巨噬细胞线粒体自噬调控ROS/NLRP3炎症小体活化对糖尿病难愈性创面炎症反应的影响及机制研究

Sustained activity of the NLRP3 inflammasome in diabetic wounds contributes to the persistent inflammatory response and impaired healing characteristic of these wounds. Our finding is that NLRP3 inflammasome is a key regulatory pathway in the chronic inflammasome response in diabetic wound. However, little is known about the regulation and function of these proteins. Previous studies have found the activity of NLRP3 inflammasome is typically associated with mitophagy in macrophages. Our pre-early experiments show inhibited autophagy, excessive mitochondrial ROS production and high level of NLRP3 and IL-1β protein from wound of diabetic human. Pretreatment with Rapamycin suppresses ROS accumulation. Therefore, we hypothesize that autophagy inhibition contributes to ROS-producing NLRP3 dependent inflammasome activation and cytokines secretion, which promotes a persistent inflammatory response and impairs diabetic wound healing. In this way, we aim to evaluate the roles of autophagy inhibition, ROS generation and NLRP3 inflammasome formation in development of diabetic wound inflammation, with the use of gene silence, western blot, immunofluorescence, Real time-PCR, ELISA and histological examination in vitro and in vivo studies. It may provide a new approach for improvement of chronic wound healing process in diabetes mellitus.

糖尿病（DM）创面迁延难愈，呈现为持续性慢性炎症反应，NLRP3炎症小体活化在其中起重要作用，但机制不明。NLRP3炎症小体与巨噬细胞线粒体自噬关系密切，我们预实验发现DM创面自噬标志蛋白LC3Ⅱ表达下调，ROS水平增高，NLRP3炎症小体高表达，促炎巨噬细胞和IL-1β增加，而促进自噬能抑制ROS产生。因此我们假设，线粒体自噬与NLRP3炎症小体参与DM创面炎症反应的相关信号通路，巨噬细胞线粒体自噬下调促进ROS产生，致NLRP3炎症小体异常活化并释放IL-1β，加剧炎症免疫应答，影响DM创面愈合。本研究拟通过DM创面动物模型和高糖细胞模型，应用siRNA、免疫印迹、免疫荧光、RT-PCR、Elisa、组织学检测等手段，从形态学、组织学、细胞学和分子水平探讨巨噬细胞线粒体自噬调控ROS/NLRP3炎症小体活化对DM难愈性创面炎症反应的影响及机制，为DM难愈性创面的防治提供新思路和新靶点。

糖尿病创面迁延难愈，呈现为持续性慢性炎症反应，NLRP3炎症小体活化在其中起重要作用，但机制不明。NLRP3炎症小体与巨噬细胞线粒体自噬关系密切，我们预实验发现糖尿病创面自噬标志蛋白LC3Ⅱ表达下调，ROS水平增高，NLRP3炎症小体高表达，促炎巨噬细胞和IL-1β增加，而促进自噬能抑制ROS产生。通过糖尿病创面动物模型和高糖细胞模型，研究证实了巨噬细胞线粒体自噬与NLRP3炎症小体参与糖尿病难愈性创面炎症反应的相关信号通路，高糖环境中巨噬细胞线粒体自噬下调促进ROS产生，致NLRP3炎症小体异常活化并释放IL-1β，加剧炎症免疫应答，影响糖尿病创面愈合。除完成原实验计划外，研究证实晚期糖基化终末产物（AGEs）通过ROS/NLRP3炎症通路诱导成纤维细胞凋亡。此外，我们结合临床研究了维生素D与糖尿病足溃疡的相关性，研究自体血PRP和自体干细胞能治疗糖尿病足溃疡，分析糖尿病足多重耐药菌感染的危险因素，这些研究成果为研究糖尿病难愈性创面修复提供了新的思路。