Hypoxia is an important factor in the development of synovitis in rheumatoid arthritis (RA). The previous study of the research group found that monomeric derivatives of paeoniflorin (MDP) can alleviate joint inflammation in adjuvant-induced arthritis (AA) rats by inhibiting macrophage pyroptosis. This study revealed increased levels of hypoxia-inducible factor- (HIF-) 1α and N-terminal p30 fragment of GSDMD (GSDMD-N) in fibroblast-like synoviocytes (FLS) of RA patients and AA rats, while MDP significantly inhibited their expression. Subsequently, FLS were exposed to a hypoxic environment or treated with cobalt ion in vitro. Western blot and immunofluorescence analysis showed increased expression of G protein-coupled receptor kinase 2 (GRK2), HIF-1α, nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), ASC, caspase-1, cleaved-caspase-1, and GSDMD-N. Electron microscopy revealed FLS pyroptosis after exposure in hypoxia. Next, corresponding shRNAs were transferred into FLS to knock down hypoxia-inducible factor- (HIF-) 1α, and in turn, NLRP3 and western blot results confirmed the same. The enhanced level of GSDMD was reversed under hypoxia by inhibiting NLRP3 expression. Knockdown and overexpression of GRK2 in FLS revealed GRK2 to be a positive regulator of HIF-1α. Levels of GRK2 and HIF-1α were inhibited by eliminating excess reactive oxygen species (ROS). Furthermore, MDP reduced FLS pyroptosis through targeted inhibition of GRK2 phosphorylation. According to these findings, hypoxia induces FLS pyroptosis through the ROS/GRK2/HIF-1α/NLRP3 pathway, while MDP regulates this pathway to reduce FLS pyroptosis.

Background: Synovial hypoxia is a crucial pathological characteristic of rheumatoid arthritis (RA) that significantly contributes to synovitis and synovial hyperplasia. Fibroblast-like synoviocytes (FLS) respond to hypoxic conditions by undergoing adaptive changes that involve the modulation of gene expression, with hypoxia inducible factors (HIF) playing a pivotal role. The aim of this study was to investigate the molecular mechanism underlying the abnormal activation of FLS in response to hypoxia. Methods: The study employed Western blot and immunohistochemistry techniques to identify the presence of mitophagy in synovial tissue affected by rheumatoid arthritis. Mitophagy was further confirmed through the use of Western blot, immunofluorescence, qPCR, and CUT&Tag assays conducted under hypoxic conditions. Pyroptosis was observed through electron microscopy, fluorescence microscopy, and Western blot analysis. The level of ROS was assessed using flow cytometry and immunofluorescence. The silencing of HIF-1α and BNIP3 was achieved through the transfection of short hairpin RNA (shRNA) into cells. Chloroquine was utilized to inhibit mitochondrial autophagy. Results: The present study observed a significant increase in the expressions of BNIP3 and LC3B in the synovial tissue of patients with rheumatoid arthritis (RA). Following exposure to hypoxia, BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis were activated in fibroblast-like synoviocytes (FLS) of RA. Furthermore, the activation of mitophagy was found to significantly inhibit hypoxia-induced pyroptosis by reducing the intracellular content of reactive oxygen species (ROS). Conclusion: In summary, BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis are both involved in the activation of FLS in RA patients under hypoxia. And mitophagy could inhibits hypoxia-induced FLS pyroptosis by clearing ROS and inhibiting HIF-1α/NLRP3 pathway.

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