中药虎杖通过NLRP3炎症小体抑制急性肺损伤炎症反应的机制研究

Uncontrolled inflammatory reaction evoked by infective or non-infective agents has been considered as the cause of acute lung injury (ALI), but pathogenesis of the disease remains poor understood. Reactive oxygen species (ROS) has been shown to activate NLRP3 inflammasome to generate inflammation. We found that both LPS and ischemic reperfusion caused the elevation of inflammasome-specific IL-18 and IL-33 levels in the blood plasma, ROS level in the alveolar macrophages of ALI mice and confirmed the anti-inflammatory effect of Polygonum cuspidatum. So we hypothesized that Polygonum cuspidatum is likely to reduce the inflammation reaction by NLRP3 inflammasome in ALI. In this research project, we use the LPS-or ischemic reperfusion-based ALI mice and LPS- or I/R-treated mouse macrophage model to demonstrate the anti-inflammatory effect of Polygonum cuspidatum. With the methods such as immunohistochemistry, immunofluorescence, ELISA, signaling pathway phosphorylation detection and signal blocking, we detect the ROS level elevation and NLRP3 inflammasome activation of alveolar macrophages, Nrf2 expression, cytokine level in plasma and bronchoalveolar lavage fluid, and lung tissue pathology to elucidate the molecular mechanism of Polygonum cuspidatum anti-inflammatory effect. The aim of this project is to illuminate a novel mechanism of ALI as well as to provide a new idea for traditional Chinese medicines to treat ALI.

感染或非感染因素所致失控性炎症反应是ALI的病因，其机制未完全明了。已知活性氧(ROS)能激活NLRP3炎症小体引起炎症反应，我们发现LPS及缺血再灌注均使ALI小鼠肺泡巨噬细胞ROS及炎症小体特征性因子IL-18和IL-33升高，前期实验证实虎杖具有较好的抗炎作用，因此我们推测虎杖极有可能通过NLRP3炎症小体减轻ALI炎症反应。本项目中，我们先采用LPS及缺血再灌注制备ALI小鼠模型，再以小鼠巨噬细胞为对象制备ALI细胞模型，采用免疫组织化学、免疫荧光、ELISA、信号通路磷酸化水平检测及信号阻断等技术，观察虎杖治疗前后肺泡巨噬细胞ROS和NLRP3活化，Nrf2表达、血浆和肺泡灌洗液相关炎症因子水平变化及肺组织病理改变，明确虎杖通过Nrf2抗氧化系统抑制ROS/NLRP3炎症小体途径，从而抑制ALI炎症反应，为中医药治疗ALI提供思路。

失控性炎症反应是急性肺损伤的发病主因，前期实验表明中药虎杖具有较好的抗炎效果，因此我们推测虎杖可通过调节NLRP3炎症小体影响HMGB1的转位释放来抑制ALI中的炎症反应。. 项目研究内容包含：.1..感染性小鼠ALI相关炎症模型制备及鉴定、巨噬细胞ALI炎性模型制备及鉴定.2..中药虎杖体内抗NLRP3炎症小体介导ALI相关炎症反应的作用.3..中药虎杖含药血清通过NLRP3炎症小体抗ALI相关炎症反应的作用及机制研究.根据项目申请书，项目组使用LPS诱导的方法成功建立ALI小鼠，通过对肺泡灌洗液，小鼠血清，肺组织病理检测，明确虎杖及其有效成分白藜芦醇对急性肺损伤的疗效。然后在细胞水平，成功建立ALI细胞模型（巨噬细胞ALI和肺血管上皮细胞ALI），通过药物血清的制备和细胞培养，明确了虎杖抗细胞炎症反应的效果。最后在细胞水平探索虎杖及其有效成分对抗ALI的分子机制，通过ROS/NLRP3炎症小体发挥抗炎作用。..总体而言，我们完成了研究内容中1、2、3的内容，其中对巨噬细胞的分子机制研究做了详细的机理，在分子水平探索了虎杖通过ROS/NLRP3炎症小体途径抗炎作用，另外增加了虎杖的主要有效成分——白藜芦醇通过p38MAPK/NF-κB和JAK/STAT1/3途径抗ALI炎症反应的作用，增加了白藜芦醇通过抑制ENaC和AQP-1减轻ALI肺水肿的机理研究。