Xuemei Li Chao Xiong Siyi Wang ^* Zhangjun Ren ^* Qi Jin ^* Jinhai Yu Yunxiu Chen ^* Puying Gan ^* Qihua Xu ^* Yaohua Wang ^* Hongfei Liao ^*

• Affiliated Eye Hospital of Nanchang University, Nanchang, China

Background: Thyroid-associated orbitopathy (TAO) is an autoimmune inflammatory disorder affecting orbital adipose tissue, leading to oxidative stress and tissue remodeling. Ferroptosis, a form of programmed cell death, is driven by reactive oxygen species (ROS) accumulation, iron metabolism disorders, and lipid peroxidation. This study aims to identify and validate optimal feature genes (OFGs) of ferroptosis with diagnostic and therapeutic potential in TAO orbital adipose tissue using bioinformatics analysis and to assess their correlation with immune cell infiltration. Methods: The GSE58331 dataset was selected from the Gene Expression Omnibus for differential gene expression analysis. WGCNA identified key disease modules and hub genes, which were intersected with ferroptosis-related genes to find key ferroptosis genes. Machine learning algorithms identified OFGs. Additionally, comparisons of FRG expression in orbital adipose tissue and orbital fibroblasts (OFs) from healthy controls and TAO patients, along with macrophage-OF co-culture experiments, explored the influence of macrophages on FRGs in OFs. CIBERSORT was used to analyze immune cell proportions, and Spearman correlation examined the relationship between OFGs and immune cells. GSEA determined the function of each key biomarker based on OFG expression levels. Results: Three TAO FRGs (ACO1, MMD, and HCAR1) were screened in the dataset.The ROC results of ACO1 showed that the AUC value was greater than 0.8 in all the datasets, which was the strongest for disease specificity and diagnostic ability.Validation results showed that, in addition to MMD, the expression of ACO1 and HCAR1 in orbital adipose tissue of TAO patients was significantly down-regulated, while M2-type macrophages might be involved in regulating the expression of ACO1 in orbital adipose-derived OFs. CIBERSORT immune cell infiltration analysis showed that in orbital adipose tissue of TAO patients, memory B-lymphocytes, T regulatory cells, NK-cells, M0-type macrophages, M1-type macrophages, resting dendritic cells, activated mast cells, and neutrophils infiltration levels were significantly elevated. Conclusion: Through bioinformatics analysis, this study identified and validated two OFGs of ferroptosis with diagnostic and therapeutic potential in TAO orbital adipose tissue, suggesting that the downregulation of ACO1 and HCAR1 may be potential molecular targets in the pathogenesis of TAO.