Targeting EZH2 attenuates the ferroptosis-mediated osteoblast-osteoclast imbalance in rheumatoid arthritis.

Objective: The enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) can regulate osteogenesis and osteoclastogenesis. This study aimed to further explore the effects of EZH2 modification on ferroptosis and the osteoblast-osteoclast balance in rheumatoid arthritis (RA) in vitro and in vivo.
Methods: Bone marrow mesenchymal stromal cells were transfected with EZH2 overexpression (oeEZH2) and EZH2 shRNA (shEZH2) plasmids with or without ferrostatin-1 (Fer-1) treatment and subjected to an osteoblast differentiation assay. The cells were then cocultured with bone marrow-derived macrophages and subjected to an osteoclast differentiation assay. Collagen-induced arthritis (CIA) mice were generated and injected with shEZH2 adeno-associated virus (AAV).
Results: OeEZH2 repressed osteoblast differentiation, as reflected by decreased ALP and Alizarin Red S staining and increased OPN, RUNX2, OPG and RANKL; however, shEZH2 had the opposite effects. Besides, oeEZH2 promoted osteoblast ferroptosis, as suggested by increased MDA, Fe ²⁺ , ROS, and PTGS2 but decreased GPX4 and SLC7A11; these effects could be attenuated by Fer-1 treatment. In contrast, shEZH2 ameliorated osteoblast ferroptosis. OeEZH2 subsequently increased osteoclast differentiation, as indicated by increased TRAP ⁺ multinucleated cells, NFATC1, CTSK, and c-FOS; however, shEZH2 had the opposite effect, except that it did not regulate CTSK. In CIA mice, shEZH2 AAV decreased the clinical symptom score, histological score of cartilage, and systemic inflammation (TNF-α and IL-6) and repressed bone ferroptosis and restored the osteoblast-osteoclast balance to some extent, as reflected by immunohistochemical staining of related markers.
Conclusion: Targeting EZH2 attenuates the ferroptosis-mediated osteoblast-osteoclast imbalance in RA, revealing its potential as a treatment target.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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