Role of Exosomal microRNAs in Alcohol-induced Liver Injury

摘要 在全球范围内，酒精性肝病（ALD）仍然是发病率和死亡率的主要原因。慢性酒精性肝病 炎症和氧化应激以及肝细胞死亡，其释放多种细胞损伤和相关损害 分子、凋亡小体、微泡（MV）和外来体（EXO）进入肝脏微环境，激活其他 肝细胞类型。这种慢性损伤导致以过量胶原蛋白为特征的伤口愈合反应加剧 沉积可导致纤维化/肝硬化和肝细胞癌。酒精是肝毒性的，但它的新陈代谢驱动 导致肝脏病理的氧化应激。肝脏中的多种细胞类型需要协调对 这种慢性损伤。肝星状细胞（HSC）从静止型转分化为活化的肌纤维母细胞型细胞 这是胶原沉积的主要效应物。肝脏巨噬细胞、枯否细胞（KC）调节先天性炎症 反应和肝重塑。修复和重塑的协调需要细胞内通讯。的 这些细胞在肝微环境中的紧密排列促进了外泌体（EXO）在肝微环境之间的转移。 细胞群EXO是由所有活性或静息细胞产生的30 - 100 nm的囊泡， microRNA（miR）、mRNA和蛋白质货物。EXO可以在组织、血液和其他体液中找到， 功能性地将其内容物转移到受体细胞，诱导基因表达和活性的变化。EXO介导 miR的转移可能在酒精诱导的损伤过程中对HSC活化和KC功能发挥局部控制作用。miR，小 非编码RNA是影响所有细胞生理功能的基因表达的关键调节因子， 是EXO货物的主要组成部分我们已经确定，在活化的HSC中，EXO分泌增加，并且miR-125表达增加。 配置文件在激活时更改。此外，来自活化的HSC的EXO与巨噬细胞相互作用以抑制细胞增殖。 暴露于细菌内毒素的细胞的炎症免疫应答。基于这些数据，我们假设EXO 在酒精诱导的肝损伤期间由HSC分泌的递送可调节KC反应的独特miR货物 并在肝损伤期间协调组织修复。在目标1中，我们将鉴定来自HSC的miR谱的变化- 酒精诱导损伤模型中的EXO。在目标2中，我们将鉴定膜和内部蛋白质和RNA (mRNA、miR等）来自HSC-EXO的货物可能促进细胞摄取并定义miR：mRNA相互作用 可以指导和协调KC功能。关于细胞交换和EXO在细胞内的作用， ALD病理学本申请中的方法是创新的，因为它将从活化的HSC中鉴定外泌体miR， 用于局部调节KC功能。此外，使用酒精性肝损伤模型，采用一种新的方法， 提取组织特异性外泌体，我们将更直接地确定它们在肝脏病理学中的作用。我们预计这些 研究将揭示新的诊断/预后指标，并确定新的治疗靶点。

Abstract Globally alcoholic liver disease (ALD) remains a leading cause of morbidity and mortality. ALD results from chronic inflammation and oxidative stress and hepatocyte death which releases a variety of cellular injury and damage associated molecules, apoptotic bodies, microvesicles (MV) and exosomes (EXOs) into the hepatic microenvironment activating other hepatic cell types. This chronic injury results in an exacerbated wound-healing response characterized by excess collagen deposition that can lead to fibrosis/cirrhosis and hepatocellular carcinoma. Alcohol is hepatotoxic, but its metabolism drives oxidative stresses contributing to hepatic pathology. Multiple cell types in the liver are required to coordinate a response to this chronic injury. Hepatic stellate cells (HSCs) transdifferentiate from a quiescent to an activated myofibroblast-type cell that is the primary effector of collagen deposition. Hepatic macrophages, Kupffer cells (KC) regulate innate inflammatory responses and hepatic remodeling. Coordination of repair and remodeling requires intracellular communication. The intimate arrangement of these cells in hepatic microenvironment facilitates the transfer of exosomes (EXOs) between the cell populations. EXOs are vesicles of 30-100 nm that are produced by all active or resting cells and are packaged with microRNA (miR), mRNA and protein cargo. EXOs can be found in tissues, blood and other bodily fluids where they can functionally transfer their contents to recipient cells inducing changes in gene expression and activity. EXO-mediated transfer of miRs may exert local control on HSC activation and KC function during alcohol-induced injury. miRs, small non-coding RNAs, are critical regulators of gene expression that influence all cellular physiological functions, and they are a major component of EXO cargo. We have identified that EXO secretion is increased in activated HSCs, and that the miR profiles change on activation. In addition, EXOs from activated HSCs interact with macrophages to suppress the inflammatory immune response in cells exposed to bacterial endotoxin. Based on these data we hypothesize that EXOs secreted by HSCs during alcohol-induced liver injury deliver unique miR cargo that can modulate KC responses and coordinate tissue repair during hepatic injury. In aim 1 we will identify changes in miRs profiles from HSC- EXOs in models of alcohol-induced injury. In aim 2 we will identify membrane and internal protein and RNA (mRNA, miR, etc.) cargo from HSC-EXOs that may facilitate cellular uptake and define miR:mRNA interactions that may direct and coordinate KC function. Much remains unknown about the cellular exchange and role of EXOs in ALD pathology. The approach in this application is innovative as it will identify exosomal miRs from activated HSCs that serve to locally modulate KC function. In addition, using models of alcohol-induced hepatic injury with a novel method of extracting tissue specific exosomes we will more directly determine their role in hepatic pathology. We anticipate these studies will reveal novel diagnostic/prognostic indicators and identify new therapeutic targets.