CoNPs通过ROS-NLRP3-GSDMD途径诱导巨噬细胞焦亡在假体周围炎性损伤和骨溶解中的作用

CoNPs have been shown to trigger an innate immune response that lead to aseptic chronic inflammation and osteolysis, which are closely associated with pain and implant failure. They are detected and phagocytosed mainly by tissue-resident macrophages. While CoNPs could not be fully digested by macrophages, it has been shown by many studies that these particles induce apoptosis in a variety of cell types. Apoptosis is a regulated process for eliminating unwanted cells without eliciting inflammatory response. However, the response to a CoNPs has been reported to be immune cell predominant infiltration of soft tissue failure, which is characterized by chronic tissue lesion and excess bone loss around hip prothesis. Therefore, it is possible that apoptosis is not the only form of cell death triggered by CoNPs. Based on our preliminary data collected from patients who have received revision THR, we found that the genes related to the transcription of pyroptosis-associated proteins (NLRP3, Caspase-1, ASC and GSDMD) are highly upregulated compared with those from patients undergoing primary hip replacement. Furthermore, we revealed that CoNPs induce macrophage pyroptosis in vitro. Further pyroptosis-related protein analysis is also consistent with the genetic findings. Pyroptosis are lytic, inflammatory types of programmed cell death that mainly require the activation of NLRP3 inflammasome that promote the cleavage of Caspase-1, which in turn cleave the membrane damaging protein: GSDMD and the release of inflammatory mediators including IL-1β and IL-18. The NLRP3 inflammasome is known to be activated by a wide range of danger signals including ROS. We also published in our previous research that CoNPs stimulate ROS production in macrophage. Thus, we hypothesized that CoNPs induce periprosthetic tissue lesion and osteolysis by triggering macrophage pyroptosis via ROS-NLRP3-GSDMD pathway. By applying in vivo subcutaneous air-pouch chronic inflammation model, calvaria osteolysis model in mice and in vitro experiments using bone marrow derived macrophages (BMDMs), we aim to unravel the underling molecular mechanism of CoNPs-induced macrophage pyroptosis and its role in tissue lesion and osteolysis. The results of this project may serve as therapeutic targets to mitigate adverse tissue reaction due to CoNPs and improve performance of implanted joints.

髋关节金属假体周围软组织炎症反应和骨溶解与钴金属纳米微粒（CoNPs）所诱导的细胞毒性反应有关。目前认为，CoNPs主要引发巨噬细胞凋亡，但细胞凋亡触发的炎症反应一般较轻微，通常不会导致明显的炎性损伤。焦亡属于伴有大量促炎因子释放的炎性细胞坏死。我们前期研究发现细胞焦亡标志物（NLRP3、GSDMD）在金属假体周围滑膜组织中高表达以及CoNPs可诱导巨噬细胞发生焦亡。此外，我们已明确CoNPs可促进活性氧（ROS）的生成，而ROS正是细胞焦亡的重要诱导因子之一。据此我们提出假说：CoNPs通过ROS-NLRP3-GSDMD途径引起巨噬细胞焦亡伴炎症因子的成熟和释放，同时刺激破骨细胞活化，从而导致局部软组织炎症损伤和骨溶解。本项目拟利用动物模型和细胞学实验，阐明CoNPs介导的巨噬细胞焦亡机制及其与软组织炎症损伤和骨溶解的关系，以期为假体周围炎性损伤和骨溶解提供新的治疗靶点。

人工髋关节置换是目前治疗严重髋关节退变性疾病及其他终末期髋关节病患最有效的外科手段。钴金属纳米微粒（CoNPs）所诱导的假体周围炎性骨溶解是制约人工关节使用寿命的重要因素。在前期研究基础上，本项目已基本按研究计划顺利完成，并有如下发现：1、细胞焦亡相关标志物在髋关节金属假体周围滑膜组织中表达升高。2、金属磨损纳米颗粒CoNPs可在体外诱导巨噬细胞焦亡；BMDMs来源的巨噬细胞吞噬CoNPs后出现特征性的形态学改变和产生大量焦亡小体，并伴有Cleaved-Caspase-1、Caspase-1、Gasdermin-D和N-Gasdermin-D的高表达以及IL-1β的细胞外分泌。通过进一步采用活性氧探针检测、ROS相关抑制剂预处理CoNPs激活的巨噬细胞，我们验证了CoNPs通过提高巨噬细胞内ROS水平和CASP1/GSDMD 信号轴诱导巨噬细胞焦亡。3、通过构建Gsdmd基因敲除小鼠和颅骨骨溶解模型，我们发现抑制细胞焦亡有助于减轻金属磨损颗粒介导的炎性骨溶解反应。综上所述，本课题揭示了CoNPs诱导巨噬细胞焦亡这一现象并阐明了相关机理；同时结合动物实验验证了细胞焦亡在金属磨损颗粒介导的软组织炎性损伤和骨溶解中的重要作用。