BET蛋白对超负荷压力诱导的大鼠颞下颌关节骨关节炎样病变的表观遗传调控

Temporomandibular joint (TMJ) osteoarthritis (OA)-liked pathological change can be induced by overloading mechanical stress, which including condylar cartilage degradation and subchondral bone resorption. In our previous study, we found mandibular chondrocytes death induced by mechanical stress is closely related to the local inflammation, but the molecular mechanism of up-regulated inflammatory factors is unclear. Recently, the epigenetic regulation of inflammation is becoming the concern in this field. As a reader of histone post-translational modification (PTM), BET family can epigenetically regulate the downstream genes of NF-kB which are related to inflammatory factors and osteoclast differentiation and it is proved that BET inhibitor JQ1 plays an important role in many inflammatory diseases. We found that JQ1 can effectively restore cartilage thickness and inhibit expression of inflammatory factors after overloading mechanical stress, but the mechanism is unclear. We hypothesize that BET plays an important role in mechanical stress induced TMJ OA-like changes and inhibiting BET can relieve inflammation and alleviate TMJ OA-like changes on the epigenetic level. In this present study, we will use existed compressive mechanical stress model combined with RNAi and inhibitor to study the mechanism and the clinical significance of BET in the TMJ OA-like pathological changes trying to explore new epigenetic therapy target in treating TMJ disease.

超负荷压应力可以诱发颞下颌关节（TMJ）骨关节炎样病变，多表现为关节软骨和软骨下骨的破坏。在前期研究中，我们观察到压力刺激下髁突软骨细胞的死亡和炎症反应密切相关，但炎症因子上调的分子机制尚不明确。近来，受表观遗传调控的炎症反应受到了密切关注。作为组蛋白翻译后修饰的“阅读者”， BET家族能调控NF-κB下游炎性因子表达及破骨过程，其特异性抑制剂JQ1在许多炎症性疾病中表现出强大作用。据此，我们提出了科学假说，BET蛋白能在表观遗传水平上调控超负荷压力诱导的TMJ骨关节炎样病变，抑制BET能减轻炎症反应和骨吸收。我们的预实验结果也初步证实了JQ1能有效地恢复压力源性变薄的髁突软骨厚度，但其具体作用机制仍不清楚。因此，本课题拟采用动物模型、细胞培养，结合小分子抑制剂、RNAi等技术研究BET在压力源性TMJ骨关节炎样病变过程中的具体作用机制，阐明其临床意义，寻找新的表观遗传治疗手段。

溴区结构域额外末端蛋白家族成员BRD4是表观遗传信息的阅读者，近年来研究证明其在炎症及骨吸收相关疾病中具有一定的保护作用。本课题旨在探索BRD4抑制对超负荷压应力介导的颞下颌关节骨关节炎（temporomandibular joint osteoarthritis, TMJ OA）的保护作用及其机制。体内实验部分，我们使用大鼠TMJ压应力加载模型，配合注射BRD4抑制剂JQ1。利用HE染色及Micro-CT对髁突的组织形态及骨小梁结构进行评估，利用免疫组化和qPCR来检测炎症因子表达的变化。我们还进行了ChIP-seq高通量的测序来比较加力后BRD4和H3K27ac与DNA的结合变化。体外实验部分，我们采用IL-1β处理大鼠原代TMJ髁突软骨细胞，配合使用siBRD4以及siTREM1，利用qPCR来检测炎症因子的变化。我们发现BRD4抑制可以显著缓解超负荷应力介导的髁突软骨变薄、软骨下骨骨吸收以及炎症因子TNF-α、IL-1β、IL-6表达。ChIP-seq结果显示在加力后BRD4更多的富集在与炎症相关的基因的启动子区域，其中包括Trem1。Trem1作为一种促炎基因，其启动子区域可以同时结合BRD4和H3K27ac。体内及体外实验都证实Trem1受到BRD4的调控。体外实验我们进一步证实TREM1对IL-1β诱导的软骨细胞炎症因子表达具有调控作用。综上所述，我们认为BRD4抑制可以缓解超负荷应力介导的TMJ OA病理变化，并且缓解TREM1介导的软骨细胞炎症损伤。