Artemisinin Derivatives Modulate KEAP1-NRF2-xCT Pathway to Alleviate Sjögren's Disease: Insights from ScRNA-seq and Systems Biology

Yong Luo^1*Liuting Zeng^1,2*Yanan Wang¹Qianyue Yang³Chang Liu⁴Genhong Yao⁴Xiaojun Tang⁴Lingyun Sun^1,4,5*

• ¹Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, China
• ²Nanjing Drum Tower Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China
• ³Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
• ⁴Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, Nanjing, China
• ⁵Department of Rheumatology and Immunology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

Objective: This study explores the therapeutic effects of Artesunate (ART) on Sjögren's Disease in NOD/Ltj mice by modulating ferroptosis in salivary gland epithelial cells (SGECs). Methods: Female NOD/Ltj mice at 8 weeks of age were randomly divided into two groups: a model group treated with saline and an ART group receiving oral gavage of ART. Daily water intake, weekly salivary flow rate, and body weight were monitored. At the end of the 8-week treatment period, mice were euthanized, and spleen and submandibular gland indices were assessed. scRNA-seq was used to analyze the characteristic profiles of SJD patients, while RNA-seq evaluated inflammatory pathway signaling responses following ART treatment. Histological examination included HE staining of submandibular glands, scoring based on lymphocytic infiltration. Western blotting detected KEAP1, TFRC, xCT, NRF2, and GPX4 expression levels in submandibular glands and SGECs. Immunofluorescence was employed to examine IgG, C3, KEAP1, TFRC, NRF2, xCT, and GPX4 expression in submandibular glands, as well as ROS and JC-1 RED/GREEN levels in SGECs. Molecular docking assessed the affinity between ART and KEAP1, and transmission electron microscopy evaluated mitochondrial morphology. Results: scRNA-seq and systems biology analyses revealed robust activation of ferroptosis signaling pathways post-ART treatment. In vitro and in vivo studies further confirmed that ART inhibited the ubiquitination and degradation of NRF2 by KEAP1, upregulated xCT and GPX4 protein expression, and suppressed TFRC levels. Consequently, ART significantly protected SGECs from ferroptosis in NOD/Ltj mice, effectively preventing glandular damage and maintaining functional integrity.