IL-17A在小鼠肝脏缺血再灌注损伤模型中促进肝脏修复的作用与机制研究
发布时间：2018-08-22 20:49
【摘要】：肝脏缺血再灌注(Ischemia/Reperfusion, I/R)损伤是肝脏外科手术中不可避免的病理生理过程。缺血器官早期大量中性粒细胞浸润,蛋白水解酶释放,氧自由基大量产生以及细胞因子激活等导致组织损伤,启动肝细胞的死亡与固有免疫应答,是发生急性再灌注损伤的重要原因。随着对肝内免疫微环境的重新认识,研究者发现这些激发损伤作用的因素却是启动后期肝脏修复的重要因素。如何界定其作用和寻求合适的干预时机是目前肝脏I/R损伤以及后期修复研究的热点和难点。IL-17A已被证实是肝脏I/R损伤过程中重要炎的症因子,而新近的研究发现IL-17A同时也是参与组织修复(肝脏、神经、小肠粘膜和骨等)的重要启动因子。在肝脏I/R损伤早期,IL-17A可由以下细胞分泌：CD4+T细胞、NKT细胞、γδT细胞、NK细胞和中性粒细胞。目前,对肝脏I/R后确切的IL-17A+细胞亚群仍有争议；但是对于减轻早期肝脏IR损伤及后期修复而言,明确具体的细胞来源具有指导临床治疗的价值。在肝脏I/R损伤后早期阶段便促进库否细胞(Kupffer Cells, KCs)分泌细胞因子白介素-6、肿瘤坏死因子、肝细胞生长因子等细胞因子。这些蛋白恰恰是促进肝脏修复的经典细胞因子,已经被证实能够加快肝细胞再生进程。研究证明,IL-17A可以促进炎症反应,那么,IL-17A的出现是否能够活化KCs,从而激活肝脏再生信号。详细了解IL-17A信号在肝脏损伤和修复中的作用机制对于指导临床治疗具有重要的意义。Olrl基因编码了凝集素样氧化型低密度脂蛋白受体-1(Lectin-like Oxidized Low-density Lipoprotein Receptor-1, LOX-1),LOX-1是一种清道夫受体。本课题旨在围绕IL-17A与LOX-1的相互联系,进一步揭示LOX-1在I/R损伤中的作用及上下游作用机制。本课题采用小鼠70%肝脏I/R(缺血90min)模型,利用IL-17A-/-和IL-17RA-/-小鼠,分析I/R后缺乏IL-17A信号时肝脏损伤和再生的变化。博士研究生期间主要获得以下结果：1.小鼠肝脏I/R损伤后急性期,即I/R后24小时内,IL-17A/IL-17RA信号的缺失会减轻I/R造成的肝脏损害和炎症反应；后期,约在损伤后48小时-72小时,IL-17A/IL-17RA信号的缺失削弱了肝脏的修复进程。2.I/R损伤早期,IL-17A加重肝脏炎症反应可能与肝脏内大量中性粒细胞的浸润相关。而此阶段,中性粒细胞成为肝内IL-17A的主要来源。在损伤修复阶段,肝内IL-17A主要来源于ROR γt+细胞。这一结果为干预炎症损伤、促进修复提供了重要的干预时间点与靶点。3.证实LOX-1与IL-17A介导的肝脏I/R损伤后修复相关。其机制可能是IL-17A/IL-17RA信号通过TRAF6、NF-κB调节LOX-1的表达。在下游,LOX-1与IL-17A一起激活库否细胞内的丝裂原活化蛋白激酶(Mitogen-activated Protein Kinase,MAPK)信号通路。4.IL-17A信号通过LOX-1间接地活化KCs,使其释放一系列促进肝脏修复的正调节信号,如HGF,IL-6等。综上所述,我们的研究表明,IL-17A/IL-17RA信号通路在肝脏I/R损伤以及修复过程中发挥核心作用,一方面,中性粒细胞分泌的IL-17A可以放大急性期肝脏的炎症反应,证明了IL-17A/IL-17RA信号在肝脏缺血再灌注损伤中居于炎症调控网络的中心地位；另一方面,循环/组织中高表达的IL-17A通过与其受体IL-17RA的结合,促进LOX-1基因的表达,直接或间接地激活下游KCs的MAPK通路相关分子的磷酸化,促进静止的KCs活化,并分泌HGF、.IL-6等细胞因子,促进肝细胞的增殖,抑制其凋亡。最终促进肝脏I/R损伤后的修复。上述这些结果有利于我们更深入地了解IL-17A在肝脏I/R损伤急性期促炎症以及后期促修复过程中作用机制,同时也为肝脏I/R损伤的干预提供时间节点以及新的靶标。
[Abstract]:Ischemia/Reperfusion (I/R) injury is an unavoidable pathophysiological process in hepatic surgery. Invasion of large numbers of neutrophils, release of proteolytic enzymes, production of oxygen free radicals and activation of cytokines in the early stage of ischemic organs lead to tissue damage, initiating death of hepatocytes and innate immune response. With the re-understanding of the intrahepatic immune microenvironment, researchers have found that these factors that trigger the injury are important factors in initiating late liver repair. How to define the role of these factors and find the right time to intervene are hot and difficult issues in the study of liver I/R injury and late liver repair. Interleukin-17A (IL-17A) has been proved to be an important inflammatory factor in liver I/R injury. Recent studies have found that IL-17A is also an important promoter of tissue repair (liver, nerve, small intestinal mucosa and bone, etc.). In the early stage of liver I/R injury, IL-17A can be secreted by CD4 + T cells, NKT cells, gamma delta T cells, NK cells and neutral cells. Granulocytes. At present, the exact subpopulation of IL-17A+ cells after liver I/R is still controversial; however, it is valuable to identify specific cell sources for clinical treatment in alleviating early liver IR injury and late repair. Kupffer Cells (KCs) secrete cytokine interleukin-6 at the early stage after liver I/R injury. Tumor necrosis factor, hepatocyte growth factor and other cytokines. These proteins are classical cytokines that promote liver repair and have been shown to speed up the process of hepatocyte regeneration. The mechanism of