RAGE is a key regulator of ductular reaction-mediated fibrosis during cholestasis

Ductular reaction (DR) is the hallmark of cholestatic diseases manifested in the proliferation of bile ductules lined by biliary epithelial cells (BECs). It is commonly associated with increased risk of fibrosis and liver failure. The Receptor for Advanced Glycation End Products (RAGE) was identified as a critical mediator of DR during chronic injury. Yet, the direct link between RAGE-mediated DR and fibrosis as well as the mode of interaction between BECs and hepatic stellate cells (HSCs) to drive fibrosis remains elusive. In this study, we aimed to delineate the specific function of RAGE on BECs during DR and its potential association with fibrosis in the context of cholestasis. Employing a biliary lineage tracing cholestatic liver injury mouse model, combined with whole transcriptome sequencing and in vitro analyses, we revealed the central role of BEC-specific Rage activity in fostering a pro-fibrotic milieu. RAGE is predominantly expressed in BECs and contributes to DR. Notch ligand Jagged1 is secreted from activated BECs in a Rage-dependent manner and signals HSCs in trans, eventually enhancing fibrosis during cholestasis. Overall design: A mouse line harboring a biliary lineage tracing reporter gene and generated R26TomHnf1b-CreRagefl/fl mice with BEC-specific deletion of Rage (Rage?BEC) was employed. Cholestasis was induced in vivo by the ad libitum Choline-deficient Ethionine-supplemented (CDE) diet. Whole transcriptome sequencing was performed on FACS-sorted primary BECs from CDE-challenged mice to identify Rage-dependent signaling pathways during cholestasis. Using this genetic mouse model, combined with immunohistological analyses, co-culture assays and mass spectrometry analysis, the interplay between BECs and hepatic stellate cells (HSCs) during chronic liver damage was evaluated.

胆管反应（Ductular reaction, DR）是胆汁淤积性疾病的标志性病理特征，表现为胆管上皮细胞（biliary epithelial cells, BECs）所衬覆的胆管小叶增殖。该反应通常与纤维化及肝衰竭风险升高密切相关。晚期糖基化终末产物受体（Receptor for Advanced Glycation End Products, RAGE）已被证实为慢性损伤过程中DR发生的关键介导因子。然而，RAGE介导的DR与纤维化之间的直接关联，以及BECs与肝星状细胞（hepatic stellate cells, HSCs）相互作用驱动纤维化的具体模式，目前仍不明确。本研究旨在明确DR过程中RAGE在BECs中的特异性功能，及其在胆汁淤积背景下与纤维化发生的潜在关联。本研究采用胆管谱系示踪的胆汁淤积性肝损伤小鼠模型，结合全转录组测序与体外分析实验，揭示了BEC特异性Rage活性在促纤维化微环境形成中的核心作用。RAGE主要在BECs中表达并参与DR过程；活化的BECs以Rage依赖的方式分泌Notch配体Jagged1，并通过跨细胞信号传导激活HSCs，最终在胆汁淤积过程中加剧纤维化。整体实验设计：本研究使用携带胆管谱系示踪报告基因的小鼠品系，构建了BEC特异性敲除Rage的R26TomHnf1b-CreRagefl/fl（RageΔBEC）小鼠。通过自由进食胆碱缺乏、乙硫氨酸补充（Choline-deficient Ethionine-supplemented, CDE）饮食的方式在体内诱导胆汁淤积模型。对经FACS（荧光激活细胞分选，Fluorescence Activated Cell Sorting）分选的原代BECs进行全转录组测序，以鉴定胆汁淤积过程中Rage依赖的信号通路。本研究结合该遗传小鼠模型、免疫组织化学分析、共培养实验与质谱分析，对慢性肝损伤过程中BECs与HSCs的相互作用进行了系统评估。