Spi1-Soat1 Transcriptional axis drives microglial lipid droplet accumulation and secondary brain injury after intracerebral hemorrhage

Abstract Background and Purpose Intracerebral hemorrhage (ICH) is a devastating stroke subtype with persistently high morbidity and mortality, and no approved disease-modifying therapies exist. Pathological microglial activation drives secondary brain injury post-ICH. While lipid droplet accumulation in activated microglia is tightly linked to this process, the key metabolic drivers and upstream transcriptional regulators of this process in the perihematomal niche remain poorly defined. Here, we define the role of cholesterol esterification in microglial lipid droplet biogenesis after ICH and identify a critical transcriptional regulatory axis governing this process. Methods We integrated two independent single-cell transcriptomic datasets of murine ICH perihematomal tissues to profile differentially expressed genes and enriched pathways. The functional role of sterol O-acyltransferase 1 (SOAT1), the rate-limiting enzyme for cholesterol esterification, was validated using myeloid-specific Soat1 conditional knockout mice and in vitro microglial models. We further combined SCENIC regulatory network analysis with JASPAR and CISTROME database screening to identify upstream transcription factors of Soat1 , and validated the transcriptional regulatory effect of Spi1 on Soat1 via gain/loss-of-function assays and dual-luciferase reporter experiments. Results Single-cell transcriptomics revealed robust activation of cholesterol metabolic pathways in perihematomal microglia at day 7 post-ICH, which we identified as the major source of pathological lipid droplet formation. Soat1 was among the most highly upregulated cholesterol metabolism genes, with expression restricted almost exclusively to activated microglia. Genetic inhibition of Soat1 markedly suppressed microglial lipid droplet accumulation, and SOAT1 levels correlated directly with lipid droplet burden. Mechanistically, we identified SPI1 as a potential upstream transcription factor of Soat1 : Spi1 knockdown reduced SOAT1 protein expression, while Spi1 overexpression significantly enhanced Soat1 transcription. Dual-luciferase assays confirmed that Spi1 positively regulates Soat1 transcriptional activity. Conclusions Our findings demonstrate that SOAT1-mediated cholesterol esterification is a critical driver of microglial lipid droplet accumulation after ICH. We establish the Spi1 - Soat1 axis as a potential transcriptional regulator of this pathological process, providing a mechanistic candidate target for further investigation of post-ICH secondary brain injury.