Hepatocyte Exosomes for Therapy of Ethanol-Induced Liver Injury

摘要 广泛的长期目标是改进肝脏疾病的治疗方法。酒精性肝病 (ALD) 由过量饮酒引起，是相当大的发病率和死亡率的原因 全世界。在美国，ALD 是胃肠道相关死亡的主要原因，75,000 例死亡中约有一半是由 ALD 引起的。 每年的肝病死亡都与饮酒有关。尽管治疗选择很少，但一种新的 我们对正常健康肝细胞产生的外泌体的研究发现了铅。我们的 初步数据表明，这些外泌体 (i) 减弱调节纤维发生或 培养的原代肝星状细胞（HSC；肝脏中产生纤维化的主要细胞类型）中的激活； (ii) 在体内肝纤维化实验模型中抑制纤维化途径并逆转纤维化； (三) 与对照肝脏中的肝细胞相比，与受损肝脏中的肝细胞的结合更强； (iv) 减弱 原代培养的肝细胞中乙醇诱导的细胞毒性。我们的总体目标是建立治疗 外泌体在治疗肝病中的用途。我们的中心假设是来自肝细胞的外泌体 在 ALD 小鼠模型中具有治疗作用。检验这一假设的具体目标是： 具体目标 1：确定肝细胞外泌体可减轻乙醇诱导的肝损伤 外泌体将 (i) 给予接受乙醇 25 天（Lieber deCarli 饮食）或 10 天的小鼠 随后几天进行急性乙醇灌胃（暴食模型）以确定其对脂肪变性的治疗效果， 炎症、细胞损伤或巨噬细胞参与；或 (ii) 添加至培养的肝细胞或库普弗细胞 (KC) 以确定它们分别逆转乙醇或脂多糖 (LPS) 对活力的影响， 细胞凋亡、MAPK 或半胱天冬酶的激活以及炎症细胞因子、ROS 或损伤的产生 诱发危险信号。 具体目标 2：鉴定肝细胞外泌体中的治疗性 microRNA 通过无偏分析，将鉴定出 miR 之间表达差异最大的 miR。 来自正常肝细胞与乙醇损伤肝细胞的外泌体，其中表达水平较高 来自正常肝细胞的外泌体。通过 PT-PCR 验证的候选 miR 将接受测试 乙醇/TNF-α 处理后肝细胞中或 LPS 后 KC 中损伤诱导途径的调节 治疗。 预期结果将是建立一种新的基于外泌体的疗法来治疗酒精诱导的细胞 损伤和免疫功能改变是 ALD 发病机制的关键特征。其背后的基本原理 建议认为，正常未受损肝细胞产生的外泌体含有分子货物，包括 miR，反映了他们的整体健康状况。这些研究的积极影响在于它们将提供新的 改善全球数百万 ALD 或其他慢性肝病患者健康的知识

ABSTRACT The broad long term objective is to improve methods of disease treatment in the liver. Alcoholic liver disease (ALD) results from excessive alcohol consumption and is the cause of considerable morbidity and mortality world-wide. In the United States, ALD is a leading cause of GI-related deaths, with about half of the 75,000 liver disease deaths each year being related to alcohol use. Although there are few therapeutic options, a new lead has emerged from our studies of exosomes that are produced by normal healthy hepatocytes. Our Preliminary Data show that these exosomes (i) attenuate expression of genes that regulate fibrogenesis or activation in cultured primary hepatic stellate cells (HSC; the principal fibrosis-producing cell type in the liver); (ii) suppress fibrogenic pathways and reverse fibrosis in experimental models of hepatic fibrosis in vivo; (iii) bind more strongly to hepatocytes in injured livers than hepatocytes in control livers; and (iv) attenuate ethanol-induced cytotoxicity in primary cultured hepatocytes. Our overall objective is to establish therapeutic uses of exosomes for treating liver disease. Our central hypothesis is that exosomes from hepatocytes are therapeutic in mouse models of ALD. The Specific Aims to test this hypothesis are: Specific Aim 1: Determine that hepatocyte exosomes attenuate ethanol-induced liver injury Exosomes will be (i) administered to mice that receive ethanol for either 25 days (Lieber deCarli diet) or for 10 days followed by acute ethanol gavage (binge model) to determine their therapeutic effect on steatosis, inflammation, cell damage, or macrophage involvement; or (ii) added to cultured hepatocytes or Kupffer cells (KC) to determine their reversal of the effects of, respectively, ethanol or lipopolysaccharide (LPS), on viability, apoptosis, activation of MAPK or caspases, and production of inflammatory cytokines, ROS, or damage- induced danger signals. Specific Aim 2: Identify therapeutic microRNAs in hepatocyte exosomes Using an unbiased analysis, miRs will be identified that are the most differentially expressed between exosomes from normal versus ethanol-injured hepatocytes and for which the higher level of expression occurs in exosomes from normal hepatocytes. Candidate miRs, validated by PT-PCR, will be tested for their modulation of damage-inducing pathways in hepatocytes after ethanol/TNF-α treatment or in KC after LPS treatment. The expected outcome will be to establish a new exosome-based therapy for treating alcohol-induced cell damage and altered immune function that are key features of ALD pathogenesis. The rationale underlying the proposal is that exosomes produced by normal non-injured hepatocytes contain a molecular cargo, including miRs, that reflects their overall healthy status. The positive impact of these studies is that they will provide new knowledge to improve the health of millions of people world-wide with ALD or other chronic liver diseases