Cholesterol toxicity in alcohol-associated hepatitis

摘要 酒精性肝炎是一种亚急性肝功能衰竭，死亡率高，治疗有限。 选项，以及难以捉摸的发病机制。新出现的证据表明，AH与一种深刻的 肝细胞胆固醇稳态受损。肝细胞是胆固醇的中枢 新陈代谢。同时，AH肝细胞的Fc含量和途径也发生了深刻的变化 胆固醇的酯化、运输和转化为胆汁酸。RNA测序研究表明， 肝细胞转录主因子hnf4α的失调是胆固醇受损的原因 动态平衡。小分子激活核糖核酸上调HNF4α可减轻血管内皮细胞胆固醇毒性 在体外，可以作为治疗急性肝炎的方法。 我们的长期目标是确定以疾病为中心的急性肝炎的途径，并开发出急需的 急性胰腺炎患者的治疗学。我们假设HNF4a依赖的胆固醇损伤 肝细胞内稳态是急性肝炎肝细胞功能障碍的直接原因，可以作为靶点。 从治疗上讲。我们的方法是绘制人类AH中胆固醇代谢的调节通路 单细胞分辨率，在功能上询问小鼠酒精模型中的胆固醇代谢途径，以及 通过靶向依赖HNF4a的胆固醇毒性来测试一种新的治疗策略。 AIM 1将使用多模式方法在单细胞分辨率下绘制AH中的胆固醇代谢图。A三- 分叉策略将使用单个核RNA绘制与AH相关的胆固醇稳态变化图 测序、超高分辨率成像和靶向空间转录。不带偏见的参考 MAP将指导细胞和小鼠AH模型中胆固醇代谢途径的询问。 目的2明确胆固醇失调对小鼠酒精性肝损伤的影响。 我们将使用慢性+酗酒喂养在与AH相关的3种条件下模拟胆固醇毒性： (1)急性胆固醇超负荷，(2)胆汁酸合成受损，(3)胆固醇分泌受损 胆汁淤积症。这一努力将产生优化的小鼠模型，概括总结急性肝炎中的胆固醇毒性。 目的3将评估靶向操作HNF4依赖的α途径的胆固醇毒性作为一种新的 急性胰腺炎的治疗策略。我们使用sRNA来调节hnf4α亚型的表达和胆固醇。 新陈代谢。GalNAc偶联的HNF4α-Sarna将作为一种肝细胞靶向疗法进行测试 利用分化的人肝细胞和小鼠模型减轻急性肝炎模型中的胆固醇毒性。 该项目建立在一支强大的多学科科学家团队的基础上，结合了 脂肪学、临床前肝脏模型，以及最先进的单细胞和空间技术。这件事的成功 该项目将进一步加深我们对AH发病机制的理解，并可能导致新型AH的开发 治疗准备好进行临床前和首个人体试验。

SUMMARY Alcohol-associated hepatitis (AH) is a form of subacute liver failure with high mortality, limited treatment options, and elusive pathogenesis. Emerging evidence suggests that AH is associated with a profound impairment in hepatocellular cholesterol homeostasis. Hepatocytes are the central hub for cholesterol metabolism. Meanwhile, AH hepatocytes exhibit profoundly altered FC content and pathways responsible for cholesterol esterification, transport, and conversion to bile acids. RNA sequencing studies indicate that the dysregulation of HNF4α, a master hepatocyte transcription factor, is responsible for impaired cholesterol homeostasis. Upregulation of HNF4α using small activating RNA (saRNA) could mitigate cholesterol toxicity in vitro and can be developed as a treatment for AH. Our long-term objective is to define the disease-centric pathways of AH and develop much-needed therapeutics for AH patients. We hypothesize that the HNF4a-dependent impairment of cholesterol homeostasis in hepatocytes directly contributes to hepatocellular dysfunction in AH and can be targeted therapeutically. Our approach is to map regulatory pathways of cholesterol metabolism in human AH at a single cell resolution, functionally interrogate cholesterol metabolic pathways in murine alcohol models, and test a novel therapeutic strategy by targeting HNF4a-dependent cholesterol toxicity. Aim 1 will map cholesterol metabolism in AH at single cell resolution using a multimodal approach. A three- pronged strategy will map AH-associated changes in cholesterol homeostasis using single nuclear RNA sequencing, ultra-high multiplexed imaging, and targeted spatial transcriptomics. The unbiased reference maps will guide the interrogation of cholesterol metabolic pathways in cell and murine AH models. Aim 2 will define the impact of cholesterol dysregulation on alcohol-induced liver injury in murine models. We will model cholesterol toxicity in 3 conditions relevant to AH using the chronic-plus-binge alcohol feeding: (1) acute cholesterol overload, (2) impaired bile acid synthesis, and (3) impaired cholesterol secretion in cholestasis. This effort will generate optimized murine models that recapitulate cholesterol toxicity in AH. Aim 3 will evaluate targeted manipulation of HNF4α-dependent pathways of cholesterol toxicity as a novel therapeutic strategy in AH. We use saRNA to modulate HNF4α isoform expression and cholesterol metabolism. GalNAc-conjugated HNF4α-saRNA will be tested as a hepatocyte-targeted therapeutics to mitigate cholesterol toxicity in models of AH using differentiated human hepatocytes and murine models. This project is built upon a strong team of multidisciplinary scientists combining unique expertise in lipidology, preclinical liver models, and state-of-the-art single cell and spatial technologies. The success of this project will further our understanding of AH pathogenesis and could lead to the development of a novel AH treatment ready for preclinical and first-in-human trials.