Hepatocyte Exosomes for Therapy of Ethanol-Induced Liver Injury

摘要 广泛的长期目标是改善肝脏疾病的治疗方法。酒精性肝病 (ALD)结果是过量饮酒，是相当大的发病率和死亡率的原因 世界范围内。在美国，ALD是GI相关死亡的主要原因， 每年死于肝病的人都与饮酒有关。虽然有一些治疗选择，一个新的 我们对正常健康肝细胞产生的外来体的研究中发现了铅。我们 初步数据显示，这些外来体（i）减弱调节纤维发生的基因的表达，或 培养的原代肝星状细胞（HSC;肝脏中主要的纤维化产生细胞类型）中的活化; (ii)在体内肝纤维化实验模型中抑制纤维化发生途径并逆转纤维化;（iii） 与对照肝脏中的肝细胞相比，损伤肝脏中的肝细胞与肝细胞的结合更强;以及（iv）减弱 乙醇诱导的原代培养肝细胞毒性。我们的总体目标是建立治疗性的 外来体用于治疗肝病的用途。我们的中心假设是来自肝细胞的外泌体 治疗ALD的小鼠模型。检验这一假设的具体目的是： 具体目的1：确定肝细胞外泌体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（Lieber deCarli饮食）的小鼠 天后进行急性乙醇管饲（暴饮暴食模型）以确定它们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枯否细胞 (KC)为了确定它们分别逆转乙醇或脂多糖（LPS）对生存力的影响， 细胞凋亡、MAPK或半胱天冬酶的活化以及炎性细胞因子、ROS或损伤的产生- 发出危险信号。 具体目标2：鉴定肝细胞外泌体中的治疗性microRNA 使用无偏分析，将鉴定miR，所述miR在以下细胞之间差异表达最大： 来自正常肝细胞与乙醇损伤肝细胞的外泌体，并且其表达水平更高 来自正常肝细胞的外来体。将测试通过PT-PCR验证的候选miR的 调节乙醇/TNF-α处理后肝细胞或LPS处理后KC中的损伤诱导途径 治疗 预期的结果将是建立一种新的基于外泌体的治疗酒精诱导的细胞凋亡的方法。 损害和改变的免疫功能是ALD发病机制的关键特征。的基本原理， 有人提出，正常非损伤肝细胞产生的外泌体含有分子货物，包括 这反映了他们的整体健康状况。这些研究的积极影响是，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