Angong Niuhuang Wan reduces hemorrhagic transformation and mortality in ischemic stroke rats with delayed thrombolysis: involvement of peroxynitrite-mediated MMP-9 activation

Background: Hemorrhagic transformation (HT) is a common complication of delayed tissue plasminogen activator (t-PA) treatment for ischemic stroke. Peroxynitrite plays an important role in the breakdown of blood–brain barrier (BBB) and the development of HT. We tested the hypothesis that Angong Niuhuang Wan (AGNHW), a traditional Chinese medicinal formula, could be used in conjunction with t-PA to protect the BBB, minimize HT, and improve neurological function by suppressing peroxynitrite-mediated matrix metalloproteinase-9 (MMP-9) activation. Methods: We first performed quality control study and chemical identification of AGNHW by using UPLC. In animal experiments, male Sprague–Dawley rats were subjected to 5 h of middle cerebral artery occlusion (MCAO) followed by 19 h of reperfusion plus t-PA infusion (10 mg/kg) at 5 h of cerebral ischemia. AGNHW (257 mg/kg) was given orally at 2 h after MCAO. Hemorrhagic transformation was measured using hemorrhagic scores and hemoglobin levels in ischemic brains. Evans blue leakage was utilized to assess the severity of the blood–brain barrier (BBB) damage. The modified neurologic severity score (mNSS) test was used to assess neurological functions. Peroxynitrite and superoxide was detected by using fluorescent probes. MMP-9 activity and expression were examined by gelatin zymography and immunostaining. The antioxidant effects were also studied by using brain microvascular endothelial b.End3 cells exposed to 5 h of oxygen and glucose deprivation (OGD) plus 5 h of reoxygenation with t-PA treatment (20 µg/ml). Results: AGNHW significantly reduced the BBB damage, brain edema, reduced hemorrhagic transformation, enhanced neurological function, and reduced mortality rate in the ischemic stroke rats with t-PA treatment. AGNHW reduced peroxynitrite and superoxide in vivo and in vitro and six active chemical compounds were identified from AGNHW with peroxynitrite scavenging activity. Furthermore, AGNHW inhibited MMP-9 activity, and preserved tight junction protein claudin-5 and collagen IV in the ischemic brains. Conclusion: AGNHW could be a potential adjuvant therapy with t-PA to protect the BBB integrity, reduce HT, and improve therapeutic outcome in ischemic stroke treatment via inhibiting peroxynitrite-mediated MMP-9 activation. Graphical Abstract: [Figure not available: see fulltext.].