Glycyrrhizin Attenuated Hemorrhagic Transformation and Improved Neurological Outcomes in Ischemic Stroke with Delayed t-PA Treatment, Possibly through Inhibiting the Interaction of Peroxynitrite and HMGB1 Signaling Pathways

Abstract

Objectives: Tissue plasminogen activator (t-PA) has restricted therapeutic window of 4.5 hours for ischemic stroke treatment and hemorrhagic transformation (HT) is one of the major complications. Our works and others revealed that peroxynitrite and high mobility group box protein 1 (HMGB1) could be critical targets for reducing HT, which activate matrix metalloproteinase-9 (MMP-9). But it remains unclear whether these two factors interact with each other in contributing to HT. Glycyrrhizin is a specific HMGB1 inhibitor isolated from Chinese Medicine Glycyrrhiza glabra. We hypothesize that glycyrrhizin could inhibit the interaction of HMGB1 and peroxynitrite signaling, subsequently reduce HT and improve neurological outcomes in ischemic stroke with delayed t-PA treatment. Methods: Male SD rats were subjected middle cerebral artery occlusion (MCAO) for 5 hours plus 19 hours of reperfusion. T-PA (10 mg/kg) was intravenously administrated at 4.5 hours after MCAO and continuously injected for 30 min. Glycyrrhizin (15, 30, 60 mg/kg) or peroxynitrite decomposition catalyst FeTmPyP (3 mg/kg) was administrated at the onset of t-PA treatment and saline was used as a control. In naive rats, peroxynitrite donor SIN-1 was intracerebroventricularly injected. For in vitro study, BV2 microglial cells were treated with tissue plasminogen activator (20 ug/ml) for 24 hours under normoxia condition. The potential interaction of glycyrrhizin with t-PA was assessed by determining the t-PA fibrinolytic activity with an assay kit. Results: (l) Delayed t-PA treatment significantly increased mortality rate, brain swelling, hemorrhagic transformation, and brain cell apoptosis with the increase of peroxynitrite, HMGB1 and its receptor TLR2, and MMP-9 activation. (2) Both inhibiting HMGBI with glycyrrhizin and scavenging peroxynitrite with FeTmPyP treatment significantly inhibited 3-NT (footprint of peroxynitrite). HMGB1 and its receptor TLR2 and MMP-9 activity in ischemic brains, subsequently attenuated HT, brain edema, cell apoptosis, decreased mortality and improved the neurological outcomes. (3) In naive rats, peroxynitrite donor SIN-1 directly induced the HMGB1 and TLR2 expression in brain tissues. (4) In BV2 microglial cells, t-PA treatment directly induced the 3-NT, inducible nitric oxide synthase (iNOS). HMGB1 and IL-1 beta expression. (5) Glycyrrhizin did not affect t-PA fibrinolytic activity in vitro. Conclusion: Glycyrrhizin could be an adjuvant therapy with t-PA to improve the therapeutic outcome for ischemic stroke, possibly through targeting the peroxynitrite-HMGB1 signaling pathways.