FISH probe sequences in this study.

Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized primarily by hepatocellular steatosis and lipid accumulation, which leads to hepatocyte apoptosis, autophagy, inflammation, and intracellular oxidative stress. NAFLD is recognized as one of the most prevalent and complex chronic liver diseases globally, with its occurrence and associated mortality rates rising swiftly each year. Due to the high similarity between chicken fatty liver syndrome (FLS) and NAFLD, as well as the easy availability of diseased chickens, the chicken is considered an ideal model for studying the pathogenesis of NAFLD. Previous studies have pinpointed several circular RNAs (circRNAs) implicated in the pathogenesis of NAFLD, yet the underlying functions and mechanisms of numerous circRNAs continue to remain elusive. In this experiment, we utilized circRNA sequencing of chicken livers to identify a novel circRNA, named circACACA, and discovered that it disrupts the metabolic homeostasis of lipids within hepatocytes. Consequently, this disruption leads to oxidative stress and the induction of autophagy, ultimately exerting an adverse effect on chicken liver health. Mechanistically, circACACA functions as a molecular sponge for miR-132b-5p and miR-101-2-5p to modulate the expression of the downstream CBFB/PIM1 complex. Consequently, it influenced the activity of the AKT/mTOR and PPAR-γ signaling pathways to perform its physiological functions. Crucially, we noticed substantial sequence similarity of circACACA across diverse species by comprehensively searching databases. Further, our research with a mouse model confirmed that the functional conservation of circACACA across livers of different species. Overall, this study built a mechanistic network for circACACA and confirmed its sequence conservation and functional relevance across various species. Our results not only provide new targets for the prevention and treatment of NAFLD but also present fresh perspectives for progress in healthy production of laying hens.非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）是一类以肝细胞脂肪变性与脂质蓄积为主要特征的临床综合征，可引发肝细胞凋亡、自噬、炎症及细胞内氧化应激。NAFLD被认为是全球范围内最流行且最复杂的慢性肝脏疾病之一，其发病率与相关死亡率逐年快速攀升。由于鸡脂肪肝病综合征（FLS）与NAFLD具有高度相似性，且病鸡易于获取，鸡被视为研究NAFLD发病机制的理想动物模型。既往研究已明确多种环状RNA（circRNAs）参与NAFLD的发病过程，但多数环状RNA的具体功能与作用机制仍有待阐明。本实验通过对鸡肝脏组织进行环状RNA测序，鉴定得到一种新型环状RNA circACACA，并发现该环状RNA可破坏肝细胞内的脂质代谢稳态，由此引发氧化应激并诱导细胞自噬，最终对鸡肝脏健康造成不良影响。机制层面上，circACACA可作为miR-132b-5p与miR-101-2-5p的分子海绵，调控下游CBFB/PIM1复合物的表达，进而影响AKT/mTOR与PPAR-γ信号通路的活性，发挥其生理功能。值得注意的是，通过全面检索各类数据库，我们发现circACACA在多个物种间存在显著的序列同源性。进一步通过小鼠模型开展的研究证实，circACACA在不同物种的肝脏组织中具有功能保守性。综上，本研究构建了circACACA的作用机制网络，验证了其在多个物种间的序列保守性与功能相关性。本研究结果不仅为NAFLD的防治提供了新的靶点，也为蛋鸡健康养殖的发展提供了全新视角。