Targeting Microglial HDAC5 Suppresses NLRP3 Inflammation via NF ‐ κB Deacetylation to Alleviate Ischemic Brain Injury

ABSTRACT Objective Histone deacetylases are implicated in ischemic stroke, yet the role of HDAC5 remains poorly defined. This study aimed to determine the pathological significance of HDAC5 after cerebral ischemia, clarify its involvement in post‐ischemic NF‐κB p65/NLRP3 signaling, and evaluate an HDAC5‐targeted extracellular vesicle (EV)‐based therapeutic strategy. Methods HDAC5 expression after ischemic injury was examined in a mouse middle cerebral artery occlusion (MCAO) model by western blotting and immunofluorescence. Its function was assessed by gain‐ and loss‐of‐function experiments in BV2 cells and MCAO mice. Infarct volume, cerebral blood flow, and neurological recovery were evaluated by TTC staining, magnetic resonance imaging (MRI), color Doppler imaging, and behavioral tests. Co‐immunoprecipitation, acetylation analysis, and cytoplasmic/nuclear fractionation were used to explore the underlying mechanism. Human neural stem cell‐derived EVs loaded with miR‐9‐5p and modified with RGD peptide were further tested as a targeted therapeutic approach. Results HDAC5 was markedly upregulated after MCAO, localizing mainly to neurons in the ischemic core and microglia in the penumbra, together with increased NLRP3 expression. In BV2 cells, HDAC5 overexpression increased NLRP3 expression, whereas HDAC5 knockdown reduced it. In vivo, AAV‐mediated HDAC5 knockdown decreased NLRP3 levels, reduced infarct volume, improved blood flow recovery, and ameliorated motor deficits after MCAO. Mechanistically, HDAC5 interacted with NF‐κB p65 rather than NLRP3. HDAC5 knockdown increased p65 acetylation and reduced its nuclear translocation, consistent with suppression of NLRP3‐associated inflammatory signaling. Moreover, engineered RGD‐EV:miR‐9‐5p was efficiently taken up by microglia, suppressed HDAC5 and NLRP3 expression, and improved histological and functional outcomes after MCAO. Conclusion These findings identify HDAC5 as a previously underappreciated regulator of post‐ischemic inflammatory injury and support a role for the HDAC5/NF‐κB p65/NLRP3 axis in cerebral ischemia. Engineered RGD‐EV:miR‐9‐5p may therefore represent a promising targeted therapeutic strategy for ischemic stroke.