Regulation of IRGM1-Mediated Pyroptosis and Neuroinflammation by Transcription Factor NR3C1 Alleviates Early Brain Injury after Subarachnoid Hemorrhage

OBJECTIVE: Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is critical for patient prognosis, and neuroinflammation and pyroptosis play core roles in its pathological process. Immunity-related GTPase 1 (IRGM1) is downregulated after SAH. Nuclear Receptor Subfamily 3, Group C, Member 1 (NR3C1) is closely associated with inflammatory responses and cellular injury in central nervous system injury. However, the interaction between the two and their mechanism of action in SAH lacks experimental verification. This study aimed to explore the regulatory mehcanism of the NRC31/IRGM1 axis in EBI after SAH. METHODS: An in vitro SAH model was established using hemin-treated BV2 microglial cells. Hemin is hemoglobin's main degradation product post-SAH; its accumulation drives SAH-induced EBI, so hemin-stimulated BV2 cells are a validated model for SAH-related microglial injury. Gene and protein expressions were detected via quantitative real-time polymerase chain reaction and Western blot, respectively; cell viability was assessed by cell counting kit-8 assay, apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, oxidative stress indicators (reactive oxygen species, malondialdehyde, and superoxide dismutase) by corresponding kits, and inflammatory cytokines (interleukin-1 beta and interleukin-18) by enzyme-linked immunosorbent assay. NR3C1 was identified as a potential upstream transcription factor of IRGM1 via bioinformatics prediction (JASPAR), and its binding to the IRGM1 promoter was verified by chromatin immunoprecipitation and dual-luciferase reporter assays. An in vivo SAH model was established, with neuronal injury observed by Nissl staining; SAH grade, neurological function scores, and brain water content were also measured. RESULTS: SAH induction significantly downregulated IRGM1 mRNA and protein. IRGM1 overexpression in SAH-modeled BV2 cells increased cell viability, reduced TUNEL-positive cells, upregulated Bcl-2 and downregulated Bax, decreased reactive oxygen species and malondialdehyde levels, increased superoxide dismutase activity, and suppressed interleukin-1 beta/interleukin-18 release as well as cleaved gasdermin D (GSDMD-N), NOD-like receptor family pyrin domain-containing 3, and caspase-1 expression. NR3C1 mRNA and protein were decreased in SAH-induced BV2 cells; it bound to the IRGM1 promoter and regulated its transcriptional activity. NR3C1 overexpression upregulated IRGM1, ameliorated SAH-induced cell viability loss, apoptosis, oxidative stress, and inflammation/pyroptosis activation, whereas IRGM1 silencing reversed these protective effects. In vivo , NR3C1 overexpression alleviated neuronal injury, improved neurological scores, and reduced brain water content, with no significant difference in SAH grade among groups. CONCLUSION: NR3C1 transcriptionally activated IRGM1 to inhibit inflammation, pyroptosis, and oxidative stress, thereby alleviating EBI after SAH, and targeting the NR3C1/IRGM1 axis might provide a novel therapeutic strategy for SAH.