Evans Blue Might Produce Pathologically Activated Neuroprotective Effects via the Inhibition of the P2X4R/p38 Signaling Pathway.

: The main pathological features of ischemic stroke include neuronal damage and blood-brain barrier (BBB) dysfunction. Previous studies have shown that Evans Blue, a dye used to probe BBB integrity, could enter the brain only during the pathological status of ischemic stroke, indicating the potential pathologically activated therapeutic use of this chemical to treat ischemic stroke. In this study, we have reported that Evans Blue could produce in vitro neuroprotective effects against iodoacetic acid (IAA)-induced hypoxia neuronal death in HT22 cells. We further found that P2X purinoreceptor 4 (P2X4R), a subtype of ATP-gated cation channel, was expressed in HT22 cells. Evans Blue could prevent IAA-induced increase of P2X4R mRNA and protein expression. Interestingly, shRNA of P2X4R could protect against IAA-induced activation of p38, and SB203580, a specific inhibitor of p38, could reverse IAA-induced neurotoxicity, indicating that p38 is a downstream signaling molecule of P2X4R. Molecular docking analysis further demonstrated the possible interaction between Evans Blue and the ATP binding site of P2X4R. Most importantly, pre-treatment of Evans Blue could largely reduce neurological and behavioral abnormity, and decrease brain infarct volume in middle cerebral artery occlusion/reperfusion (MCAO) rats. All these results strongly suggested that Evans Blue could exert neuroprotective effects via inhibiting the P2X4R/p38 pathway, possibly by acting on the ATP binding site of P2X4R, indicating that Evans Blue might be further developed as a pathologically activated therapeutic drug against ischemic stroke.