Proteomic profile of the extracellular matrix following cerebral ischemia-reperfusion injury identified HCK as a target for ischemic stroke therapy.

Ischemic stroke is a detrimental central nervous system (CNS) disorder with high morbidity and disability rates, caused by local cerebral ischemia. Extracellular matrix (ECM) is a complex network structure secreted by cells and located in the intercellular compartment, which is significant changed following ischemic stroke. Here, we quantified the proteomic profile of the ECM of brain from young (2-month-old) and aged (18-month-old) mice underwent cerebral ischemia/reperfusion (I/R) at 24 h and 60 d post I/R. The proteomics results indicated that proteins associated with the tricarboxylic acid (TCA) cycle and neutrophil extracellular trap (NET) formation were significantly up-regulated in the brain ECM from mice underwent cerebral I/R during acute stage, while those associated with synaptic vesicle cycle were significantly down-regulated in all stage post cerebral I/R. Differently, the brain ECM from aged mice underwent I/R expressed higher levels of lysosomal proteins and lower levels of autophagy and synaptic vesicle cycle associated proteins than the brain ECM from young mice underwent I/R. Furtherly, the proteomics identified that Hematopoietic Cell Kinase (HCK) is a regulator for NET formation. Inhibition of HCK could down-regulate LPS-induced phosphorylation of ERK1/2 and IKKα/β, as well as blocking the LPS plus nigericin induced activation of NLRP3 inflammasome and NET-like trap formation in vitro. In addition, inhibition of HCK significantly ameliorated cerebral I/R-induced brain injury and NET formation in vivo, suggesting HCK is a therapeutic target for ischemic stroke treatment. SIGNIFICANCE: Our results systemically analyzed the protein profiles of ECM in the brain post- acute and chronic ischemic stroke, and identified upregulation of NET-associated proteins was a common feature of the cerebral ECM during the acute phase, while down-regulation of synaptic vesicle cycle associated proteins was a common character of the brain ECM in all stage. What's more, HCK was identified as a regulator for NET formation, inhibition of HCK could block the formation of NET by inhibiting the activation of ERK1/2, IKKα/β and NLRP3 inflammasome, suggesting HCK is a therapeutic target for ischemic stroke treatment.