Background and Purpose: Subarachnoid hemorrhage (SAH) is a life-threatening subtype of stroke with high rates of mortality. In the early stages of SAH, neuroinflammation is one of the important mechanisms leading to brain injury after SAH. In various central nervous system diseases, activation of RARα receptor has been proven to demonstrate neuroprotective effects. This study aimed to investigate the anti-inflammatory effects of RARα receptor activation after SAH.Methods: Internal carotid artery puncture method used to established SAH model in Sprague-Dawley rats. The RARα specific agonist Am80 was injected intraperitoneally 1 hour after SAH. AGN196996 (specific RARα inhibitor), Msr1 siRNA and LY294002 (PI3K-Akt inhibitor) were administered via the lateral ventricle before SAH. Evaluation SAH grade, neurological function score, blood-brain barrier permeability. BV2 cells and SH-SY5Y cells were co-cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH. RT-PCR, Western blotting, and immunofluorescence staining were used to investigate pathway-related proteins, microglia activation and inflammatory response. Results: The expression of RARα, Mafb, and Msr1 increased in rat brain tissue after SAH. Activation of the RARα receptor with Am80 improved neurological deficits and attenuated brain edema, blood brain barrier permeability. Am80 increased the expression of Mafb and Msr1, and reduced neuroinflammation by enhancing the phosphorylation of Akt and by inhibiting the phosphorylation of NF-κB. AGN196996, Msr1 siRNA, and LY294002 reversed the therapeutic effects of Am80 by reducing the expression of Msr1 and the phosphorylation of Akt. In vitro model of SAH, Am80 promoted M1-to-M2 phenotypic polarization in microglia and suppressed the nuclear transcription of NF-κB.Conclusion: Activation of the RARα receptor attenuated neuroinflammation by promoting M1-to-M2 phenotypic polarization in microglia and regulating the Mafb/Msr1/PI3K-Akt/NF-κB pathway. RARα might serve as a potential target for SAH therapy.

Background: The poor prognosis of subarachnoid hemorrhage (SAH) is often attributed to neuroinflammation. The cGAS-STING axis, a cytoplasmic pathway responsible for detecting dsDNA, plays a significant role in mediating neuroinflammation in neurological diseases. However, the effects of inhibiting cGAS with the selective small molecule inhibitor RU.521 on brain injury and the underlying mechanisms after SAH are still unclear. Methods: The expression and microglial localization of cGAS following SAH were investigated with western blot analysis and immunofluorescent double-staining, respectively. RU.521 was administered after SAH. 2’3’-cGAMP, a second messenger converted by activated cGAS, was used to activate cGAS-STING. The assessments were carried out by adopting various techniques including neurological function scores, brain water content, blood–brain barrier permeability, western blot analysis, TUNEL staining, Nissl staining, immunofluorescence, morphological analysis, Morris water maze test, Golgi staining, CCK8, flow cytometry in the in vivo and in vitro settings. Results: Following SAH, there was an observed increase in the expression levels of cGAS in rat brain tissue, with peak levels observed at 24 h post-SAH. RU.521 resulted in a reduction of brain water content and blood–brain barrier permeability, leading to an improvement in neurological deficits after SAH. RU.521 had beneficial effects on neuronal apoptosis and microglia activation, as well as improvements in microglial morphology. Additionally, RU.521 prompted a shift in microglial phenotype from M1 to M2. We also noted a decrease in the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and an increase in the level of the anti-inflammatory cytokine IL-10. Finally, RU.521 treatment was associated with improvements in cognitive function and an increase in the number of dendritic spines in the hippocampus. The therapeutic effects were mediated by the cGAS/STING/NF-κB pathway and were found to be abolished by 2’3’-cGAMP. In vitro, RU.521 significantly reduced apoptosis and neuroinflammation. Conclusion: The study showed that SAH leads to neuroinflammation caused by microglial activation, which contributes to early brain injury. RU.521 improved neurological outcomes and reduced neuroinflammation by regulating microglial polarization through the cGAS/STING/NF-κB pathway in early brain injury after SAH. RU.521 may be a promising candidate for the treatment of neuroinflammatory pathology after SAH.

Background: We have previously verified the beneficial effects of exosomes from endothelial progenitor cells (EPC-EXs) in ischemic stroke. However, the effects of EPC-EXs in hemorrhagic stroke have not been investigated. Additionally, miR-137 is reported to regulate ferroptosis and to be involved in the neuroprotection against ischemic stroke. Hence, the present work explored the effects of miR-137-overexpressing EPC-EXs on apoptosis, mitochondrial dysfunction, and ferroptosis in oxyhemoglobin (oxyHb)-injured SH-SY5Y cells. Methods: The lentiviral miR-137 was transfected into EPCs and then the EPC-EXs were collected. RT-PCR was used to detect the miR-137 level in EPCs, EXs, and neurons. The uptake mechanisms of EPC-EXs in SH-SY5Y cells were explored by the co-incubation of Dynasore, Pitstop 2, Ly294002, and Genistein. After the transfection of different types of EPC-EXs, flow cytometry and expression of cytochrome c and cleaved caspase-3 were used to detect the apoptosis of oxyHb-injured neurons. Neuronal mitochondrial function was assessed by reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) depolarization, and cellular ATP content. Cell ferroptosis was measured by lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4. Additionally, recombinational PGE2 was used to detect if activation of COX2/PGE2 pathway could reverse the protection of miR-137 overexpression. Results: The present work showed (1) EPC-EXs could be taken in by SH-SY5Y cells via caveolin-/clathrin-mediated pathways and macropinocytosis; (2) miR-137 was decreased in neurons after oxyHb treatment, and EXs^miR-137 could restore the miR-137 levels; (3) EXs^miR-137 worked better than EXs in reducing the number of apoptotic neurons and pro-apoptotic protein expression after oxyHb treatment; (4) EXs^miR-137 are more effective in improving the cellular MMP, ROS, and ATP level; (5) EXs^miR-137, but not EXs, protected oxyHb-treated SH-SY5Y cells against lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4; and (6) EXs^miR-137 suppressed the expression of the COX2/PGE2 pathway, and activation of the pathway could partially reverse the neuroprotective effects of EXs^miR-137. Conclusion: miR-137 overexpression boosts the neuroprotective effects of EPC-EXs against apoptosis and mitochondrial dysfunction in oxyHb-treated SH-SY5Y cells. Furthermore, EXs^miR-137 rather than EXs can restore the decrease in miR-137 levels and inhibit ferroptosis, and the protection mechanism might involve the miR-137-COX2/PGE2 signaling pathway.