Molecular mechanism of Sickle Cell Hepatic Crisis

项目摘要/摘要 这项建议的目标是延长我作为一名独立科学家在肝脏表现领域的培训 镰状细胞病(SCD)。为此，我选择了加州大学血液肿瘤学系 匹兹堡继续我的过渡，成为一家学术机构的独立调查员。这 提案概述了广泛的研究战略，并辅之以几个培训领域，这些领域 包括几门与我在特定目标上的学习直接相关的课程，与我的初级教员研究会面 委员会，以及出席和参与匹兹堡大学的多个研讨会 医学院。我的研究策略将确定SCD导致肝脏危象的分子机制。 SCD是一种常染色体隐性单基因遗传病，影响大约10万美国人和数百万人 世界各地的人。血窦血管闭塞和溶血被认为是镰刀的主要因素。 肝脏危象。10%-40%的住院SCD患者出现肝危象，其特征是肝损伤 和镰状细胞性肝内胆汁淤积症(SCIC)，可进展为致命的肝功能衰竭。目前的治疗方法是 肝危象主要是支持性的，分子机制尚不清楚，提示预防性 基于对导致SCIC的分子通路的更好理解的治疗是必要的。 在这项研究中，我们使用了一种专门表达镰刀的转基因人源化SCD小鼠模型。 人类血红蛋白。初步结果表明，SCD小鼠随着年龄的增长而出现慢性肝损伤，这是 主要表现为持续炎症、高胆红素血症和胆汁淤积。使用我们最新开发的 实时活体成像活体小鼠完整的肝脏，我们发现存在肝窦缺血和 SCD小鼠肝细胞顶膜胆汁转运受损。胆汁转运受损 与肝细胞顶端胆汁转运蛋白(BSEP、ABCG5和ABCG8)的丢失有关。RNA序列 分析发现，与炎症和胆汁分泌有关的编码蛋白的基因调节失调。 SCD小鼠的肝脏。此外，我们观察到核因子-κB在SCD小鼠肝脏中的激活抑制了FXR 信号及其下游靶点，导致胆汁分泌受损。这些发现构成了我的 关于缺血和溶血引起炎症、组织损伤和氧化应激的主要假说 促进镰状肝细胞核因子-kB活化，抑制FXR信号导致胆汁分泌受损 在SCD中，激活FXR或挽救胆汁分泌可改善SCIC。这一假设将在 目的如下：1)确定缺血再灌注损伤和溶血是否促进肝细胞特异性 核因子-kB在SCD中的激活以及2)确定肝细胞特异性激活的核因子-kB是否促进丢失 FXR信号导致SCD胆汁分泌受损和胆汁淤积。成功地完成这项工作 NIH-K01培训奖将为RO1提案奠定基础，旨在阐明如何操纵 炎症途径、FXR信号转导和胆汁分泌可挽救SCD的SCIC和进行性肝损伤。

Project Summary/Abstract The goal of this proposal is to extend my training as an independent scientist in the field of hepatic manifestations of Sickle cell disease (SCD). To this end, I have selected the division of Hematology-Oncology at University of Pittsburgh to continue my transition to become an independent investigator at an academic institution. This proposal outlines an extensive Research Strategy that is complemented by several areas of training, which includes several courses directly related to my studies in Specific Aims, meeting with my junior faculty research committee, and attendance and participation of multiple seminars throughout the University Of Pittsburgh Medical School. My Research Strategy will determine the molecular mechanisms of SCD induced hepatic crisis. SCD is an autosomal-recessive monogenic disorder that affects approximately 100,000 Americans and millions of people worldwide. Sinusoidal vaso-occlusion and hemolysis are considered as chief contributors of sickle hepatic crisis. Hepatic crisis affects 10-40% of hospitalized SCD patients which is characterized by liver injury and sickle cell intrahepatic cholestasis (SCIC) that can progress to fatal liver failure. The current treatment for hepatic crisis is primarily supportive, and the molecular mechanism is unknown, suggesting that preventive therapies based on the improved understanding of the molecular pathways that enable SCIC are needed. In this study, we have used a transgenic, humanized mouse model of SCD that exclusively expresses sickle human hemoglobin. Preliminary findings reveal that SCD mice developed chronic liver injury with age, which was manifested by sustained inflammation, hyperbilirubinemia and cholestasis. Using our recently developed real-time in vivo imaging of the intact liver of live mice, we discovered the presence of sinusoidal ischemia and impaired bile transport across the apical membrane of hepatocytes in SCD mice. The impaired bile transport was associated with loss of apical bile transporters (BSEP, ABCG5 and ABCG8) from hepatocytes. RNA-seq analysis identified dysregulation of genes encoding proteins responsible for inflammation and bile secretion in the liver of SCD mice. Furthermore, we observed NF-κB activation in the liver of SCD mice inhibited FXR signaling and its downstream targets, leading to impaired bile secretion. These findings form the basis for my overarching hypothesis that ischemia and hemolysis induced inflammation, tissue injury and oxidative stress promotes NF-kB activation in sickle hepatocytes which inhibits FXR signaling leading to impaired bile secretion in SCD, and activating FXR or rescuing bile secretion can ameliorate SCIC. This hypothesis will be tested in the following aims: 1) To determine whether ischemia-reperfusion injury and hemolysis promotes hepatocyte specific activation of NF-kB in SCD and 2) To determine whether hepatocyte-specific activation of NF-kB promotes loss of FXR-signaling leading to impaired bile secretion and cholestasis in SCD. The successful completion of this NIH-K01 training award will set the stage for an RO1 proposal aimed at elucidating how the manipulation of inflammatory pathways, FXR signalling and bile secretion can rescue SCIC and progressive liver injury in SCD.