Peroxynitrite is an important target mediating hemorrhagic transformation induced by delayed tissue plasminogen activator (t-PA) treatment in ischemic stroke: potential application of natural compound baicalin

Abstract

Aim: Hemorrhagic transformation (HT) is a major complication of delayed tissue plasminogen activator (t-PA) treatment in ischemic stroke. We aimed to explore whether peroxynitrite plays an important role in mediating HT and whether natural compound baicalin could reduce HT via scavenging peroxynitrite. Methods: For the first part, Male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) with t-PA (10 mg/kg) or t-PA plus FeTMPyP (3 mg/kg, a representative peroxynitrite decomposition catalyst) at MCAO for 2 or 5 h and reperfusion for 22 or 19 h, respectively. For the second part, SD rats were subjected to MCAO with t-PA (10 mg/kg) or t-PA plus baicalin (50, 100, 150 mg/kg) at MCAO 5 h and reperfusion for 19 h. HT was assessed with hemoglobin assay and HT scores. Blood-brain barrier damage was assessed with Evans blue leakage assay. Cell apoptosis in cortex and striatum was examined by TUNEL assay. Neurological deficit was evaluated with Modified Neurological Severity Score (mNSS). Peroxynitrite formation was examined by detecting 3-nitrotyrosine (3-NT) formation in brains and direct detection with our newly developed highly sensitive and specific probe HK-Yellow-AM. The expression and activity of MMP-9/MMP-2 were assessed by Western blotting, immunostaining, and gelatin zymography. Tight junction protein ZO-1 was assessed by Western blotting. Interaction of baicalin and t-PA activity was assessed by a t-PA activity assay kit. Results: t-PA treatment at 2 h of MCAO did not induce HT but attenuated neurological deficit, whereas treatment at 5 h significantly induced HT and worsened the neurological outcome. Such complications were prevented by FeTMPyP co-treatment. Early t-PA treatment inhibited 3-NT and MMP-9/MMP-2 expression, whereas delayed treatment induced 3-NT and MMP-9/MMP-2 expression and activity. FeTMPyP treatment downregulated 3-NT and inhibited MMP-9/MMP-2 in both time points. Baicalin co-treatment with t-PA significantly reduced HT, BBB damage, cell apoptosis and improved the neurological outcomes. HK-Yellow AM probe staining showed significant induction of peroxynitrite signal in ischemic brains after delayed t-PA treatment and attenuated by baicalin co-treatment. Baicalin co-treatment significantly reduced MMP-9 expression and activity, and protected tight junction ZO-1. In addition, baicalin did not inhibit t-PA’s fibrinolytic function. Conclusion: Peroxynitrite mediates hemorrhagic transformation induced by delayed t-PA treatment in ischemic stroke. Targeting peroxynitrite with natural compound baicalin significantly reduced hemorrhagic transformation and improved the functional outcomes possibly via down-regulating MMPs activation.