Transcriptomic Identification and Validation of NDUFS2 in Acute Rat TMJ Synovitis and Its Functional Role in LPS-Induced Synovial Fibroblast Inflammation

Abstract

Purpose: Temporomandibular joint (TMJ) synovitis persistently drives TMJ osteoarthritis (TMJOA) progression. Aberrant reactive oxygen species (ROS) accumulation triggers chronic oxidative stress, amplifying synovial inflammation. This study aimed to identify candidate ROS-related genes through transcriptomic screening and explore their potential functional involvement in TMJ synovitis. Methods: Acute TMJ synovitis was induced in male Sprague-Dawley rats via intra-articular injection of Complete Freund’s Adjuvant (CFA). RNA sequencing of inflamed and normal synovial tissues was performed to screen target genes by intersecting differentially expressed genes (DEGs) with ROS-related genes (ROS-RGs). The expression of the candidate gene was validated in vivo and in vitro, and in vitro functional assays were performed using lipopolysaccharide (LPS)-induced synovial fibroblasts (SFs). The functions of NDUFS2 were assessed via siRNA knockdown or plasmid overexpression, supplemented by rescue experiments using the mitochon-dria-targeted antioxidant Mito-TEMPO. Intracellular ROS, mitochondrial ROS (mtROS), mitochondrial membrane potential (ΔΨm), and pro-inflammatory markers (MMP13, IL-1β, TNF-) were evaluated. Results: NDUFS2 was identified as a candidate ROS-related gene through transcriptomic screening, and Gene Set Enrichment Analysis (GSEA) linked it to signaling pathways including oxidative phosphorylation. NDUFS2 was markedly upregulated in inflamed TMJ synovial tissues and LPS-stimulated SFs. In the LPS-induced model, NDUFS2 knockdown attenuated ROS/mtROS accumulation, partially restored ΔΨm, and reduced pro-inflammatory marker expression, whereas NDUFS2 overexpression could increase ROS/ mtROS levels, reduce ΔΨm, and elevate pro-inflammatory marker expression. Furthermore, rescue experiments demonstrated that scavenging mtROS with Mito-TEMPO reversed NDUFS2-mediated oxidative stress and inflammation. Conclusion: Our findings identify NDUFS2 as a key ROS-related gene associated with acute TMJ synovitis through transcriptomic screening. In vitro evidence indicates that NDUFS2 modulates inflammatory responses in synovial fibroblasts via mtROS-mediated mitochondrial oxidative stress. These findings provide an experimental basis for further exploration of TMJ synovitis pathogenesis.