THE RELAXIN RECEPTOR GR/RXFP1 SIGNALING IN LIVER TRANSPLANT ISCHEMIA-REPERFUSION INJURY AND THE INFLAMMATION RESOLUTION

项目摘要/摘要 肝脏缺血/再灌注损伤(IRI)是一种先天免疫驱动的炎症反应，是一种主要障碍 限制终末期肝病患者原位肝移植(OLT)的成功 患有肝源性肿瘤。尽管在更好地了解肝脏方面已经取得了重大进展 IR应激引起的炎性级联反应，对其消退知之甚少，这可能不仅影响 不仅是组织损伤本身的严重程度，更重要的是长期结果。最新研究文献 重组人松弛素-2对小鼠原位肝移植模型IR应激的保护作用 通过肝细胞糖皮质激素受体(GR)信号；而通过Notch1极化巨噬细胞激活 增强了对IRI-OLT的抗性。这些实验发现，得到了增强的临床证据的支持 人原位肝移植IRI抵抗所需的GR/Notch1表型提示rh RLX可能发挥作用 作为一种新的GR激动剂和糖皮质激素(GC)模拟物在肝移植中的应用。试点研究也指出了反 同源RXFP1受体的纤维化功能，它是一种非GR依赖的RLX-2结合伙伴。作为传统的 小鼠原位肝移植模型提供的翻译功能有限，本项目将解剖GR-RXFP1分子 在表达人RXFP1基因的小鼠中的相互作用；以及测试RXFP1驱动的肝脏的新概念 在低温机器保存过程中废弃的人类肝脏的复壮。一种新发现的发散型 GR-RXFP1信号轴在IRI-OLT炎症的“急性期”和“缓解期”中的作用 提出一个新的、迄今为止未经检验的总体假设，即：1/药理学上的重组人RLX诱导GR 增强功能将使OLT免于急性IR损伤；同时通过ITS利用不依赖GR的rhRLX信号 同源受体RXFP1将在炎症消退阶段促进体内平衡/抗纤维化功能。 两个相互关联的目标探索了这一假设： 目的1：探讨重组人RLX-GR在肝移植中保护肝细胞的分子机制 阶段)。目的1.1：假设肝细胞Keap1依赖的Nrf2信号对重组人RLX是不可或缺的 -GR轴，防止冷藏供体肝脏中的湿气释放和先天炎症。目标1.2：测试 假设SIRT1增强IR应激原位肝移植中GR诱导的肝细胞再生功能/自噬。 目的2.阐明Notch1/RXFP1在原位肝移植(IRI-炎症)中抗纤维化作用的分子机制 “解决”阶段)。目的2.1：测试假设Notch1(巨噬细胞)-RXFP1(T细胞)交叉调节是 对IRI-OLT炎症的消退至关重要。目标2.2：检验假设人类RLX的激活 受体在人源化RXFP1“敲门”的IRI-OLT炎症消退中的抗纤维化作用 鼠标系统。目的2.3：测试假设RXFP1受体在低温机械过程中被激活 保存可以减轻炎症，使废弃的人类供体肝脏恢复活力。

PROJECT SUMMARY/ABSTRACT Hepatic ischemia/reperfusion injury (IRI), an innate immune-driven inflammation response, is a major obstacle limiting the success of orthotopic liver transplantation (OLT) in patients with end-stage liver disease and those with tumors of hepatic origin. Although significant progress has been made in better appreciation of the liver inflammatory cascade by IR-stress, much less is known about its resolution, which may affect not only the severity of tissue injury itself but also, more importantly, the long-term outcomes. Recent studies document striking cytoprotective functions of hrRLX (recombinant human relaxin-2) against IR-stress in mouse OLT models via hepatocyte glucocorticoid receptor (GR) signaling; while polarizing macrophage activation via Notch1 promoted IRI-OLT resistance. These experimental findings, supported by a clinical evidence of enhanced GR/Notch1 phenotype needed for IRI resistance in human OLT, prompted to propose that rhRLX may function as a novel GR agonist and glucocorticoid (GC) mimetic in liver transplantation. Pilot studies also point to anti- fibrotic functions of the cognate RXFP1 receptor, a GR-independent RLX-2 binding partner. As the conventional murine OLT model offers a limited translational utility, this project will be dissecting GR – RXFP1 molecular interplay in mice expressing human RXFP1 gene; as well as testing new concepts of RXFP1-driven hepatic rejuvenation of discarded human livers during hypothermic machine preservation. A newly discovered divergent role of GR–RXFP1 signaling axis in the “acute” and “resolution” phase of IRI-OLT inflammation, has prompted to put forth a novel and heretofore untested overall hypothesis, that: 1/ pharmacological rhRLX-induced GR enhancement will rescue OLT from acute IR-insult; while 2/ harnessing GR-independent rhRLX signaling via its cognate receptor, RXFP1, will promote homeostatic/anti-fibrotic functions in the inflammation resolution phase. Two interlocked aims explore this hypothesis: Aim 1: Delineate molecular mechanisms of rhRLX – GR hepatocellular protection in OLT (acute IRI-inflammation phase). Aim 1.1: Test hypothesis that hepatocellular Keap1-dependent Nrf2 signaling is indispensable for rhRLX – GR axis to prevent DAMPs release and innate inflammation in cold-stored donor livers. Aim 1.2: Test hypothesis that SIRT1 enhances GR-induced hepatocyte regenerative functions/autophagy in IR-stressed OLT. Aim 2. Delineate molecular mechanisms of Notch1 / RXFP1 anti-fibrotic functions in OLT (IRI-inflammation “resolution” phase). Aim 2.1: Test hypothesis that Notch1 (macrophage) – RXFP1 (T cell) cross-regulation is essential in the resolution of IRI – OLT inflammation. Aim 2.2: Test hypothesis that the activation of human RLX receptor exerts anti-fibrotic functions in the resolution of IRI-OLT inflammation in humanized RXFP1 “knockin” mouse system. Aim 2.3: Test hypothesis that activation of RXFP1 receptor during hypothermic machine preservation attenuates inflammation and rejuvenates discarded human donor livers.