ZL006-05 prevents tPA-induced haemorrhagic transformation after stroke via inhibiting neutrophil myeloperoxidase-related pathway

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Background Haemorrhagic transformation (HT) is the most feared complication of thrombolytic therapy in acute ischaemic stroke (AIS). This study aims to determine whether bornyl-2-hydroxy-4-(2-hydroxy-3,5-dichlorobenzyl) aminobenzoate (ZL006-05), a neuroprotectant that mitigates cerebral ischaemia/reperfusion injuries in animals and improves functional outcome in AIS patients, prevents recombinant tissue plasminogen activator (rtPA)-induced HT in transient middle cerebral artery occlusion (tMCAO) mice through affecting neutrophil myeloperoxidase (MPO). Methods The tMCAO was induced in adult mice. The recanalisation after rtPA thrombolysis within or beyond the clinical therapeutic window was simulated based on 110-minute-tMCAO and 5-hour-tMCAO, respectively. ZL006-05 was injected before or after rtPA administration. Haemoglobin and Evans blue contents in the brain were measured by spectrophotometric assay. MPO activity was measured with an MPO peroxidation activity assay kit. Mobilisation and infiltration of neutrophils were detected by flow cytometry or immunofluorescence. Neutrophil extracellular traps (NETs) were quantified by DNA stained with Hoechst. Neutrophils were depleted by anti-Ly6G mAb clone 1A8. Pro-inflammatory cytokines and MPO expressions were measured by Western blot analysis. Results ZL006-05 prevented rtPA-induced HT and blood-brain barrier (BBB) damage in 110-minute-tMCAO mice and reduced animal mortality caused by rtPA in 5-hour-tMCAO mice. ZL006-05 suppressed the activation of neutrophil MPO by the combination of rtPA with high mobility group protein B1 and prevented rtPA-induced MPO activation in the ischaemic brain. Molecular docking predicted that ZL006-05 is an MPO inhibitor, and ZL006-05 dose-dependently inhibited MPO enzymatic activity in purified MPO protein and cultured inflammatory cells. In the MPO deficient (MPO −/− ) mice, rtPA-induced HT was significantly reduced. Moreover, ZL006-05 reversed rtPA-induced neutrophil mobilisation, infiltration and NET formation. Importantly, the inhibitory effect of ZL006-05 on rtPA-induced HT disappeared in the mice with MPO knockout or depletion of neutrophils. In addition, ZL006-05 inhibited expressions of pro-inflammatory cytokines in wild-type but not in MPO −/− neutrophils. Conclusion ZL006-05 can prevent rtPA-induced BBB disruption and HT by interfering with neutrophil MPO activation.

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