活性氧（ROS）激活NLRP3炎症小体在滑膜炎介导的下颌骨髁突软骨细胞焦亡中的作用

Degeneration of condylar cartilage and synovial inflammation are the most prominent pathological features of osteoarthritis (OA) in the temporomandibular joint (TMJ). The exact mechanism of cartilage degeneration in inflammatory environment remains elusive. Less populated chondrocytes caused by cell death are a key feature of cartilage degeneration. Recent studies suggest that pyroptosis, a form of cell death related to inflammation, might contribute to the decrease of the number of chondrocytes and the mechanism remains yet unknown. Based on previous studies, this project hypothesizes that sustained synovial inflammation induces massive release of proinflammatory cytokines such as IL-1β and TNF-α, which in turn cause accumulation of reactive oxygen species (ROS) via various pathways in chondrocytes. Accumulated ROS activate NLRP3 inflammasome, leading further to pyroptosis of chondrocytes through activation of caspase-1 cleaving its substrate GSDMD. To validate the aforementioned hypothesis, this project intends to investigate and reveal the participation of ROS in the pyroptosis of the mandibular condylar chondrocytes mediated by synovial inflammation, the participation of NLRP3 inflammasome in the pyroptosis of the mandibular condylar chondrocytes, the protective effect of NLRP3 inhibitor on mandibular chondylar chondrocytes in CFA-induced rat temporomandibular synovial inflammation and the clinical evidences of activation of NLRP3 inflammation and subsquent pyroptosis by animal, cell experiments and clinical sample assays. If successful, it will reveal the mechanism of the degeneration of the mandibular condylar cartilage in synovial inflammation from a new perspective and provide new targets for the prevention and treatment of OA.

髁突软骨退行性变和滑膜炎是颞下颌关节骨关节炎重要的病理改变，炎性环境下软骨退变机制目前还不清楚。软骨细胞死亡导致的软骨细胞数目减少是软骨退变的重要特点，研究提示一种与炎症相关的细胞死亡方式——焦亡可能参与其中但机制不明。本项目基于前期研究，提出持续性滑膜炎时IL-1β、TNF-α等炎症因子大量释放，炎症因子通过多种途径造成软骨细胞中活性氧(ROS)的积聚；积聚的ROS激活NLRP3炎症小体,进而通过caspase-1切割其底物GSDMD引发细胞焦亡并致软骨退变。本研究拟通过体内外实验和临床研究，揭示ROS在滑膜炎介导的髁突软骨细胞焦亡中的作用、NLRP3炎症小体在ROS致髁突软骨细胞焦亡中的作用、NLRP3特异性抑制剂对滑膜炎动物髁突软骨细胞的保护作用及NLRP3炎症小体活化以及焦亡的临床证据，从新角度揭示滑膜炎中髁突软骨退行性变的机制，为颞下颌关节骨关节炎的预防和治疗提供新靶标。

背景：颞下颌关节（temporomandibular joint, TMJ）骨关节炎(osteoarthritis, OA)是口腔颌面外科临床常见病与多发病。关节软骨的退行性变是其重要病理改变。关节软骨的退行性变包括软骨细胞死亡和软骨基质的异常降解。本课题组前期的研究检测到TMJ OA关节滑液中炎症因子的异常。通过文献复习，我们提出炎症因子可能通过活性氧（ROS）-NLRP3炎症小体-caspase-1轴促进软骨细胞的焦亡和软骨基质的降解导致关节软骨的退行性变。我们通过临床样本、动物模型和软骨细胞来探究其可能的分子机制。..主要研究内容：1.颞下颌关节骨关节炎（TMJ OA）患者和髁突骨折患者（No-OA）关节滑膜组织学染色，比较滑膜炎症及炎性因子表达差异；抽取颞下颌关节紊乱病患者滑液，研究滑液中炎症因子、软骨代谢标志物及细胞焦亡标志物浓度相关性。2.小鼠颞下颌关节腔注射碘乙酸钠（MIA)构建TMJ OA模型，观察模型中滑膜炎症和软骨退行性变；研究ALPK1基因敲除减轻滑膜炎后，对碘乙酸钠所致的TMJ OA的保护作用。3.利用巨噬细胞活化后上清、IL-1β+TNF-α模拟炎症环境，观察其对软骨细胞焦亡及软骨细胞基质降解酶类表达的影响，通过提高或抑制ROS、NLRP3炎症小体探究其具体机制。..重要结果：1.颞下颌关节骨关节炎患者滑膜炎症明显，IL-1β、TNF-α、ALPK1和NF-κB p65等炎性因子表达提高；颞下颌关节紊乱病患者关节滑液中炎症因子、软骨代谢标志物及细胞焦亡标志物浓度呈正相关关系。2.关节腔注射碘乙酸钠后小鼠TMJ呈现严重的滑膜炎症及髁突软骨的退行性变，电镜下可见软骨细胞呈现焦亡特征；ALPK1基因敲除能减轻碘乙酸钠注射所致TMJ OA模型的滑膜炎症，并减轻软骨退化和软骨细胞的焦亡。3.重组人ALPK1（rhALPK1）刺激活化的巨噬细胞上清、IL-1β+TNF-α模拟的炎症环境均能促进软骨细胞中ROS产生及细胞焦亡；炎症因子经过ROS、NLRP3信号促进软骨细胞焦亡和软骨细胞基质降解酶类表达。..科学意义：活性氧（ROS）激活NLRP3炎症小体在滑膜炎介导的颞下颌关节软骨退行性变中具有重要作用，具有成为治疗靶标的潜力。