PERK-eIF2α介导的内质网应激-自噬通路参与调控正畸牙移动压力区骨细胞（osteocyte）损伤研究

Orthodontic tooth movement (OTM) is clinically performed to correct malocclusion, and its rate-limiting step is considered to be bone resorption at the compression side of alveolar bone. Recent studies have demonstrated that the orthodontic force-induced injuries of osteocyte, which is believed to be the primary sensor of mechanical stimuli in bone, initialized the bone resorption at the compression side. However, the underline mechanism is still unclear. Our preliminarily results showed that orthodontic force activated both the PERK/eIF2α arm of endoplasmic reticulum stress (ERS) response and the autophagy at the OTM compression side. It also indicated that the compressive force caused apoptotic cell death of osteocyte, while the selective inhibitor of eIF2α (salubrinal) could restore the compressive force-induced autophagy in MLO-Y4 osteocytes. Accordingly, in order to elucidate the damage of osteocytes suffered from orthodontic force, we will observe the effect of orthodontic force on osteocyte function and analyze the level of key proteins of both ERS and autophagy at the compression side in vivo or under compressive force in vitro. Then, we will also explore the role of PERK-eIF2α mediated ERS-autophagy signaling cascade in the orthodontic force-induced osteocyte injuries and alveolar bone resorption by blocking such pathway with RNA interference or pharmacological inhibitors. This study will provide new strategies for promoting bone resorption at the compression side of OTM as well as accelerating of tooth movement via selective regulation of the PERK/eIF2α-mediated ERS-autophagy pathway in osteocyte.

应用矫治器产生正畸力、实现牙移动是治疗错颌畸形的重要手段，而压力区牙槽骨骨吸收缓慢是正畸牙移动的限速步骤。新近研究证实正畸力致力学敏感细胞—骨细胞损伤是压力区牙槽骨骨吸收的关键启动事件，但其调控机制不明。我们前期研究表明正畸力激活压力区骨细胞PERK-eIF2α内质网应激（ERS）通路、活化自噬并诱导细胞凋亡，且eIF2α抑制剂可减轻压力致骨细胞自噬。据此，本项目拟从动物、细胞和分子水平观察正畸力在牙移动初期对压力区骨细胞功能的影响，分析ERS-自噬通路关键蛋白表达，明确正畸力致牙移动压力区骨细胞损伤；通过RNA干扰或小分子抑制剂阻断PERK-eIF2α介导的ERS-自噬通路，研究干预该通路对牙移动压力区骨细胞损伤和牙槽骨骨吸收的影响，阐明ERS-自噬通路参与调控机理。本研究将为以骨细胞为靶细胞，通过调控PERK-eIF2α介导的ERS-自噬通路促进压力区骨吸收、提高临床正畸效率提供新思路

在正畸治疗中，正畸力作用于牙齿和牙周组织，诱导牙槽骨压力区的骨吸收使牙齿产生移动。然而，牙槽骨骨吸收缓慢造成的患者牙齿移动慢是正畸临床面临的一大难题。因此，有效调控压力区牙槽骨骨吸收进程将有助于加速牙齿移动、提高临床正畸效率。骨细胞是牙槽骨内含量最丰富的力学敏感细胞，我们前期研究显示正畸力可造成压力区骨细胞损伤，但正畸力通过何种机制调控压力区骨细胞损伤尚未完全阐明。本项目应用正畸镍钛拉簧建立大鼠实验性牙移动在体模型，通过组织学染色和Micro-CT等方法观察牙移动压力区的组织学和结构变化，利用ELISA和化学比色法检测血清中骨细胞相关细胞因子骨硬化蛋白（Sclerostin，SOST）、核因子κB受体活化因子配体（Receptor Activator for Nuclear Factor-κB Ligand，RANKL）水平和总抗氧化能力、脂质氧化等氧化损伤指标的改变。另外，构建体外骨细胞压力加载模型，并利用该模型观察压力对骨细胞的损伤作用，检测PERK-eIF2α介导的内质网应激-自噬通路蛋白表达的变化。同时探讨该通路的抑制剂和激活剂干预对压力造成的骨细胞损伤的影响。动物实验结果显示，与对照组相比，大鼠OTM模型压力区内因骨细胞死亡造成的空骨陷窝数量随时间和正畸力增大而上升，压力区骨密度下降；SOST和RANKL等骨吸收相关细胞因子表达上升，提示正畸力可导致压力区骨细胞损伤和牙槽骨骨吸收。体外压力加载实验表明，压力能导致骨细胞的细胞活性、细胞膜完整性和线粒体膜电位下降，并造成细胞凋亡率及促凋亡蛋白表达上升。该压力还能激活PERK-eIF2α介导的内质网应激-自噬通路，表现为GRP78、p-PERK、p-eIF2α、LC3II/LC3I和P62蛋白水平上升。内质网应激抑制剂能减轻压力所致的骨细胞细胞活性和细胞膜完整性下降、抑制自噬流受阻和细胞凋亡；自噬抑制剂3-MA和激活剂Rapamycin分别加重和减轻了压力所致的骨细胞损伤。该项目研究结果基本证实了我们所提出的假说：在正畸牙移动过程中，压力通过PERK-eIF2α介导的内质网应激-自噬通路调控骨细胞损伤，进而调控压力区牙槽骨骨吸收和牙移动进程。因此，我们认为骨细胞参与牙移动压力区牙槽骨骨吸收调控，研究成果为揭示压力区牙槽骨骨吸收调控的相关机制提供了数据支持，并为临床调节牙槽骨骨吸收进程提供了可能的靶细胞和靶点。