ROS/TRPM2/NLRP3炎性体信号通路对机械通气所致肺损伤的调节作用

Mechanical ventilation has been used to support acutely ill patients for several decades. But it has a serious potential complication, ventilation-induced lung injury (VILI). NLRP3 Inflammasomes associates with numerous biological processes of infectious diseases induced by innate immune response, but the the exact pathways involved are not elucidated. In our study, Stretch can induce oxidative stress in alveolar macrophages, which triggers NLRP3 inflammasome-mediated interleukin 1β(IL-1β) secretion to initiate undesirable inflammatory responses. Although it is clear that mitochondrial reactive oxygen species (ROS) play a central role in NLRP3 inflammasome activation, the precise mechanism underlying ROS-induced NLRP3 inflammasome activation remains poorly understood. Based on our previous work, we put forward the hypothesis: Ventilation can activate NLRP3 Inflammasomes in Alveolar Macrophages via mitochondria ROS dependent TRPM2 mediated calcium influx manner . We will further address the role of ROS/ TRPM2/ NLRP3 Inflammasomes signal pathway in Ventilator-induced lung injury in Mice. To test the hypothesis, we will use TRPM2 and NLRP3 knockout mice, NLRP3 agonist and ROS inhibitors with immunology and biochemistry methods to explore the molecular mechanism of ROS/TRPM2//NLRP3 inflammasomes signaling pathways mediating Ventilator-Induced Lung Injury.? Our findings will provide a new insight into the role of ROS/TRPM2//NLRP3 inflammasomes signaling pathways in the pathophysiology of VILI.

机械通气作为治疗手段可导致肺损伤。申请者证实机械牵张激活肺泡巨噬细胞线粒体ROS/NLRP3炎性体信号通路并导致炎性因子IL-1β和IL-18释放，但ROS如何激活NLRP3炎性体尚不明确。课题组预实验发现机械牵张引发肺泡巨噬细胞瞬时感受器电位阳离子通道2(TRPM2)调节的钙离子内流，该通道受线粒体ROS影响并调节NLRP3炎性体活性。据此，我们假设: 机械牵张可能通过线粒体ROS依赖性TRPM2通道激活NLRP3炎性体/炎性因子通路。本课题拟通过肺泡巨噬细胞、基因敲除小鼠、小鼠VILI模型等研究对象，采用Western Blot、siRNA干扰、激光共聚焦显微镜、流式细胞术、ELISA、膜片钳等手段，多层面探讨TRPM2在ROS/NLRP3炎性体/细胞因子通路的调节作用及机械通气对TRMP2及其调节通路的影响，为探寻防治机械通气肺损伤对策提供新的理论依据。

背景：呼吸机是一种重要的生命支持，但也会带来急性肺损伤和呼吸机所致肺损伤(VILI)等并发症。本研究通过ROS/TRPM2/NLRP3信号通路揭示VILI的调节机制，并利用巨噬细胞(BMDMs)进行牵张实验。.方法：用siRNAs对NLRP3基因、Caspase-1基因进行下调，或用SS-31、鱼藤酮和YVAD处理细胞，进行牵张。分析ASC、NLRP3、Caspase-1、IL-1β、IL-18、ROS和TRPM2通道电流的表达及水平。对VILI模型小鼠进行通气处理，并对这些参数进行分析。.结果：NLRP3和Caspase-1下调，SS-31和YVAD抑制ASC、NLRP3和Caspase-1的表达，降低IL-1、β和IL-18水平。除鱼藤酮处理外，其他处理均抑制ROS的产生，降低TRPM 2通道电流，激活ASC、NLRP 3和Caspase-1的表达，使IL-1β、IL-18、ROS和TRPM 2通道电流显著升高(p<0.0 5)。潮气量通气可明显上调肺组织IL-1、β、IL-18的表达及NLRP3、ASC、Caspase-1的表达，增加肺湿/干重、肺组织MPO和总蛋白的比值。与WT小鼠相比，Nlrp3-/-和TRPM 2-/-小鼠上述指标显著降低(P<0.05)。.结论：机械拉伸肺泡巨噬细胞激活NLRP-3-ASC炎症复合体的表达，诱导线粒体ROS的产生，导致下游炎症因子IL-1、β和IL-18的表达及肺炎症和损伤。因此，线粒体ROS/TRPM2/NLRP3信号通路在VILI早期起着关键作用。