Aspartate beta-hydroxylase and DNA damage in chronic liver diseases

慢性肝病中的天冬氨酸 β-羟化酶和 DNA 损伤

• 批准号: 10667881
• 负责人: Chiung-Kuei Huang
• 金额: $ 21.96万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667881
• 关键词: Affect; Alcohol abuse; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcoholic steatohepatitis; Alcohols; Animal Experiments; Animal Feed; Aspartate; CRISPR/Cas technology; Cause of Death; Cell Death; Cell Death Induction; Cessation of life; Chronic; Cirrhosis; DNA; DNA Damage; Data; Development; Disease Progression; Disease model; Down-Regulation; Ectopic Expression; Ethanol; Gamma-H2AX; Goals; Hepatic; Hepatocyte; Homeostasis; Human; Knock-out; Knowledge; Laboratories; Link; Liver; Liver Cirrhosis; Liver Fibrosis; Maintenance; Malignant neoplasm of liver; Mediating; Mediator; Microinjections; Mixed Function Oxygenases; Molecular; Mus; Patients; Phosphorylation; Pilot Projects; Plasmids; Proteomics; Regulation; Research; Rodent; Role; Severity of illness; System; Testing; Therapeutic; Toxin; Transgenic Mice; Up-Regulation; Viral; advanced disease; cell injury; chronic alcohol ingestion; chronic liver disease; comparison control; dietary control; effective therapy; feeding; in vivo; innovation; insight; knock-down; liver injury; novel; overexpression; problem drinker; protective effect; response; therapeutic target

Project summary Chronic alcohol drinking has been linked to the development and progression of alcoholic liver disease (ALD) by means of damaging cellular DNA, which results in cell death of hepatocytes, contributing to alcoholic steatohepatitis, liver fibrosis, and potentially liver cancers. However, it remains unclear how alcohol promotes DNA damage responses in ALD development and understanding the underlying mechanisms may yield insight into therapeutic approaches toward treating ALD. In the pilot studies, it was demonstrated that aspartate beta- hydroxylase (ASPH) is down-regulated in the rodent livers derived from the ethanol-fed group. In addition, DNA damage markers, including γ-H2aX (a phosphorylated form of H2aX) and GADD45α, were found elevated when compared to the control-diet fed group. The liver is predominantly composed of hepatocytes whose function is critical in the maintenance of homeostasis thereof. Thus, we investigated whether there is a correlation between ASPH and DNA damage in human hepatocytes. We ectopically expressed ASPH and challenged hepatocytes with DNA damage inducers. Intriguingly, the expression of ASPH almost eliminated γ-H2aX expression; however, knockdown of ASPH substantially promoted DNA damage. These observations suggest a protective role of ASPH in the DNA damage response. Based on these findings, we hypothesize that chronic alcohol consumption leads to ASPH down-regulation in the liver contributing to DNA damage responses and hepatocyte cell death in chronic liver disease progression. Our long-term goal is to understand how ASPH is involved in ethanol-induced hepatocyte DNA damage and the consequent impacts on ALD progression. To test our hypothesis and achieve this goal, two specific aims are proposed. In aim 1, we will investigate how the expression of ASPH alters ALD progression in vivo. We will determine if targeting hepatic ASPH using an AAV strategy will exacerbate DNA damage and cell death in the hepatocytes of ethanol-fed mice. Furthermore, we investigate if overexpression of ASPH protects hepatocytes in an ALD model. In aim 2, we will decipher the mechanism by which ASPH protects hepatocytes from DNA damage induced cell death. We will determine how ASPH is involved in ethanol-mediated DNA damage by using CRISPR/Cas9 to knock out ASPH or ectopically expressing ASPH via a lentiviral system. To delineate the mechanisms involved in this regulation, we will determine how the enzymatic functions of ASPH are associated with the ASPH-mediated DNA damage responses and cell death. Using the proteomic approach, we will also illustrate innovative ASPH downstream targets upon ethanol treatment. The results will significantly advance our understanding of the molecular mechanisms in hepatocyte cell death and pathophysiological mechanisms underlying ALD progression. We also anticipate that it will broaden our knowledge of ASPH expression and its relationship to hepatocyte function in general.

项目摘要 长期饮酒与酒精性肝病（ALD）的发展和进展有关 破坏细胞DNA的手段，导致肝细胞的细胞死亡，导致酒精 脂肪性肝炎，肝纤维化和潜在的肝癌。但是，目前尚不清楚酒精如何促进 ALD开发和理解潜在机制中的DNA损伤反应可能会产生洞察力 进入治疗ALD的治疗方法。在试点研究中，证明天冬氨酸β- 羟化酶（ASPH）在源自乙醇喂养基团的啮齿动物肝中下调。另外，DNA 损害标记物，包括γ-H2AX（H2AX的磷酸化形式）和GADD45α，在 与对照组联邦组相比。肝脏主要由肝细胞组成，其功能为 维持体内平衡至关重要。那我们调查了是否之间存在相关性 人类肝细胞中的ASPH和DNA损伤。我们在异位表达ASPH并挑战肝细胞 与DNA损伤诱导者。有趣的是，ASPH的表达几乎消除了γ-H2AX的表达。然而， ASPH的敲低基本上促进了DNA损伤。这些观察结果表明 在DNA损伤反应中ASPH。根据这些发现，我们假设慢性酒精 消费导致肝脏的ASPH下调，导致DNA损伤反应和 慢性肝病进展中的肝细胞死亡。我们的长期目标是了解Asph 参与乙醇诱导的肝细胞DNA损伤以及随之而来的对ALD进展的影响。到 检验我们的假设并实现这一目标，提出了两个具体目标。在AIM 1中，我们将调查如何 Asph的表达改变了体内ALD进展。我们将确定是否使用肝脏ASPH使用 AAV策略会加剧乙醇喂养小鼠的肝细胞中DNA损伤和细胞死亡。此外， 我们研究ASPH的过表达是否在ALD模型中保护肝细胞。在AIM 2中，我们将破译 ASPH保护肝细胞免受DNA损伤诱导细胞死亡的机制。我们将 通过使用CRISPR/CAS9击败ASPH，确定ASPH如何参与乙醇介导的DNA损伤 或通过慢病毒系统进行生态表达ASPH。要描述该法规所涉及的机制， 我们将确定ASPH的酶函数如何与ASPH介导的DNA损伤相关 反应和细胞死亡。使用蛋白质组学方法，我们还将在下游说明创新的asph 靶向乙醇治疗。结果将大大提高我们对分子的理解 肝细胞细胞死亡和病理生理机制的机制。我们也是 预计它将扩大我们对Asph表达的了解及其与肝细胞功能的关系 一般的。