WTAP promotes fibroblast-like synoviocyte pyroptosis in Rheumatoid arthritis by upregulating N6-methyladenosine modification of NLRP3.

Rheumatoid arthritis (RA) is a chronic condition characterized by inflammation and an abnormal immune response. N6-methyladenosine (m6A) methylation has altered nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing (NLRP) 3. This change is implicated in the regulation of cell pyroptosis and inflammation. WTAP has a crucial role in regulating NLRP3 m6A. In this work, we used a rat model of collagen-induced arthritis (CIA) to investigate the involvement of WTAP in the evolution of inflammation in RA. The purpose of silencing or overexpressing WTAP in RA-fibroblast-like synoviocytes (RA-FLSs) treated with TNF-α was to identify its impact on pyroptosis, NLRP3 inflammasome-related proteins, the secretion of pro-inflammatory cytokines and migration. Bioinformatics techniques were used to pinpoint the exact target controlled by WTAP. To assess WTAP and NLRP3's role in RA-FLSs, we used methylated RNA immunoprecipitation, LDH test, flow cytometry, RT-qPCR, Western blotting, and Transwell. Our results show that WTAP expression is upregulated in both RA rats and cell models. Cell pyroptosis, NLRP3-related pro-inflammatory cytokines, and migration were reduced in TNF-α-treated RA-FLSs when WTAP was knocked down, whereas overexpression of WTAP displayed the opposite effect in RA-FLSs. WTAP mediated m6A modification in the NLRP3 mRNA and enhanced its mRNA stability. These results suggested that WTAP promoted FLSs pyroptosis and related inflammatory response via NLRP3 and identified WTAP as a potential target for treating RA.

Quantitative proteomic analysis of circulating exosomes reveals the mechanism by which Triptolide protects against collagen-induced arthritis.

INTRODUCTION: Triptolide (TP), a natural product derived from the herbal medicine Tripterygium wilfordii, exhibits potent immunosuppressive activity. However, the mechanisms underlying its effects in rheumatoid arthritis remain incompletely understood. METHODS: Collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by immunization with bovine type II collagen, and TP was administrated as treatment. The therapeutic effect of TP was evaluated based on paw swelling, histopathology, and serum levels of inflammatory factors. Exosomes isolated from rat serum were characterized by transmission electron microscopy, dynamic light scattering, and western blot analysis. Proteomic profiling of exosomes was analyzed by direct DIA quantitative proteomics analysis. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes databases were employed for enrichment analysis related to molecular function, biological processes, and signaling pathways. Western blot analysis was used to analyze differentially expressed proteins. RESULTS: TP treatment ameliorated arthritic phenotypes in CIA rats as evidenced by reduced arthritis score, paw swelling, pathological injury severity scores, and serum levels of inflammatory cytokines. The proteomic analysis revealed that TP treatment significantly inhibited complement and coagulation cascades, interleukin-17 signaling pathway, and cholesterol metabolism, which were reactivated in CIA rats. Importantly, lipocalin 2 (LCN2) and myeloperoxidase (MPO) levels were markedly upregulated in the CIA group but suppressed upon TP administration. Furthermore, in synovial tissues, LCN2 and MPO expression levels were also elevated in the CIA group but decreased following TP treatment. CONCLUSION: Our findings demonstrate that TP alleviates CIA, possibly through modulation of exosomal LCN2 and MPO proteins.

USP2 Promotes the Proliferation and Inflammation of Fibroblast-Like Synovial Cells in Rheumatoid Arthritis Through Deubiquitination of TRAF2.

Rheumatoid arthritis (RA) is a prevalent inflammatory disorder affecting about 1% of the global population. The ubiquitin-specific protease 2 (USP2) is known to have a substantial influence on the regulation of several cellular processes. Both in vivo (using rats with collagen-induced arthritis, CIA) and in vitro (using human fibroblast-like synoviocytes, HFLS-RA) models of RA were used to examine the role of USP2 in RA. The proliferation of HFLS-RA cells was assessed using the cell counting kit 8 test and EdU staining. The technique used for the assessment of gene expression was quantitative real-time PCR. Protein expression was quantified using Western blot (WB) analysis, while the quantities of inflammatory factors and matrix metalloproteinases were assessed using an ELISA test. The co-immunoprecipitation and ubiquitination tests investigated the relationships between proteins and the underlying molecular pathways. The results of this study demonstrate an upregulation of USP2 expression in both vivo and vitro models of RA. In addition, our findings indicate that the overexpression of USP2 notably exacerbates both proliferation and inflammation. The consistent downregulation of USP2 resulted in a reduction in the secretion of inflammatory cytokines and a suppression of cellular proliferation. Furthermore, it was shown that USP2 interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2) and facilitates the removal of ubiquitination chains from TRAF2, enhancing its stability. Our findings propose that USP2 functions as a favorable modulator of proliferation and inflammatory reactions in HFLS-RA, thereby indicating its potential as a therapeutic target for the treatment of RA.