IL-33/ST2在减体积肝脏缺血再灌注损伤的作用和机制

Living donor liver transplantation (LDLT), as an effective surgical alternative to whole liver transplantation, has been applied to expand the size of donor pool for the treatment of end-stage liver disease. However, ischemia/reperfusion (I/R) injury which is inevitable occurred in reduced-size liver (RSL) either in donors or recipients during LDLT, may impair liver regeneration and lead to serious graft damage. I/R injury during LDLT contributes to a higher incidence of acute primary graft non-function or late chronic graft rejection, especially in marginal liver grafts have a higher susceptibility to the ischemic insult. Liver sinusoidal endothelial cells (LSECs) have been implicated as principal cells in the immune response during liver I/R. Interleukin-33 (IL-33), locates in epithelial or endothelial cells as nuclear protein, has been shown to drive neutrophil infiltration during inflammatory responses through its receptor ST2. We recently found that in response to danger associated molecular patterns (DAMPs) released from hepatocytes, infiltrating neutrophils form neutrophil extracellular trap (NET) which exacerbate sterile inflammatory injury in hepatic I/R. However, whether NETs participates in reduced-size liver I/R injury and what their potential roles remain unknown. In this proposal, we hypothesize that IL-33，released from damaged LSECs, can both activate neutrophil to form NETs，and stimulate hepatocytes to actively release HMGB1, via ST2 signaling pathway, exacerbate organ damage and sterile inflammation in reduced-size liver I/R, consequently impair liver regeneration. By using neutrophil or hepatocytes primary culture and animal experiments, we will intend to adopt overexpression or silencing blocking IL-33 or its receptor ST2 to study the mechanism of IL33 mediated neutrophil forming NETs, and hepatocytes actively releasing HMGB1. The information gained by studying the mechanisms by which IL-33 can induce NET formation and hepatocytes damage during liver I/R could be of therapeutic value. The data yielded from these studies will increase our understanding of the molecular pathophysiology of liver I/R injury and provide significant insight into the mechanisms by which ischemic tissues notify the immune system, in particular neutrophils, of impending cell damage.

在活体肝移植（LDLT）术中，供体和受体都会不可避免的发生缺血/再灌注损伤（IRI），导致急性原发性小肝综合征或慢性移植物排斥。在肝IRI中，肝窦内皮细胞介导损伤及免疫应答。在细胞受损后，白介素（Interleukin ，IL)-33作为危险相关分子模式（DAMP）因子被释放到胞外警告免疫系统。我们前期研究提示，在肝脏IRI中，受损肝细胞释放DAMP，刺激中性粒细胞形成中性粒细胞胞外诱捕网（NET），促进炎症反应和肝细胞损伤。因此我们推测在减体积肝脏I/R中，受损的肝窦内皮细胞释放IL-33，通过ST2信号通路诱导中性粒细胞形成NET，刺激肝细胞主动释放HMGB1，继而加重剩余肝脏损伤和I/R炎症，进一步损害肝再生。故本课题拟采用IL-33 KO、ST2 KO、TLR4KO及WT 小鼠，通过中性粒细胞或肝细胞原代共培养和小鼠减体积肝脏I/R模型，这一精准模拟活体肝移植I/R损伤的在体动物实

在活体肝移植（LDLT）术中，供体和受体都会不可避免的发生缺血/再灌注损伤（IRI），导致急性原发性小肝综合征或慢性移植物排斥。在肝IRI中，肝窦内皮细胞介导损伤及免疫应答。在细胞受损后，白介素（Interleukin ，IL)-33作为危险相关分子模式（DAMP）因子被释放到胞外警告免疫系统。我们既往研究提示，在肝脏IRI中，受损肝细胞释放DAMP，刺激中性粒细胞形成中性粒细胞胞外诱捕网（NETs），促进炎症反应和肝细胞损伤。因此我们推测在减体积肝脏I/R中，受损的肝窦内皮细胞释放IL-33，通过ST2信号通路诱导中性粒细胞形成NET，刺激肝细胞主动释放HMGB1，继而加重剩余肝脏损伤和I/R炎症，进一步损害肝再生。故本课题采用IL-33 KO、ST2 KO、TLR4KO及WT 小鼠，通过中性粒细胞或肝细胞原代共培养和小鼠减体积肝脏I/R模型，这一精准模拟活体肝移植I/R损伤的在体动物实验，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，发现，1）体外中性粒细胞在IL-33的刺激下形成NETs的过程，结合在体实验进而证实IL-33参与调控中性粒细胞表面NETs形成，从而加重肝叶切除时肝脏IRI小鼠模型的损伤;2）通过抑制剂及基因缺陷小鼠等一系列阻断手段进一步证明IL-33通过中性粒细胞胞膜上其受体ST2调控NETs形成的机制，确定IL-33刺激NETs 形成是通ST2介导，其次讨论在肝叶切除时肝脏IRI损伤的小鼠模型中肝窦内皮细胞作为IL-33释放的主要细胞类型与NETs 形成的关系；3）使用过继移植的方法将ST2基因敲除的小鼠中性粒细胞注射至中性粒细胞删除的WT小鼠体内，发现具有减少NETs形成及保护肝脏缺血再灌注损伤的作用, 进一步证实IL-33/ST2轴参与调控中性粒细胞表面NETs形成。 据此，我们证实了“IL-33—ST2轴参与调控中性粒细胞表面NETs形成，从而加重肝叶切除时肝脏IRI小鼠模型的损伤”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，肝细胞miR-210与SMAD4形成负反馈环路促进促进肝肝脏缺血再灌注损伤，据此，我们开展了进一步的的研究，目前实验结果理想。