β-arrestin负调控阿片与TLR4通路减轻肝脏缺血再灌注损伤

Hepatic ischemia reperfusion injury(HIRI) restricts the progress of liver surgery for a long period, to explore the effective treatment and mechanisms of HIRI are the most urgent and clinical meaningful task. We have demonstrated firstly that down regulation of TLR4 inflammatory pathways by opioid agonist conferred ideal hepatic protection, and this work has received wide spread attention. Recently we also confirmed that beta-arrestin participated both down regulation of TLR4 pathway and opioid agonist induced activation as well as translocation of membrane. We have reasons to believe that beta-arrestin can bridge opioid and TLR4 pathway for revealing opioid's anti-inflammatory mechanism as a key molecule. In order to further clarify the role of beta-arrestin on opioid inhibiting TLR4 inflammatory pathways and its signal processes from cell membrane cytoplasm to nucleus in detail, The present project will demonstrate the key target sites and mechanisms of beta-arrestin on opioid agonist reducing HIRI and its relations between opioid receptors and TLR4 pathways by using transgenic technology, RNA interference and animal models of gene knockout, these will also be viewed from the overall level of animal, cells and molecules. The present research will further clarify the immune mechanisms of inflammatory response after liver IR injury, and provide important theoretical evidence and experimental basis for the clinical practicability of opioid agonist's liver protection therapy.

肝脏缺血再灌注(IR)损伤长期制约着肝脏外科的发展，探索其有效治疗手段和作用机制一直是临床最迫切和有意义的任务。我们率先发表的阿片激动剂下调TLR4炎症通路具有非常理想肝保护作用的论文引起国内外广泛关注，近来我们还证实了β-arrestin既参与下调TLR4通路，其又能够被阿片激动剂诱导激活和膜易位。我们有理由相信β-arrestin既能桥接阿片与TLR4通路更是揭示阿片抗炎机制的关键分子。为了进一步阐明该分子在阿片下调TLR4炎症通路中扮演的角色及其从胞膜、胞浆至核内的详细信号传递过程，本课题拟采用转基因技术、RNA干扰技术和基因敲除动物模型，从整体动物、细胞和分子等多个水平观察β-arrestin在阿片激动剂减轻肝脏IR损伤中调控阿片和TLR4通路的关键作用靶位和机制。本研究将为进一步阐明肝IR损伤后炎症反应的免疫机制，并为临床实用性的阿片激动剂肝保护治疗提供重要的理论佐证和实验依据。

研究背景：.肝脏缺血再灌注(IR)损伤作为一种常见的临床病理生理过程，探索其有效治疗手段和作用机制一直是临床最迫切和有意义的任务。前期研究发现，阿片激动剂下调TLR4炎症通路具有非常理想的肝保护作用，而β-arrestin2作为G蛋白偶联受体(GPCRs)的重要负调节因子，既参与下调TLR4通路，又能被阿片激动剂诱导激活和膜易位。本项目拟探讨β-arrestin2负调控阿片与TLR4通路减轻肝脏缺血再灌注损伤之间的相互作用，进一步阐明肝IR损伤后炎症反应的免疫机制。 .研究内容：首先，使用肝脏IR小鼠模型和细胞缺氧复氧模型，探究不同TLR4活性对阿片预处理减轻肝脏IR损伤中作用，明确阿片激动剂预处理减轻小鼠肝脏缺血再灌注损伤依赖于TLR4抑制；其次，明确阿片激动剂预处理及肝IR损伤后β-arrestin2表达和细胞定位，使用慢病毒感染下调β-arrestin2的表达，探究β-arrestin2对阿片信号的负调控及Raw264.7细胞系LPS刺激后炎症反应性的影响；最后，对于β-arrestin2与TLR4通路介导阿片肝保护效应的机制进行了相关研究。.研究结果： .1. 阿片激动剂瑞芬太尼可以有效抑制肝脏IR后TLR4蛋白的表达，并减轻肝脏损伤。而在TLR4-KO小鼠肝IR损伤后，不能有效激活TLR4通路，从而使肝脏炎症级联反应得到有效抑制，明显减轻肝损害，阿片激动剂瑞芬太尼的预处理不能在TLR4-KO小鼠上体现出保护效应。.2. 瑞芬太尼预处理能够促进β-arrestin2的表达、增加LPS抑制的细胞活力并减轻细胞的凋亡。阐明了β-arrestin2基因对阿片信号负调控及Raw264.7细胞系炎症反应性的影响，初步明确β-arrestin2在阿片信号的胞内传递和抗炎效应的作用。.3. 证实瑞芬太尼作用下的β-arrestin2核移位及其与TRAF6特异结合现象，明确β-arrestin2对肝细胞TLR4通路相关分子表达及核内炎症相关转录因子活性的影响。 .本项目科学意义：.本课题采用转基因技术、慢病毒感染技术和基因敲除动物模型，从多个水平研究了β-arrestin2在阿片激动剂减轻肝脏IR损伤中调控阿片和TLR4通路的关键作用靶位和机制，进一步揭示肝IR损伤后炎症反应和获得性免疫的分子机制，为临床实用性的阿片激动剂肝保护治疗提供重要的理论佐证和实验依据。