Targeting Ninjurin‐1‐Mediated Microglial Pyroptosis Attenuates Neuroinflammation and Neuronal Injury Following Intracerebral Hemorrhage

OBJECTIVE: This study aims to delineate the pathophysiological contribution of Ninjurin-1 (Ninj1) to secondary neural injury following intracerebral hemorrhage (ICH), specifically elucidating its function in microglial pyroptosis and its upstream transcriptional governance by nuclear factor-kappa B (NF-κB). METHODS: Candidate targets were initially identified via in silico transcriptomic profiling. We subsequently employed a collagenase-induced murine ICH paradigm alongside hemin-challenged microglia. Mechanistic pathways were interrogated utilizing targeted genetic silencing (in vivo siRNA and in vitro shRNA) and pharmacological blockade. Disease phenotypes-encompassing pyroptotic execution, neuroinflammation, and collateral neuronal death-were quantified via immunoblotting, molecular assays, and histopathology. The upstream regulatory architecture was validated through ChIP-qPCR, EMSA, and dual-luciferase reporter assays. RESULTS: Bioinformatic screening highlighted Ninj1 as a prominently upregulated candidate transcript post-ICH, a finding corroborated by robust induction in both in vivo and in vitro hemorrhagic models. Through prophylactic in vivo target validation, Ninj1 depletion restricted perihematomal edema and preserved sensorimotor function. Mechanistically, suppressing Ninj1 abrogated the microglial pyroptotic cascade and mitigated collateral neurotoxicity. Furthermore, NF-κB was shown to transcriptionally activate NLRP3 and Ninj1, although direct promoter enrichment in the ChIP-qPCR assay was statistically significant only for the Nlrp3 locus. CONCLUSIONS: Ninj1 functions as a terminal executioner of microglial pyroptosis and a critical amplifier of post-ICH neuroinflammation. Governed by a coordinated NF-κB-dependent transcriptional program alongside NLRP3, this signaling axis exacerbates secondary brain injury, positioning Ninj1 as a viable molecular candidate for neuroprotective target validation.