Rheumatoid arthritis (RA) is characterized by synovial inflammation and progressive joint destruction. Neutrophil extracellular traps (NETs) and Toll-like receptor 4 (TLR4) signaling form an inflammatory amplification circuit that contributes to RA progression. Exosome-mediated delivery of non-coding RNAs plays a critical regulatory role within the joint microenvironment; however, the function of circular RNAs (circRNAs) derived from synovial mesenchymal stem cell (SMSC)-derived exosomes (SMSCs-Exo) in the TLR4/NETs-associated feedback axis remains unclear. This study aimed to elucidate the regulatory mechanism and pathological significance of SMSCs-Exo circRNF20 in the TLR4/NETs/IL-6 (interleukin-6) positive feedback loop.

Exosomes were isolated from SMSCs following lipopolysaccharide (LPS) stimulation and characterized by morphology, particle size distribution, and marker protein expression. The circular characteristics of circRNF20 were validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in combination with RNase R digestion and transcriptional inhibition assays. SMSCs-Exo were co-cultured with fibroblast-like synoviocytes (FLSs), and delivery dependency was verified using GW4869 and small interfering RNA targeting circRNF20 (si-circRNF20). Molecular targeting relationships were confirmed through miR-921 overexpression or inhibitor-based rescue experiments and dual-luciferase reporter assays targeting the wild-type or mutant TLR4 3′ untranslated region (TLR4-3′UTR). Western blot (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) were employed to assess TLR4 expression, NETs-related markers, IL-6 production, and activation of the NF-κB and STAT3 signaling pathways. FLS phenotypic changes were evaluated using CCK-8, EdU incorporation, cell cycle analysis, and TUNEL or Annexin V-propidium iodide (PI) assays. A collagen-induced arthritis (CIA) rat model was established, in which DNase I treatment or intra-articular injection of circRNF20-silenced synovial mesenchymal stem cell-derived exosomes (si-circRNF20-Exo) was administered to assess synovial pathology and pathway activity.

SMSCs-Exo were internalized by FLS and efficiently delivered circRNF20, a process inhibited by GW4869. Exosomal circRNF20 upregulated TLR4 expression and increased NETs-related markers and IL-6 levels, concomitant with activation of the NF-κB and STAT3 signaling pathways; silencing exosomal circRNF20 markedly attenuated these effects. Mechanistically, exosome treatment reduced miR-921 expression, and dual-luciferase reporter assays together with expression analyses confirmed that miR-921 directly targeted the TLR4-3′UTR; miR-921 overexpression reversed exosome-induced activation of the TLR4/NETs/IL-6 axis. Functionally, exosomes promoted FLS proliferation and cell-cycle progression while conferring resistance to apoptosis and enhancing pro-inflammatory secretion; DNase I significantly weakened these effects and simultaneously reduced TLR4 and IL-6 expression. In vivo experiments demonstrated that TLR4 expression, NETs-associated markers, and IL-6 levels were markedly elevated in CIA synovium; treatment with DNase I or si-circRNF20-Exo reduced NET burden, suppressed TLR4/IL-6 signaling, and alleviated synovial pathological damage.

SMSCs-Exo circRNF20 relieved miR-921-mediated negative regulation of TLR4, thereby activating the TLR4/NETs/IL-6 positive feedback loop and triggering the NF-κB/STAT3 signaling cascade, which promoted aberrant FLS proliferation, inflammatory phenotypes, and RA progression. Targeting circRNF20 or disrupting NET formation effectively suppressed this amplification circuit and mitigated CIA-associated pathological injury, providing experimental support for potential therapeutic strategies in RA.