Zhen Qi,1,2,* Kongli Lu,1,* Mengxi Huang,3 Haixia Wang,4 Rongheng Liao,1,5 Ri Tang4 

1Department of Cardiovascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 2Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, People’s Republic of China; 3Jinshan Hospital, Fudan University, Shanghai, People’s Republic of China; 4Department of Critical Care Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 5Department of Cardiology, Erasmus Medical Center, Rotterdam, 3015GD, The Netherlands

*These authors contributed equally to this work

Correspondence: Ri Tang; Rongheng Liao, Email raytong7@163.com; ronanliao@163.com

Background: This study aimed to investigate the role of Radical S-adenosyl Methionine Domain-Containing 2 (RSAD2) in regulating the apoptosis of LPS-stimulated RAW264.7 macrophages via the NF-κB signaling pathway.
Methods: Differentially expressed genes in LPS-stimulated macrophages were identified using a gene expression dataset from the Gene Expression Omnibus (GEO) database and analyzed with R software. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to predict the biological functions of the identified genes. Key genes involved in NF-κB-mediated apoptosis regulation were selected for further investigation. The expression levels of Bcl-2, cleaved caspase-3, and NF-κB p65 were assessed by Western blotting. TUNEL staining was used to evaluate apoptosis.
Results: RSAD2 knockdown significantly improved cell viability and reduced apoptosis in LPS-stimulated RAW264.7 cells. Downregulation of RSAD2 increased Bcl-2 expression and inhibited cleaved caspase-3 activity, thus inhibiting apoptosis. Mechanistically, the downregulation of RSAD2 suppressed the NF-κB signaling pathway in vitro. Treatment with phorbol 12-myristate 13-acetate (PMA), an NF-κB agonist, reversed the protective effects of RSAD2 knockdown in LPS-stimulated RAW264.7 cells.
Conclusion: Our findings suggested that RSAD2 knockdown alleviated LPS-induced apoptosis in RAW264.7 macrophages by suppressing the NF-κB signaling pathway, highlighting RSAD2 as a potential therapeutic target for sepsis-related macrophage dysfunction.

Keywords: sepsis, apoptosis, NF-κB pathway, RAW264.7, lipopolysaccharide