NOX4调控破骨细胞活化在假体周围骨溶解中的作用及机制研究

Osteoclasts play important roles in the development of peri-prosthesis osteolysis. Extensive evidence has showed that reactive oxygen species (ROS) stimulates receptor activator of nuclear factor-κB-induced osteoclast differentiation and bone resorption. It has been demonstrated that NADPH oxidase (NOXs) is one of the major enzymes for the generation of ROS in skeletal system. Our preliminary experimental results showed that the protein level of NADPH-oxidase 4 (NOX4) was significantly increased in the tissue around loosening implant. In addition, siRNA-NOX4 obviously attenuates titanium particle-induced osteolysis in the murine osteolysis model. Thus, we propose the hypothesis: NOX4 mediates the generation of ROS in the process of PPO, and subsequently promotes the activation of osteoclast, and then makes osteolysis seriously. In the current study, the molecular, cellular, animal model and clinical samples were used to verify this hypothesis. First, we explore the expression and location of NOX4 in osteolytic tissue obtained from PPO patients and titanium particle-induced murine osteolysis model. Second, gene transfection and siRNA were used to discover the target gene which regulates the expression and function of NOX4 in the process of PPO. Third, NOX4 transgenic mice, NOX4 overexpression/knockdown or strategies of drug interference were used to investigeate the role of NOX4 in ROS generation, osteoclast activation and wear particle-induced osteolysis in vivo. And finally, western blot and the double immunofluorensence were used to investigate the mechanism of NOX4 in osteoclast activation and bone resorption. This project will clarify the relationship between NOX4 and osteoclast activation and bone destruction in the process of PPO, and provide a promising therapeutic target for treating or preventing of PPO.

破骨细胞（OC）在假体周围骨溶解（PPO）发展中发挥重要作用，ROS是诱导OC活化的关键因素。NADPH氧化酶（NOXs）是骨骼系统介导ROS产生的主要酶系之一。我们的预研结果证实NOX4在PPO模型中高表达，调控NOX4能减轻磨损颗粒引起的骨溶解。据此，我们提出假说： PPO后，NOX4介导ROS的产生，激活OC，加剧骨溶解。为验证该假说，本项目从细胞、组织和活体层面研究以下内容：①利用小鼠骨溶解模型及临床标本，检测PPO后NOX4的表达及定位变化；②利用siRNA干扰及基因转染技术，明确调控NOX4功能及表达变化的关键因子；③利用转基因小鼠、药物干预和过表达/沉默NOX4，评估NOX4对ROS水平和骨溶解的影响；④应用western blot和免疫荧光染色等技术，阐述PPO后NOX4调控OC活化的作用机制。期望通过本研究，阐明NOX4与PPO的关系，为PPO的防治提供新思路和新靶点。

假体周围骨溶解引起的无菌性松动是导致人工关节置换术失败的重要原因，磨损颗粒引起的级联反应可导致假体周围组织中活性氧水平升高，破骨细胞过度活化，最终导致假体周围骨溶解和无菌性松动的发生。NADPH氧化酶4（NOX4）是介导ROS产生的重要酶之一，对破骨细胞活化也有重要的调节作用；因此，NOX4可能是治疗骨溶解的合适靶点。我们的研究结果发现：NOX4在松动假体周围组织中及破骨细胞分化过程中表达增加，而靶向干预NOX4能抑制破骨细胞分化并减轻活性氧的产生，这一过程主要依赖Nrf2信号通路；动物实验结果显示，抑制NOX4可减轻钛颗粒导致的小鼠颅骨骨质破坏。上述结果表明，NOX4通过介导氧化应激和破骨细胞活化参与PPO，抑制NOX4能通过减少破骨细胞生成和ROS的产生减轻钛颗粒诱导的骨溶解。因此，抑制NOX4可能是预防和治疗假体周围骨溶解的潜在手段。