Ms4a4c/Stat3/Gpx4 Regulates Microglial Iron Homeostasis In Pulpitis Pain

Understanding the central mechanisms of pulpitis pain and developing targeted analgesic drugs. Adult male C57BL/6 mice (20-26g) were used to establish pulpitis models, with pain thresholds assessed via filaments and medullary dorsal horn (MDH) Fe²⁺ levels quantified by ferrozine assay. . Western blotting and immunofluorescence analyzed Iba-1, GPX4, HIF-1α, IL-1β, and TNF-α expression. In vitro, LPS-stimulated microglia were evaluated for iron content, LPO and GPX4 activity, followed by Fer-1 treatment validation. Single-cell RNA sequencing (10x Genomics) of MDH tissues identified cell clusters via Seurat, with GO enrichment and DEG analysis. Lentiviral shRNA knockdown of Ms4a4c/STAT3 preceded GPX4 measurement. Finally, engineered Ms4a4cscFv-BNVGPX4-saRNA vesicles selectively targeted Ms4a4c-high microglia to restore GPX4 and assess analgesia. (Ethics number: 2023JCZD56) Following pulp exposure, mice exhibited reduced pain thresholds, accompanied by increased microglial activation, elevated iron levels, and decreased GPX4 expression in the MDH, alongside upregulation of HIF-1α, IL-1β, and TNF-α. Single-cell RNA sequencing identified nine microglial subpopulations, with the Ms4a4c-high C0 cluster showing the most pronounced changes during pulpitis pain and significant enrichment of oxidative stress pathways. Lentiviral silencing of Ms4a4c elevated STAT3 and GPX4 expression, while STAT3 inhibition reduced GPX4, and the engineered Ms4a4cscFv-BNVGPX4-saRNA vesicles are efficiently internalized by microglia with high Ms4a4c expression. After pulpitis, elevated Ms4a4c expression in MDH microglial C0 subgroup suppresses the STAT3/GPX4 pathway, triggering iron accumulation and microglial activation. This subsequently promotes IL-1β/TNF-α release, ultimately driving pulpitis-associated nociception.