Inhibiting EZH2 Alleviates Osteoarthritis and Pain in an Experimental Murine Model Through Modulating Synovial and Macrophage Inflammation, Axon Guidance, and Osteoclastogenesis

Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, has gained attention as a promising therapeutic target in osteoarthritis (OA) due to its central role in modulating inflammation, catabolism, and hypertrophy within chondrocytes. Previous studies have further demonstrated that EZH2 inhibition can slow OA progression in surgically induced mouse models, highlighting its potential in reducing joint degradation. However, the precise mechanisms by which EZH2 influences other key cell types in OA pathology remain poorly understood. In this study, we aimed to evaluate the effects of EZH2 inhibition in an alternative OA model and investigate its broader impact on cellular and molecular pathways across various tissues involved in OA progression and joint pain. OA was induced in mice via intra-articular injection of monosodium iodoacetate (MIA), with disease progression evaluated by histological and behavioral assessments. In parallel, human synoviocytes and bone marrow-derived cells were isolated from OA patients. Synoviocytes were stimulated with interleukin-1β (IL-1β) in the presence or absence of the EZH2 inhibitor EPZ-6438 (Tazemetostat), and ChIP-Seq and proteomic analyses were conducted to identify genomic and proteomic targets of EZH2. Additionally, the effects of EZH2 inhibition on M1 macrophage polarization and osteoclast differentiation were analyzed. Results revealed that EZH2 inhibition attenuated both OA progression and joint pain in the MIA-induced mouse model. IL-1β stimulation significantly upregulated EZH2 expression in synoviocytes, and treatment with the EZH2 inhibitor reduced the expression of genes linked to inflammation, pain, and catabolism while promoting autophagy. Proteomic analysis highlighted significant alterations in pathways related to IL-1β signaling, matrix metalloproteinase (MMP) activation, and autophagy, as well as changes in proteins associated with metabolic regulation and axon guidance. Importantly, EZH2 inhibition decreased M1 macrophage polarization and osteoclast formation, cellular processes that contribute to OA pain and inflammation. In conclusion, this study underscores the pivotal role of the histone methyltransferase EZH2 in the pathophysiology of osteoarthritis and associated joint pain. Our findings reveal that EZH2 inhibition not only attenuates inflammation in synovial cells and macrophages but also modulates axon guidance and osteoclastogenesis, both critical in OA progression and pain. These insights position EZH2 inhibition as a promising, multi-targeted therapeutic approach for addressing the complex cellular interactions underlying osteoarthritis, offering new hope for effective treatment strategies in this debilitating condition. ChIP-seq of EZH2 in fibroblast-like synoviocytes stimulated or not with IL-1b. Cells were collected from 2 different patients.

zeste同源增强子2（Enhancer of zeste homolog 2, EZH2）是一种组蛋白甲基转移酶（histone methyltransferase），因其在软骨细胞（chondrocytes）炎症、分解代谢与肥大调控中发挥核心作用，已成为骨关节炎（Osteoarthritis, OA）领域极具潜力的治疗靶点。既往研究进一步证实，在手术诱导的小鼠模型中，抑制EZH2可延缓OA进展，凸显其在减轻关节退变方面的应用价值。然而，EZH2如何影响OA病理过程中其他关键细胞类型的具体机制仍不甚明晰。
本研究旨在评估EZH2抑制在另一OA模型中的效应，并探究其对OA进展及关节疼痛相关多种组织内细胞与分子通路的广泛影响。
研究通过关节腔内注射碘乙酸单钠盐（monosodium iodoacetate, MIA）诱导小鼠OA模型，并利用组织学与行为学评估方法监测疾病进展。同时，从OA患者体内分离培养人滑膜细胞（human synoviocytes）及骨髓来源细胞（bone marrow-derived cells）。采用白细胞介素-1β（interleukin-1β, IL-1β）刺激滑膜细胞，同时设置EZH2抑制剂EPZ-6438（他泽司他，Tazemetostat）处理组与对照组，并通过染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-Seq）和蛋白质组学分析，鉴定EZH2的基因组与蛋白质组靶点。此外，本研究还分析了EZH2抑制对M1型巨噬细胞极化（M1 macrophage polarization）及破骨细胞分化（osteoclast differentiation）的影响。
研究结果显示，在MIA诱导的小鼠OA模型中，抑制EZH2可同时减轻OA进展与关节疼痛。IL-1β刺激可显著上调滑膜细胞中EZH2的表达，而EZH2抑制剂处理则可降低与炎症、疼痛及分解代谢相关的基因表达，同时促进自噬（autophagy）过程。蛋白质组学分析显示，IL-1β信号通路、基质金属蛋白酶（matrix metalloproteinase, MMP）激活及自噬相关通路均发生显著改变，同时代谢调控与轴突导向（axon guidance）相关蛋白的表达也出现变化。尤为重要的是，EZH2抑制可减少M1型巨噬细胞极化与破骨细胞生成，这两类细胞过程均与OA疼痛及炎症密切相关。
综上，本研究证实了组蛋白甲基转移酶EZH2在骨关节炎及其相关关节疼痛的病理生理过程中的关键作用。研究结果表明，EZH2抑制不仅可减轻滑膜细胞与巨噬细胞的炎症反应，还可调控轴突导向与破骨细胞生成——这两个过程均是OA进展与疼痛发生的关键环节。上述发现将EZH2抑制定位为一种极具潜力的多靶点治疗策略，可靶向调控骨关节炎背后复杂的细胞相互作用，为这种致残性疾病的有效治疗方案带来新的希望。
对经IL-1β刺激或未刺激的成纤维样滑膜细胞（fibroblast-like synoviocytes）进行EZH2的ChIP-Seq测序，细胞样本来自2名不同患者。