磨损颗粒诱导巨噬细胞自噬-凋亡在关节假体周围感染中的作用机制研究

Periprosthetic joint infection (PJI) is a disastrous complication after total joint arthroplasty, which is closely associated with the damage of innate immunity around implants. Our previous study demonstrated that wear particles increase apoptosis and decrease proliferation in macrophages, resulting in the damage of innate immunity around implants. Recent studies also indicate that overactivation of autophagy in macrophages could lead to apoptosis. Our pilot experiments showed that wear particles increase the expressions of LC3-II and activate autophagy in macrophages; At the same time, the expression of cleaved-caspase 3 increased, suggesting an increase in apoptosis. Thus, we hypothesized that wear particles are able to provoke macrophage apoptosis by overactivating autophagy, thereby damaging innate immunity and increasing the risk of getting PJI. In this study, the interface membranes between bones and implants of PJI patients are collected and LC3 and apoptosis related proteins are detected. In addition, after blocking autophagy by some specific inhibitors or by silencing some autophagy-related genes, apoptosis is measured using western blot, TUNEL and Annexin V/PI. Moreover, we plan to build Atg-5 or Atg-7 knockout mice, followed by building mouse calvaria models and infected by Bioluminescent S. aureus and detect bacteria growth by IVIS system. Using these methods, we further clarify the mechanism of autophagy-apoptosis caused by wear particles in macrophages, which will contribute to the finding of the targets for the prevention of PJI.

假体周围感染(PJI)是关节置换术后灾难性的并发症，其发生与假体周围固有免疫力下降密切相关。我们前期研究证实了磨损颗粒可致巨噬细胞增殖受阻、凋亡增加，从而损害固有免疫。近期研究表明自噬过度激活可诱发细胞凋亡。进一步预实验结果显示钛颗粒可致自噬蛋白LC3-II/I及凋亡相关蛋白cleaved-caspase3同步增加。因此我们假设磨损颗粒可激活巨噬细胞自噬进而促进自身凋亡，损害固有免疫，增加PJI发生风险。本研究拟收集PJI患者假体界膜组织，检测LC3及凋亡相关蛋白表达，分析两类蛋白表达的相关性；在体外抑制巨噬细胞自噬后加入磨损颗粒，检测巨噬细胞凋亡；进而构建Atg-5/7自噬基因缺陷小鼠模型，在其颅骨表面植入磨损颗粒及生物发光金葡菌，动态监测局部细菌增殖速率，分析颅骨表面巨噬细胞自噬及凋亡的变化，明确磨损颗粒诱导巨噬细胞自噬-凋亡的具体机制，从调控自噬的角度为PJI的防治提供科学依据。

假体周围感染(PJI)是关节置换术后灾难性的并发症，其发生与假体周围固有免疫力下降密切相关。在本项目中，我们主要证实了假体磨损颗粒可以通过诱导巨噬细胞自噬-凋亡途径促进PJI的发生发展，并阐明了其中的发生机制。我们发现，（1）在临床标本中，相对于初次置换的滑膜组织，假体松动患者及晚发型PJI患者的假体周围界膜组织中自噬蛋白LC3表达显著上升，TUNEL凋亡阳性率也大大提高。（2）在体外研究中，钛颗粒刺激巨噬细胞后LC3B-Ⅱ / LC3B-I比值上升，巨噬细胞内自噬体和自噬溶酶体更为丰富；而在自噬抑制剂3-MA 和 LY294002的作用下，或敲除自噬相关基因-5（ATG-5）后，巨噬细胞凋亡显著降低。钛颗粒刺激ATG-5基因缺陷的巨噬细胞系后，产生的IL-1β、IL-6及TNF-α产生量较正常细胞减少。进一步的通路研究显示，钛颗粒诱导的巨噬细胞自噬-凋亡增加是通过PI3K-Akt通路来实现的。（3）在钛颗粒植入的小鼠颅骨溶解的动物模型中，我们发现，腹腔注射自噬抑制剂后，颅骨表面细胞凋亡显著降低、IL-1β、IL-6及TNF-α表达下降、颅骨溶解度同时降低。（4）此外，SphKs抑制剂也可抑制小鼠颅骨表面破骨细胞形成、IL-6及TNF-α的产生，是通过SphKs/S1P-TRAF2-BECN1通路实现的。以上研究结果表明，自噬抑制剂及SphKs抑制剂均可抑制假体周围骨溶剂。假体植入后，假体周围组织自噬-凋亡增加从而降低局部免疫力；抑制过度自噬后可减少巨噬细胞凋亡，从而改善假体周围局部抗感染免疫力，降低PJI发生风险。