RIP1/RIP3介导的程序性坏死在压力源性炎症及髁突软骨降解病理过程中的作用机制研究

The loss of chondrocytes is the key to induce temporomandibular joint (TMJ) condylar cartilage degradition induced by overload compressive mechanical. In our previous study, a large amount of chondrocyteS death were found in the process of condylar cartilage degradation induced by compressive mechanical stress. However,the inhibition of programmed apoptosis did not improve all pathological change during this process. Necroptosis is a novel programmed cell death mechanism which is mediated by apoptosis receptor interaction protein kinase 1(RIP1) and receptor interaction protein kinase 3 kinases(RIP3). Necroptosis is caspase independent and can be specificly inhibited by necrostatin-1(RIP1 inhibitor).The previous studies already demonstrate necroptosis play an important role in inflammation and degenerative diseases. In our pilot study, We found typical necrotic morphologocal changes occurred in the degradated mandibular cartilage, such as infiltrated inflammatory cells, upregulated RIP1,RIP3,and TNF-α, and increased reactive oxygens.We also demonstrated the inhibition of RIP1 by necrostatin-1 significantly improve the pathological change. Based on these finding, we raised an hypothesis that necroptosis mediated by RIP1/RIP3 cause inflammation reaction,then result in the mandibular condylar cartilage degradation under compressive mechanical stress stimulation. This present study will use existed compressive mechanical stress model combined with RNAi and inhibitor to study the pathomechanism of TMJ degradation. The purpose of this study is to further clarification of the mechanism of mandibular condylar cartilage degradation and explore new therapy target in treating TMJ disease.

异常压力刺激下软骨细胞的死亡是髁突软骨降解的关键因素之一。我们的研究证实了在压力源性髁突软骨降解过程出现了大量软骨细胞死亡，而凋亡的抑制并不能完全缓解髁突软骨降解。最近研究发现了一种被命名为程序性坏死的新的细胞死亡机制，它是由细胞内信号因子RIP1/RIP3介导，能被Nec-1特异性抑制的caspases非依赖性模式。目前研究已证实程序性坏死在炎症或退行性疾病中起着非常重要的作用。在预实验中我们观察到压力刺激的病变髁突软骨处存在炎性细胞浸润， RIP1,RIP3,TNFα表达上调，活性氧增加这些程序性坏死的病理特征，而采用Nec-1能有效地改善髁突软骨病变。因此我们提出压力刺激下，RIP1/RIP3介导的程序性坏死可能引起炎症反应，继而导致髁突软骨降解这一病理机制假说。本课题拟利用现有的压力模型，结合RNAi，抑制剂等技术进行深入研究，进一步明确髁突软骨降解机制,探索新的分子治疗手段。

颞下颌关节骨关节炎会给患者带来严重的关节疼痛和不适，严重影响患者的生活治疗，本研究旨在明确压应力作用下颞下颌关节软骨变薄和细胞死亡的机制，以寻找治疗颞下颌关节骨关节炎的新的靶点。本研究使用压应力动物模型和TNF-a+cycloheximide处理的细胞模型诱导大鼠颞下颌关节软骨细胞死亡，联合程序性坏死特异性抑制剂Nec-1和凋亡抑制剂Z-VAD以及程序性坏死和凋亡相关因子RIP1的siRNA处理，通过HE染色、电子显微镜和Micro CT观察软骨和软骨下骨组织学改变；通过免疫组化、western blotting和real-time PCR检测程序性坏死相关因子RIP1、RIP3以及凋亡相关因子caspase-8的表达改变。在体外试验中还检测大鼠原代软骨细胞线粒体膜电位和ROS含量的变化。结果发现，在压应力作用下的体内模型中，RIP1、RIP3和Caspase-8的表达在加力4天组明显升高，Nec-1和Z-VAD可以部分缓解压应力诱导的软骨变薄和软骨细胞死亡。此外，Nec-1+Z-VAD联合作用此缓解效果可以叠加。同样，通过关节腔注射RIP1 siRNA慢病毒载体也可以减轻压应力诱导的软骨变薄和软骨细胞死亡，以及 RIP1、RIP3和Caspase-8表达水平的升高。体外结果与体内类似，Nec-1和Z-VAD可以缓解TNF-a+cycloheximide诱导的线粒体膜电位和ROS含量的变化。通过此研究我们发现，除凋亡外，程序性坏死在压应力诱导的软骨变薄和软骨细胞死亡中同样发挥重要作用，RIP1作为一个既参与凋亡也参与程序性坏死的因子，可以被看作一种治疗颞下颌关节骨关节炎的潜在的治疗靶点。