L-17A signaling in liver injury and repair is of great significance for guiding clinical treatment. Olrl gene encodes lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which is a scavenger receptor. The purpose of this study is to explore the role of LOX-1 in I/R injury and its mechanism of upstream and downstream. The present study used 70% I/R (ischemia 90 min) model of mice liver and IL-17A-/- and IL-17RA-/- mice to analyze the changes of liver injury and regeneration in the absence of IL-17A signal after I/R. Deletion of IL-17A/IL-17RA signal attenuates liver damage and inflammation induced by I/R in the acute phase after injury, i.e. within 24 hours after I/R. Deletion of IL-17A/IL-17RA signal attenuates the repair process of liver in the late phase, about 48-72 hours after injury. 2. In the early phase of I/R injury, IL-17A may aggravate liver inflammation and a large number of neutral liver reactions. At this stage, neutrophils become the main source of intrahepatic IL-17A. At the stage of injury repair, intrahepatic IL-17A mainly comes from ROR gamma T + cells. This result provides an important intervening time point and target for intervening inflammatory injury and promoting repair. 3. I t is confirmed that LOX-1 and IL-17A mediated liver I/R repair phase after injury. Downstream, LOX-1, along with IL-17A, activates the mitogen-activated protein kinase (MAPK) signaling pathway in the Kupffer cells. 4. IL-17A signaling indirectly activates KCs through LOX-1 to release a series of enhancements to liver repair. In summary, our studies have shown that IL-17A/IL-17RA signaling pathway plays a central role in liver I/R injury and repair. On the one hand, IL-17A secreted by neutrophils can amplify inflammation in the acute phase of liver injury, which proves that IL-17A/IL-17RA signaling is involved in liver ischemia-reperfusion injury. On the other hand, IL-17A, which is highly expressed in circulatory/tissue, promotes the expression of LOX-1 gene by binding to its receptor IL-17RA, directly or indirectly activates the phosphorylation of MAPK pathway-related molecules in downstream KCs, promotes the activation of resting KCs, and secretes cytokines such as HGF, IL-6, and promotes hepatocyte proliferation. These results will help us to understand the role of IL-17A in promoting inflammation in the acute phase of liver I/R injury and in the late phase of liver repair, and provide time nodes and new targets for the intervention of liver I/R injury.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R657.3;R-332
本文编号：2198261
[Abstract]:Ischemia/Reperfusion (I/R) injury is an unavoidable pathophysiological process in hepatic surgery. Invasion of large numbers of neutrophils, release of proteolytic enzymes, production of oxygen free radicals and activation of cytokines in the early stage of ischemic organs lead to tissue damage, initiating death of hepatocytes and innate immune response. With the re-understanding of the intrahepatic immune microenvironment, researchers have found that these factors that trigger the injury are important factors in initiating late liver repair. How to define the role of these factors and find the right time to intervene are hot and difficult issues in the study of liver I/R injury and late liver repair. Interleukin-17A (IL-17A) has been proved to be an important inflammatory factor in liver I/R injury. Recent studies have found that