A Linarin Derivative Protects against Ischemia-Induced Neuronal Injury in Mice by Promoting Cerebral Blood Flow Recovery via KDELR-Dependent CSPG4 Activation

Abstract

The cerebral ischemic microvascular response and collateral circulation compensatory capacity are important for the outcome of ischemic stroke. Here, we sought to evaluate the effect of a linarin derivate 4 $\prime$ -benzylapigenin-7-β-rutinoside (BLR) on neurological function and cerebral blood flow restoration in ischemic stroke. A mouse model of middle cerebral artery occlusion (30 min) with reperfusion (24 h) was used to mimic ischemic stroke in vivo, and 2,3,5-triphenyltetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and immunofluorescence microscopy were used to assess the protective effects of BLR on infarct volume, neurological function, neuronal apoptosis, and inflammatory damage. Cerebral blood flow was assayed by laser speckle contrast imaging. Double immunostaining of GFAP-collagen IV and brain lucidification were performed to determine the protective effects of BLR on the disruption of brain vasculature. Differential gene expression was assessed by RNA sequencing. Coimmunoprecipitation and western blotting were used to explore the mechanism of BLR-induced neuroprotection. The results of in vivo experiments showed that BLR administration after reperfusion onset reduced infarct volume, improved neurological function, and decreased the neural cell apoptosis and inflammatory response in the ischemic brain, which was accompanied by increased cerebral blood flow and reduced detachment of astrocyte endfeet from the capillary basement membrane. The RNA sequencing data showed that BLR promoted the upregulation of extracellular matrix and angiogenesis pathway-related genes; in particular, BLR significantly increased the expression of the chondroitin sulfate proteoglycan 4 (CSPG4) gene, enhanced the membrane location of CSPG4, and promoted its downstream signaling protein expression, which is associated with KDEL receptor (KDELR) activation. In addition, activated KDELR further increased the phosphorylation of mitogen-activated protein kinases after BLR treatment. Taken together, our data showed that BLR could protect against ischemic brain injury and may serve as a new promising therapeutic candidate drug for ischemic stroke, and that KDELR might act as both a sensor and effector to activate CSPG4 to increase cerebral blood flow.