The HIF-1/ BNIP3 pathway mediates mitophagy to inhibit the pyroptosis of fibroblast-like synoviocytes in rheumatoid arthritis

Abstract 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.