Pax6 Drives Orbital Fibrosis in Thyroid-Associated Orbitopathy via Fatty Acid Metabolism-Mediated Epithelial-Mesenchymal Transition in Mice

Abstract Background Thyroid-associated orbitopathy (TAO) is an autoimmune disorder characterized by inflammation, fibrosis, and adipogenesis in the orbital tissues, driven primarily by activated orbital fibroblasts (OFs).This study aims to investigated the link between fatty acid metabolism, Pax6 expression, and EMT/fibrosis in TAO; determine the functional role of Pax6 in regulating OF activation, proliferation, migration, and fibrogenesis both in vivo and in vitro ; and explore Pax6 as a potential therapeutic target. Methods Our study employs single-cell RNA sequencing (scRNA-seq) to characterize cellular heterogeneity and identify pathogenic subpopulations and their metabolic profiles, aiming to elucidate the relationship between fatty acid metabolism, Pax6 expression, and EMT in the context of TAO. Results Using scRNA-seq on TAO and normal mouse orbital tissues, we identified distinct cellular populations, including a pathogenic epithelial-like cell subcluster (EPC1) marked by enhanced fatty acid metabolism and EMT markers. Pax6 expression increased along the EMT trajectory, especially in mesenchymal cells. In vivo experiments using a TAO mouse model demonstrated that Pax6 knockdown reduced orbital fibrosis, decreased pro-fibrotic markers (α-SMA, COL1A1, FN1), and inhibited OF activation and migration. Additionally, primary OF cultures showed that Pax6 regulates cellular proliferation and fibrogenesis in vitro . Conclusion Our findings suggest that dysregulated fatty acid metabolism in TAO enhances Pax6 expression. This, in turn, promotes EMT and contributes to orbital fibrosis. Targeting the Pax6–fatty acid metabolism axis represents a novel therapeutic strategy to mitigate the TAO-related orbital fibrosis. This study establishes Pax6 as a key driver of EMT and fibrosis in TAO and highlights its potential as a future therapeutic target.