IL-17A is also an important promoter of tissue repair (liver, nerve, small intestinal mucosa and bone, etc.). In the early stage of liver I/R injury, IL-17A can be secreted by CD4 + T cells, NKT cells, gamma delta T cells, NK cells and neutral cells. Granulocytes. At present, the exact subpopulation of IL-17A+ cells after liver I/R is still controversial; however, it is valuable to identify specific cell sources for clinical treatment in alleviating early liver IR injury and late repair. Kupffer Cells (KCs) secrete cytokine interleukin-6 at the early stage after liver I/R injury. Tumor necrosis factor, hepatocyte growth factor and other cytokines. These proteins are classical cytokines that promote liver repair and have been shown to speed up the process of hepatocyte regeneration. The mechanism of L-17A signaling in liver injury and repair is of great significance for guiding clinical treatment. Olrl gene encodes lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which is a scavenger receptor. The purpose of this study is to explore the role of LOX-1 in I/R injury and its mechanism of upstream and downstream. The present study used 70% I/R (ischemia 90 min) model of mice liver and IL-17A-/- and IL-17RA-/- mice to analyze the changes of liver injury and regeneration in the absence of IL-17A signal after I/R. Deletion of IL-17A/IL-17RA signal attenuates liver damage and inflammation induced by I/R in the acute phase after injury, i.e. within 24 hours after I/R. Deletion of IL-17A/IL-17RA signal attenuates the repair process of liver in the late phase, about 48-72 hours after injury. 2. In the early phase of I/R injury, IL-17A may aggravate liver inflammation and a large number of neutral liver reactions. At this stage, neutrophils become the main source of intrahepatic IL-17A. At the stage of injury repair, intrahepatic IL-17A mainly comes from ROR gamma T + cells. This result provides an important intervening time point and target for intervening inflammatory injury and promoting repair. 3. I t is confirmed that LOX-1 and IL-17A mediated liver I/R repair phase after injury. Downstream, LOX-1, along with IL-17A, activates the mitogen-activated protein kinase (MAPK) signaling pathway in the Kupffer cells. 4. IL-17A signaling indirectly activates KCs through LOX-1 to release a series of enhancements to liver repair. In summary, our studies have shown that IL-17A/IL-17RA signaling pathway plays a central role in liver I/R injury and repair. On the one hand, IL-17A secreted by neutrophils can amplify inflammation in the acute phase of liver injury, which proves that IL-17A/IL-17RA signaling is involved in liver ischemia-reperfusion injury. On the other hand, IL-17A, which is highly expressed in circulatory/tissue, promotes the expression of LOX-1 gene by binding to its receptor IL-17RA, directly or indirectly activates the phosphorylation of MAPK pathway-related molecules in downstream KCs, promotes the activation of resting KCs, and secretes cytokines such as HGF, IL-6, and promotes hepatocyte proliferation. These results will help us to understand the role of IL-17A in promoting inflammation in the acute phase of liver I/R injury and in the late phase of liver repair, and provide time nodes and new targets for the intervention of liver I/R injury.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R657.3;R-332
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