Jin Yu, Li Zhenxia, Wu Yanran, Li Hairui, Liu Zhen, Liu Lu, Ouyang Ningjuan, Zhou Ting, Fang Bing, Xia Lunguo
Department of Orthodontics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai, 200011, People's Republic of China.
J Inflamm Res. 2021 Nov 19;14:6067-6083. doi: 10.2147/JIR.S339382. eCollection 2021.
Osteoarthritis (OA) is a common disease for human beings, characterized by severe inflammation, cartilage degradation, and subchondral bone destruction. However, current therapies are limited to relieving pain or joint replacement and no effective treatment methods have been discovered to improve degenerative changes. Currently, a variety of evidences have indicated that aberrant mechanical stimuli is closely associated with articular joint pathogenesis, while the detailed underlying mechanism remains unelucidated. In the present study, we determined to investigate the impact of excessive high fluid shear stress (FSS) on primary chondrocytes and the underlying epigenetic mechanisms.
Phalloidin staining and EdU staining were used to evaluate cell morphology and viability. The mRNA level and protein level of genes were determined by qPCR, Western blot assay, and immunofluorescence staining. Mechanistic investigation was performed through RNA-sequencing and CUT&Tag sequencing. In vivo, we adopted unilateral anterior crossbites (UAC) mice model to investigate the expression of H3K4me3 and ZBTB20 in aberrant force-related cartilage pathogenesis.
The results demonstrated that FSS greatly disrupts cell morphology and significantly decreased chondrocyte viability. Aberrant FSS induces remarkable inflammatory mediators production, leading to cartilage degeneration and degradation. In depth mechanistic study showed that FSS results in more than 10-fold upregulation of H3K4me3, and the modulatory effect of H3K4me3 on cartilage was obtained by directly targeting ZBTB20. Furthermore, Wnt signaling was strongly activated in high FSS-induced OA pathogenesis, and the negative impact of ZBTB20 on chondrocytes was also achieved through activating Wnt signaling pathway. Moreover, pharmacological inhibition of H3K4me3 activation by MM-102 or treatment with Wnt pathway inhibitor LF3 could effectively alleviate the destructive effect of FSS on chondrocytes. In vivo UAC mice model validated the dysregulation of H3K4me3 and ZBTB20 in aberrant force-induced cartilage pathogenesis.
Through the combination of in vitro FSS model and in vivo UAC model, KMT2B-H3K4me3-ZBTB20 axis was first identified in aberrant FSS-induced cartilage pathogenesis, which may provide evidences for epigenetic-based therapy in the future.
骨关节炎（OA）是一种常见的人类疾病，其特征为严重炎症、软骨降解和软骨下骨破坏。然而，目前的治疗方法仅限于缓解疼痛或关节置换，尚未发现有效的治疗方法来改善退行性改变。目前，各种证据表明异常机械刺激与关节发病机制密切相关，但其详细的潜在机制仍未阐明。在本研究中，我们决定研究过高的流体剪切应力（FSS）对原代软骨细胞的影响及其潜在的表观遗传机制。
使用鬼笔环肽染色和EdU染色来评估细胞形态和活力。通过qPCR、蛋白质免疫印迹分析和免疫荧光染色来测定基因的mRNA水平和蛋白质水平。通过RNA测序和CUT&Tag测序进行机制研究。在体内，我们采用单侧前牙反合（UAC）小鼠模型来研究H3K4me3和ZBTB20在异常力相关软骨发病机制中的表达。
结果表明，FSS极大地破坏细胞形态并显著降低软骨细胞活力。异常的FSS诱导大量炎症介质产生，导致软骨退变和降解。深入的机制研究表明，FSS导致H3K4me3上调超过10倍，并且H3K4me3对软骨的调节作用是通过直接靶向ZBTB20实现的。此外，Wnt信号在高FSS诱导的OA发病机制中被强烈激活，ZBTB20对软骨细胞的负面影响也是通过激活Wnt信号通路实现的。此外，用MM-102抑制H3K4me3激活或用Wnt通路抑制剂LF3处理可有效减轻FSS对软骨细胞的破坏作用。体内UAC小鼠模型验证了H3K4me3和ZBTB20在异常力诱导的软骨发病机制中的失调。
通过体外FSS模型和体内UAC模型相结合，首次在异常FSS诱导的软骨发病机制中鉴定出KMT2B-H3K4me3-ZBTB20轴，这可能为未来基于表观遗传学的治疗提供证据。
