Exosomal let-7c-5p mediates remote ischemic preconditioning-induced neuroprotection against ischemic stroke via the ZBP1/PANoptosis-related pathway

Introduction Remote ischemic preconditioning (RIPC) confers neuroprotection against ischemic brain injury, but the underlying mechanisms remain unclear. This study investigated whether RIPC-derived exosomes inhibit PANoptosis-related neuronal cell death via the microRNA let-7c-5p/ZBP1 pathway using in vivo mouse and in vitro SHSY5Y cell models. Methods Male mice underwent middle cerebral artery occlusion (MCAO) with or without prior RIPC and were treated with or without the exosome inhibitor GW4869. Brain infarct volume and histopathological damage were assessed by triphenyltetrazolium chloride (TTC) and hematoxylin and eosin (H&E) staining, while neurological deficits were evaluated using neurological severity scores. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to evaluate the cortical expression of let-7c-5p, ZBP1, and PANoptosis-related marker proteins for pyroptosis (NLRP3 and GSDMD), apoptosis (caspase-3 and GSDME), and necroptosis (MLKL and RIPK3). Serum exosomes were isolated from healthy human volunteers before and after RIPC and let-7c-5p levels were quantified. SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were treated with RIPC-derived human serum exosomes or transduced with a let-7c-5p-overexpressing lentivirus. Dual-luciferase assays were used to validate the interaction between let-7c-5p and ZBP1-3’UTR. Results RIPC significantly reduced MCAO-induced brain injury and improved functional outcomes, but these effects were significantly reversed by GW4869 pretreatment. RIPC upregulated cortical let-7c-5p levels and downregulated ZBP1 and PANoptosis-associated proteins, whereas pretreatment with GW4869 abolished these changes. The human serum exosomal let-7c-5p levels were elevated following RIPC. In vitro , OGD/R-exposed SH-SY5Y cells treated with RIPC exosomes and overexpressing let-7c-5p exhibited reduced levels of ZBP1 and PANoptosis-related proteins. Dual-luciferase assays confirmed the direct binding of let-7c-5p to ZBP1. Conclusions These findings suggest that RIPC-associated exosomes provide neuroprotection against ischemic stroke by suppressing PANoptosis-related protein via the let-7c-5p/ZBP1 axis.