Department of Rheumatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China.
Department of Internal Medicine, The Third People's Hospital of Chongming District, Shanghai 202153, China.
Int Immunopharmacol. 2024 Dec 25;143(Pt 1):113201. doi: 10.1016/j.intimp.2024.113201. Epub 2024 Sep 30.
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.
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).
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, 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.
Targeting EZH2 attenuates the ferroptosis-mediated osteoblast-osteoclast imbalance in RA, revealing its potential as a treatment target.
增强子结合锌指蛋白 2 多梳抑制复合物 2 亚基（EZH2）可调节成骨和破骨细胞分化。本研究旨在进一步探讨 EZH2 修饰对类风湿关节炎（RA）体外和体内铁死亡和破骨细胞-成骨细胞平衡的影响。
用 EZH2 过表达（oeEZH2）和 EZH2 shRNA（shEZH2）质粒转染骨髓间充质基质细胞，并进行成骨细胞分化实验。然后将细胞与骨髓来源的巨噬细胞共培养，并进行破骨细胞分化实验。构建胶原诱导性关节炎（CIA）小鼠，并注射 shEZH2 腺相关病毒（AAV）。
oeEZH2 抑制成骨细胞分化，表现为碱性磷酸酶（ALP）和茜素红 S 染色减少，骨桥蛋白（OPN）、RUNX2、骨保护素（OPG）和核因子 κB 受体活化因子配体（RANKL）增加；而 shEZH2 则产生相反的作用。此外，oeEZH2 促进成骨细胞铁死亡，表现为丙二醛（MDA）、铁（Fe）、活性氧（ROS）和前列腺素内过氧化物合酶 2（PTGS2）增加，谷胱甘肽过氧化物酶 4（GPX4）和溶质载体家族 7 成员 11（SLC7A11）减少；这些作用可以被 Ferrostatine-1（Fer-1）治疗所减弱。相反，shEZH2 减轻了成骨细胞的铁死亡。oeEZH2 随后增加了破骨细胞的分化，表现为破骨细胞特异性抗酒石酸酸性磷酸酶（TRAP）多核细胞、核因子κB 受体活化因子（NFATC1）、组织蛋白酶 K（CTSK）和 c-FOS 增加；然而，shEZH2 产生了相反的作用，只是它没有调节 CTSK。在 CIA 小鼠中，shEZH2 AAV 降低了临床症状评分、软骨组织学评分和全身炎症（TNF-α 和 IL-6），并在一定程度上抑制了骨铁死亡，恢复了成骨细胞-破骨细胞的平衡，这反映在相关标志物的免疫组织化学染色上。
靶向 EZH2 可减轻 RA 中铁死亡介导的成骨细胞-破骨细胞失衡，为其作为治疗靶点提供了依据。
