Zhuo Zhao,1,* Ziyu Liu,2,* Qun Wang,3,4 Hao Gao,1 Nan Song,5 Xiao Yang1,2 

1The Second Clinical College of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China; 2The Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine/Liaoning Institute of Traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China; 3National and Local Joint Engineering Laboratory for Integrated Chinese and Western Medicine Prevention and Treatment Technology on Cardio-Brain Diseases, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China; 4Key Laboratory of Ministry of Education for TCM Viscera-State Theory and Applications, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China; 5College of Medical Laboratory, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Nan Song, Liaoning University of traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China, Tel +8613840575015, Email cpcool@126.com Xiao Yang, Liaoning University of traditional Chinese Medicine, Shenyang, Liaoning Province, People’s Republic of China, Tel +8613840575015, Email yangxiaodyx@aliyun.com

Purpose: Autoimmune thyroiditis (AIT) is the most common organ-specific autoimmune disease, and its pathogenesis is closely related to the inflammatory microenvironment driven by immune cell penetration. The role of the newly proposed concept of PANoptosis in immune-related diseases is gradually being revealed. However, there is currently a lack of reports on PANoptosis in AIT. This study aims to clarify the relationship between PANoptosis gene, cell subgroup distribution, immune penetration, and AIT through a comprehensive analysis of scRNA-seq and bulk RNA-seq data combined with animal and clinical validation.
Patients and Methods: Initially, we integrated bulk RNA-seq and scRNA-seq data from AIT in public databases to identify immune cell subpopulations and the distribution and abundance of PANoptosis within them. Subsequently, we applied ssGSEA to assess the association between immune cell infiltration and inflammatory responses in AIT patients versus healthy individuals. Furthermore, we utilized the WGCNA tool to integrate PANoptosis genes with immune functions and screened for gene modules most significantly correlated with the immune-inflammatory effects of AIT. Finally, an animal model was established and clinical samples were collected for RT-qPCR, immunohistochemical staining,Enzyme-linked immunosorbent assay (ELISA) and ROC curve to predict the diagnostic value for further verification.
Results: Through bioinformatics analysis, we identified 14 functionally heterogeneous cell subpopulations and 5 differentially expressed PANoptosis-related genes (AIM2, ZBP1, NLRP6, MLKL, and FAS). Experimental validation revealed that these differentially expressed genes were significantly upregulated in autoimmune thyroiditis (AIT). Moreover, they might promote the infiltration of inflammatory lymphocytes and the secretion of inflammatory cytokines in thyroid tissue through the PANoptosis pathway, with AIM2 potentially playing a central role.
Conclusion: In summary, our study reveals the characteristics of the immune microenvironment of AIT and highlights the clinical potential of PANoptosis-Related genes (AIM2, ZBP1, NLRP6, MLKL, and FAS) as diagnostic biomarkers.

Keywords: autoimmune thyroiditis, PANoptosis, single-cell RNA sequencing, bulk RNA-sequencing, immune microenvironment, biomarkers