indicated by t-PA activity assay. Taken together, baicalin could attenuate t-PA mediated HT and improve the outcomes in ischemic stroke treatment possibly via inhibiting peroxynitrite-mediated MMP-9 activation. Acknowledgement: This work was supported by Hong Kong General Research Fund (GRF No. 17102915, GRF No. 17118717), Research Grant Council, Hong Kong SAR; Health and Medical Research Fund, Hong Kong SAR (NO. 13142901).