Abstract Body: Background and Purpose：Increasing evidence has shown that lipid metabolism disorder and oxidative stress play crucial roles in pathogenesis of ischemic stroke, both of which are closely related to ferroptosis. Edaravone dexboneol (Eda.B) is clinically used in China for acute ischemic stroke management as a brain protectant. It consists of two ingredients, edaravone and (+)-dexborneol, exerting antioxidant, free radical scavenging, and anti-inflammation. This study aimed to identify their respective specific targets and to elucidate molecular mechanisms underlying their synergistically anti-ischemic stroke effects.
Methods: Ischemic stroke was induced in Male C57BL/6 mice by transient middle cerebral artery occlusion (tMCAO). Mice was injected intravenously with Eda.B upon reperfusion and then each day for consecutive 14 days. Neuroprotective effect, glial scar formation and pro-inflammatory factors levels, lipid droplets (LDs), lysosomal and mitochondrial damage were assessed. The click chemistry was applied for lysosomal heat shock protein (Hsp70) and mitochondrial voltage-dependent anion channel 2 (VDAC2) palmitoylation. The surface plasmon resonance (SPR) and biotin probe labelling and computer molecular docking were used to detect the respective target for Edaravone and dexboneol.
Results: Eda.B protects neuronal cell death, and inhibits glial scar and neuroinflammation. Mechanistically, edaravone and dexboneol synergistically suppress astrocytic ferroptosis pathway and lipid metabolism disorder. Among these two components: edaravone reverses OGD/Re-induced astrocytic lipid disorder via targeting activation of mitochondrial carnitine o-palmitoyltransferase 1A (CPT1A), thereby reducing LDs accumulation and LDs-mediated Hsp70 and VDAC2 palmitoylation, protecting lysosomal and mitochondrial damage; Dexborneol targets activation of antioxidant enzyme PRDX1, which can suppress the OGD/Re-induced NLRP3 inflammasome activation and inflammation. We unexpectedly found that CPT1A can bind to and activate PRDX1. Edaravone activating CPT1A promotes the interaction between CPT1A and PRDX1, further enhancing PRDX1 activation, producing synergistically effect with dexborneol in antioxidant and anti-inflammation.
Conclusions: This study demonstrates for the first time that CPT1A and PRDX1 are the key targets for edaravone and dexboneol, respectively. These two drugs enhance the interaction between CPT1A and PRDX1, causing synergistically anti-ischemic stroke effects.