Remote Ischemic Postconditioning-Mediated Neuroprotection against Stroke by Promoting Ketone Body-Induced Ferroptosis Inhibition.

Neuronal death resulting from ischemic stroke is the primary cause of adult mortality and disability, and effective neuroprotective agents for poststroke intervention are still lacking. Remote ischemic postconditioning (RIPostC) has demonstrated significant protective effects against ischemia in various organs; however, the specific mechanisms are not fully understood. This study investigated the potential neuroprotective mechanisms of RIPostC in the context of ischemic stroke. Using a rat model of middle cerebral artery occlusion, we found that RIPostC mitigated neurological damage, improved movement in the open-field test, and protected against neuronal apoptosis. In terms of energy metabolism, RIPostC enhanced ATP levels, suppressed lactate content, and increased the production of ketone bodies (KBs). In the ferroptosis assay, RIPostC protected against lipoperoxidation, reversed the reduction of glutathione peroxidase 4 (GPX4), and mitigated the excessive expression of long-chain acyl-CoA synthetase family member 4 (ACSL4). In oxygen-glucose deprivation/reoxygenation-treated HT22 cells, KBs maintained GPX4 levels, suppressed ACSL4 expression, and preserved the mitochondrial cristae number. However, the effect of KBs on the expression of GPX4, ACSL4, and the number of mitochondrial cristae was blocked by erastin. Moreover, both RIPostC and KBs reduced total iron and ferrous ion content by repressing iron transporters both in vitro and in vivo. In conclusion, KBs-induced mitigation of ferroptosis could represent a new therapeutic mechanism for RIPostC in treating stroke.

Healthy Serum-Derived Exosomes Improve Neurological Outcomes and Protect Blood-Brain Barrier by Inhibiting Endothelial Cell Apoptosis and Reversing Autophagy-Mediated Tight Junction Protein Reduction in Rat Stroke Model.

Blood-brain barrier (BBB) dysfunction causing edema and hemorrhagic transformation is one of the pathophysiological characteristics of stroke. Protection of BBB integrity has shown great potential in improving stroke outcome. Here, we assessed the efficacy of exosomes extracted from healthy rat serum in protection against ischemic stroke in vivo and in vitro. Exosomes were isolated by gradient centrifugation and ultracentrifugation and exosomes were characterized by transmission electron microscopy (TEM) and nanoparticle tracking video microscope. Exosomes were applied to middle cerebral artery occlusion (MCAO) rats or brain microvascular endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD) injury. Serum-derived exosomes were injected intravenously into adult male rats 2 h after transient MCAO. Infarct volume and gross cognitive function were assessed 24 h after reperfusion. Poststroke rats treated with serum-derived exosomes exhibited significantly reduced infarct volumes and enhanced neurological function. Apoptosis was assessed via terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and the expression of B-cell lymphoma-2 (Bcl-2), Bax, and cleaved caspase-3 24 h after injury. Our data showed that serum exosomes treatment strikingly decreased TUNEL+ cells in the striatum, enhanced the ratio of Bcl-2 to Bax, and inhibited cleaved caspase-3 production in MCAO rats and OGD/reoxygenation insulted bEnd.3 cells. Under the consistent treatment, the expression of microtubule-associated protein 1 light chain 3B-II (LC3B-II), LC3B-I, and Sequestosome-1 (SQSTM1)/p62 was detected by Western blotting. Autolysosomes were observed via TEM. We found that serum exosomes reversed the ratio of LC3B-II to LC3B-I, prevented SQSTM1/p62 degradation, autolysosome formation, and autophagic flux. Together, these results indicated that exosomes isolated from healthy serum provided neuroprotection against experimental stroke partially via inhibition of endothelial cell apoptosis and autophagy-mediated BBB breakdown. Intravenous serum-derived exosome treatment may, therefore, provide a novel clinical therapeutic strategy for ischemic stroke.