Integrated single-cell analysis with experimental validation reveals ANXA2 as a therapeutic target for ferroptosis in...

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by intestinal dysfunction. Ferroptosis is a critical pathogenic mechanism in IBD. However, the therapeutic targets for ferroptosis-related IBD progression remain unclear. Therefore, this study aimed to identify potential therapeutic targets associated with ferroptosis in IBD.

Single-cell RNA sequencing data (GSE134809) were analyzed using gene set scoring, cell-cell communication, pseudotime analysis, and high-dimensional gene co-expression network analysis (hdWGCNA) to screen for ferroptosis-related targets. In vitro experiments, including RT-qPCR, western blotting, flow cytometry, and ELISA, were performed to verify the regulatory role of annexin A2 (ANXA2) in ferroptosis and inflammation using its silencing or overexpression. For in vivo validation, a dextran sulfate sodium (DSS)-induced IBD mouse model was established. Immunofluorescence (IF) staining was then performed to examine ANXA2 expression and its co-localization with collagen type I alpha 2 chain (COL1A2) and 4-hydroxynonenal (4-HNE) in colon tissues.

Bioinformatic analysis of 28,974 cells identified that fibroblasts, particularly the Fibro_2 subpopulation, were highly associated with ferroptosis, with ANXA2 identified as a core target. In vitro, ANXA2 silencing significantly inhibited ferroptosis, oxidative stress, and inflammatory factors interleukin-6 (IL-6) and C-X-C Motif Chemokine Ligand 8 (CXCL8), whereas ANXA2 overexpression demonstrated the opposite effects. In vivo, ANXA2 was significantly up-regulated in the colon tissues of IBD mice, showing strong co-localization with the fibroblast marker COL1A2 and the ferroptosis marker 4-HNE.

ANXA2 is highly expressed in fibroblasts and is associated with the ferroptosis of IBD, providing a novel therapeutic target for treatment of IBD.