A novel cholinergic circuitry in alcoholic liver disease (NIAAA)

酒精性肝病（NIAAA）中的新型胆碱能回路

• 批准号: 10371184
• 负责人: Jun Wu
• 金额: $ 41.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10371184
• 关键词: Acetaldehyde; Acetylcholine; Agonist; Alcohol consumption; Alcohol dehydrogenase; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcoholic liver damage; Alcohols; Biogenesis; Cell Line; Cell membrane; Cells; Choline O-Acetyltransferase; Chronic; Chronic Hepatitis; Cirrhosis; Collaborations; Country; Data; Development; Diet; Diffuse; Enzymes; Ethanol; Fatty Liver; Fibrosis; Flow Cytometry; Gender; Gene Deletion; Genetic; Heavy Drinking; Hematopoietic; HepG2; Hepatic; Hepatocyte; Human; Immune; Impairment; In Vitro; Inflammation; Inflammatory; Injury; Intestines; Investigation; Ion Channel; Ion Channel Gating; Knockout Mice; Kupffer Cells; Lead; Ligands; Liver; Liver Cirrhosis; Liver Fibrosis; Liver diseases; Mediating; Modeling; Molecular; Mus; National Institute on Alcohol Abuse and Alcoholism; Nicotinic Receptors; Paracrine Communication; Pathogenesis; Pathway interactions; Pattern; Play; Primary carcinoma of the liver cells; Regimen; Regulation; Regulatory Pathway; Reporter; Reporting; Risk; Role; Route; Sampling; Series; Signal Pathway; Signal Transduction; Site; Spatial Distribution; Symptoms; Testing; Therapeutic Intervention; Therapeutic Uses; Tissues; Woman; alcohol exposure; cholinergic; chronic alcohol ingestion; chronic liver disease; dietary control; dysbiosis; effective therapy; endoplasmic reticulum stress; experimental study; in vivo; insight; interdisciplinary approach; lipid biosynthesis; liver function; liver injury; liver preservation; liver transplantation; loss of function; macrophage; monocyte; mortality; mouse model; new therapeutic target; novel; pathogen; recruit; screening; sexual dimorphism; single-cell RNA sequencing; small molecule; three-dimensional visualization; transcriptome; transcriptomics

Project Summary Alcoholic liver disease (ALD) is one of the major chronic liver diseases, encompassing symptoms from fatty liver, alcoholic hepatitis, chronic hepatitis with liver fibrosis and cirrhosis, and potentially hepatocellular carcinoma. Despite extensive investigations, current understanding of the pathogenesis of ALD is still limited and no effective therapies are available for late-stage ALD besides liver transplantation. Our preliminary studies revealed a previously unknown non-neuronal cholinergic signaling pathway between acetylcholine-producing immune cells residing within the liver and hepatocytes expressing the nicotinic acetylcholine receptor, alpha 2 subunit (CHRNA2). This signaling pathway was activated in the livers of mice following alcohol consumption and loss of function mouse models with genetic deletion of either Chrna2 or Chat (choline acetyltransferase, rate limiting enzyme for acetylcholine biogenesis) suffered aggravated liver damage after chronic alcohol consumption. We propose to thoroughly test the hypothesis that this novel hepatic acetylcholine-CHRNA2 signaling pathway plays an adaptive/protective role against alcohol-induced liver damage. Aim 1. We will investigate how ChAT+ hepatic non-parenchymal cells (NPCs), particularly liver-resident Kupffer cells and monocyte-derived macrophages (MDMs), are activated after alcohol consumption. In vivo regulation will be analyzed with flow cytometry using hepatic NPCs isolated from ChATBAC-eGFP mice and a double reporter mice (ChAT-Cre;tdTomato;ChATBAC- eGFP). Mechanistic insights will be investigated with cultured macrophages (BMDMs) and human macrophage cell lines. Spatial distribution of ChAT+ cells in the whole tissue will be visualized in CLARITY-prepared liver samples and single cell RNA-seq will be carried out to characterize the transcriptomic landscape of these hepatic cholinergic NPCs. Aim 2. We will investigate signaling mediated through CHRNA2 in hepatocytes using mouse primary hepatocytes, HepG2 cells and human primary hepatocytes. The composition of the CHRNA2-containing ligand-gated ion channel in hepatocytes will be investigated to enable screening for potential hepatocyte-specific small molecule agonists to activate this signaling pathway. Aim 3. Hepatocyte-specific Chrna2 knockout mice and immune specific ChAT knockout mice will be treated with three regimens of chronic alcohol challenge to reveal the functional significance of this signaling pathway in the pathogenesis of ALD. A team of leading experts in related fields have been recruited to carry out the proposed studies with interdisciplinary approaches. Ultimately, uncovering the mechanisms that underlie this novel hepatic pathway may elucidate new routes of therapeutic intervention for counteracting liver injury arising from excessive alcohol consumption.

项目概要 酒精性肝病（ALD）是主要的慢性肝病之一，包括脂肪肝、 酒精性肝炎、伴有肝纤维化和肝硬化的慢性肝炎，以及潜在的肝细胞癌。 尽管进行了广泛的研究，但目前对 ALD 发病机制的了解仍然有限，并且没有有效的治疗方法。 除肝移植外，晚期 ALD 还可以采用其他治疗方法。我们的初步研究揭示了 以前未知的乙酰胆碱产生免疫细胞之间的非神经元胆碱能信号通路 存在于表达烟碱乙酰胆碱受体、α2亚基的肝脏和肝细胞内 (CHRNA2)。小鼠饮酒和丧失肝脏功能后，该信号通路在小鼠肝脏中被激活。 具有 Chrna2 或 Chat（胆碱乙酰转移酶、限速酶）基因删除的功能小鼠模型 乙酰胆碱生物发生酶）在长期饮酒后会遭受严重的肝损伤。我们 提议彻底检验这种新型肝乙酰胆碱-CHRNA2信号通路发挥作用的假设 对酒精引起的肝损伤具有适应性/保护作用。目标 1. 我们将研究 ChAT+ 肝脏如何 非实质细胞 (NPC)，特别是肝脏驻留的库普弗细胞和单核细胞衍生的巨噬细胞 （MDM），在饮酒后被激活。将使用流式细胞术分析体内调节 从 ChATBAC-eGFP 小鼠和双报告小鼠 (ChAT-Cre;tdTomato;ChATBAC- 绿色荧光蛋白）。将利用培养巨噬细胞 (BMDM) 和人类巨噬细胞研究机制见解 细胞系。 ChAT+ 细胞在整个组织中的空间分布将在 CLARITY 准备的肝脏中可视化 将进行样本和单细胞 RNA-seq 来表征这些肝脏的转录组景观 胆碱能 NPC。目标 2. 我们将使用小鼠研究肝细胞中 CHRNA2 介导的信号传导 原代肝细胞、HepG2细胞和人原代肝细胞。含CHRNA2的组成 将研究肝细胞中的配体门控离子通道，以筛选潜在的肝细胞特异性 小分子激动剂可以激活该信号通路。目标 3. 肝细胞特异性 Chrna2 敲除小鼠 免疫特异性 ChAT 敲除小鼠将接受三种慢性酒精激发方案治疗 揭示该信号通路在 ALD 发病机制中的功能意义。领先的专家团队 已招募相关领域的专家以跨学科方法开展拟议的研究。 最终，揭示这种新的肝脏途径背后的机制可能会阐明新的途径 用于对抗过量饮酒引起的肝损伤的治疗干预。