ABSTRACT
JOURNAL/nrgr/04.03/01300535-202503000-00029/figure1/v/2024-06-17T092413Z/r/image-tiff It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke. Indeed, previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue. Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke, but its specific role and mechanism are currently unclear. To simulate stroke in vivo, a middle cerebral artery occlusion rat model was established, with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke. We found that in the early stage (within 24 hours) of ischemic stroke, neutrophils produced a large amount of hypochlorous acid, while in the recovery phase (10 days after stroke), microglia were activated and produced a small amount of hypochlorous acid. Further, in acute stroke in rats, hypochlorous acid production was prevented using a hypochlorous acid scavenger, taurine, or myeloperoxidase inhibitor, 4-aminobenzoic acid hydrazide. Our results showed that high levels of hypochlorous acid (200 µM) induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation. However, in the recovery phase of the middle cerebral artery occlusion model, a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes. This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury. Lower levels of hypochlorous acid (5 and 100 µM) promoted nuclear translocation of ß-catenin. By transfection of single-site mutation plasmids, we found that hypochlorous acid induced chlorination of the ß-catenin tyrosine 30 residue, which promoted nuclear translocation. Altogether, our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.
ABSTRACT
Peroxynitrite (ONOO-) and high mobility group box 1 protein (HMGB1) are important cytotoxic factors contributing to cerebral ischemia-reperfusion injury. However, the roles of ONOO- in mediating HMGB1 expression and its impacts on hemorrhagic transformation (HT) in ischemic brain injury with delayed t-PA treatment remain unclear. In the present study, we tested the hypothesis that ONOO- could directly mediate the activation and release of HMGB1 in ischemic brains with delayed t-PA treatment. With clinical studies, we found that plasma nitrotyrosine (NT, a surrogate marker of ONOO-) was positively correlated with HMGB1 level in acute ischemic stroke patients. Hemorrhagic transformation and t-PA-treated ischemic stroke patients had increased levels of nitrotyrosine and HMGB1 in plasma. In animal experiments, we found that FeTmPyP, a representative ONOO- decomposition catalyst (PDC), significantly reduced the expression of HMGB1 and its receptor TLR2, and inhibited MMP-9 activation, preserved collagen IV and tight junction claudin-5 in ischemic rat brains with delayed t-PA treatment. ONOO- donor SIN-1 directly induced expression of HMGB1 and its receptor TLR2 in naive rat brains in vivo and induced HMGB1 in brain microvascular endothelial b.End3 cells in vitro. Those results suggest that ONOO- could activate HMGB1/TLR2/MMP-9 signaling. We then addressed whether glycyrrhizin, a natural HMGB1 inhibitor, could inhibit ONOO- production and the antioxidant properties of glycyrrhizin contribute to the inhibition of HMGB1 and the neuroprotective effects on attenuating hemorrhagic transformation in ischemic stroke with delayed t-PA treatment. Glycyrrhizin treatment downregulated the expressions of NADPH oxidase p47 phox and p67 phox and iNOS, inhibited superoxide and ONOO- production, reduced the expression of HMGB1, TLR2, MMP-9, preserved type IV collagen and claudin-5 in ischemic brains. Furthermore, glycyrrhizin significantly decreased the mortality rate, attenuated hemorrhagic transformation, brain swelling, blood-brain barrier damage, neuronal apoptosis, and improved neurological outcomes in the ischemic stroke rat model with delayed t-PA treatment. In conclusion, peroxynitrite-mediated HMGB1/TLR2 signaling contributes to hemorrhagic transformation, and glycyrrhizin could be a potential adjuvant therapy to attenuate hemorrhagic transformation, possibly through inhibiting the ONOO-/HMGB1/TLR2 signaling cascades.
