A novel cholinergic circuitry in alcoholic liver disease (NIAAA)

项目摘要 酒精性肝病(ALD)是主要的慢性肝病之一，包括脂肪肝的症状， 酒精性肝炎、慢性肝炎合并肝纤维化和肝硬变，以及潜在的肝细胞癌。 尽管进行了广泛的研究，但目前对ald的发病机制的了解仍然有限，而且没有有效的方法。 除肝移植外，晚期ALD还有其他治疗方法。我们的初步研究显示， 此前未知的乙酰胆碱产生免疫细胞之间的非神经性胆碱能信号通路 驻留在肝脏和肝细胞中，表达烟碱型乙酰胆碱受体α2亚单位 (CHRNA2)。这一信号通路在饮酒和饮酒后小鼠肝脏中被激活 Chat(胆碱乙酰转移酶，速率限制)基因缺失的功能性小鼠模型 乙酰胆碱生物生成酶)在长期饮酒后肝损伤加重。我们 建议彻底检验这一新的肝乙酰胆碱-CHRNA2信号通路发挥作用的假设 对酒精性肝损伤的适应性/保护性作用。目标1.我们将调查Chat肝脏如何 非实质细胞，特别是驻肝的枯否细胞和单核细胞来源的巨噬细胞 (MDMS)，在饮酒后被激活。体内调节将用流式细胞仪进行分析， 从CHATBAC-EGFP小鼠和双报告小鼠(ChAT-Cre；tdTomato；ChATBAC- 绿色荧光蛋白)。我们将用培养的巨噬细胞(BMDM)和人类巨噬细胞来研究其机理 细胞系。ChAT细胞在整个组织中的空间分布将在透明准备的肝脏中可视化 样品和单细胞rna-seq将被用来描述这些肝脏的转录图谱。 胆碱能神经前体细胞。目的2.研究CHRNA2在小鼠肝细胞中介导的信号转导 原代肝细胞、HepG2细胞和人原代肝细胞。含CHRNA2的化合物的组成 将研究肝细胞中的配基门控离子通道，以筛选潜在的肝细胞特异性 小分子激动剂激活这一信号通路。目的3.肝细胞特异性chrna 2基因敲除小鼠 免疫特异性ChAT基因敲除小鼠将被三种方案的慢性酒精攻击 揭示该信号通路在ALD发病机制中的功能意义。一支顶尖的专家团队 已聘请相关领域的专家以跨学科方法开展拟议的研究。 最终，揭示这一新的肝脏途径背后的机制可能会阐明 抗过度饮酒所致肝损伤的治疗干预。

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.