游离胞外组蛋白通过TLR9介导NET形成并促进肝脏缺血再灌注损伤

Liver ischemia/reperfusion (I/R) injury is a clinically important phenomenon encountered during elective liver surgical procedures, solid organ transplantation, trauma, and hypovolemic shock. Liver transplantation (LTx) is an effective therapeutic modality for the treatment of end-stage liver disease. However, I/R injury leads to serious liver graft damage after LTx, contributes to a higher incidence of acute primary liver graft non-function or late chronic graft rejection, especially in marginal livers that have a higher susceptibility to the ischemic insult. Polymorphonuclear leukocytes (PMNs, neutrophils) are highly specialized cellular effectors in host defense and immune surveillance. Formation of neutrophil extracellular trap (NET) has been recently found to be a novel response to various stimuli. NET is composed of decondensed chromatin fibers lined with antimicrobial proteins such as neutrophil elastase and myeloperoxidase. Neutrophils are well-known to be a vital part of the innate immune response to liver I/R injury. However, whether NET participates in liver I/R injury and what their potential roles remain unknown. We hypothesize that during liver I/R injury,free histones released from damaged or necrotic hepatocytes function as danger associated molecular patterns (DAMPs) to promote peptidylarginine deiminases (PAD) 4 activation via Toll-like receptor (TLR)-9 signaling pathways, which subsequently activate NET formation. Development of NETs during liver I/R injury is detrimental, as NETs initiate inflammatory responses. By using neutrophil primary culture and animal experiments, we will intend to adopt overexpression or silencing blocking PAD4 to study the mechanism of free extracellular histone mediated PAD4 activation and NET formation. This study will serve as a basis for developing both a more comprehensive understanding of how innate immune cells are activated and mediate inflammatory injury during liver I/R, and should prove useful in the design of novel therapies to minimize liver damage after Ltx. Importantly, the mechanisms of inflammation mediated by NET formation is likely common in a number of infectious and non-infectious inflammatory conditions found within the liver and the implications of this work likely extend to a variety of other organ transplantations (e.g. lung, kidney).

中性粒细胞浸润是在肝脏缺血再灌注损伤（IRI）中引起肝脏无菌性炎症反应、导致急性原发性移植肝无功能的重要原因之一，其作用机理有待深入研究。新近研究发现，中性粒细胞还可释放由解旋染色质纤维和颗粒蛋白所组成的胞外诱捕网（NET）介导固有免疫反应促进炎症进程，且ROS和PAD4扮演重要作用。我们前期研究提示，在肝IRI中肝细胞释放的游离胞外组蛋白可激活toll样受体9（TLR9）加重肝脏损伤；而且我们在肝脏组织中观察到NET的形成。因此我们推测受损/应激肝细胞释放游离胞外组蛋白，通过TRL9信号通路介导NET形成，进而促进炎症反应和肝细胞损伤。故本课题拟采用TLR9 KO、PAD4 KO及WT小鼠，通过中性粒细胞原代共培养和在体动物实验，研究游离胞外组蛋白通过TLR9介导NET形成的机制，分析PAD4及ROS在其中的作用，探索NET促进炎症反应和损伤肝细胞机制，为临床IRI防治提供新的靶点。

项目负责人及主要参与者严格按照研究标年度工作计划开展该课题，通过为期4年的研究 ，已圆满完成项目计划各要点，证实了“游离胞外组蛋白通过TLR9介导NET形成并促进肝脏缺血再灌注损伤”这一科学假说。比照研究工作计划，本项目已达到预定目标，进度研究按照计划书执行，完成了本项目的全部研究工作计划。.与此同时，项目小组执行该研究过程中饶有兴趣地发现，危险信号相关分子模式（DAMP）如白介素33，通过ST2的相互作用介导中性粒细胞NET 的体内或体外形成。更令人感兴趣的是来自受损肝窦内皮细胞释放的白介素33通过中性粒细胞胞膜上受体ST2激活浸润的中性粒细胞能形成NET，这一信号通路是肝脏I/R后NET形成的必要条件。据此，我们开展了进一步的研究，目前实验结果理想。

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