Background Non-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. However, therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies. Results We describe here a novel method for generating 3D hepatic spheroids using HepaRG cells, vascular endothelial cells, and mesenchymal stem cells cultured on a thick layer of soft matrix in a narrow conical tube; this method improved self-organization efficiency and functional competence. We further developed a 3D hepatic steatosis model with excess glucose and palmitate, accurately recapitulating steatosis phenotypes such as neutral lipid accumulation, enhanced expression of lipogenesis and gluconeogenesis markers, increased intracellular triglyceride content, and reduced glucose uptake. The expression and activity of cytochrome P450 4A (CYP4A), a hepatic glucose and lipid homeostasis enzyme, that is highly expressed in liver tissues from NAFLD patients, was induced in our in vitro steatosis model, and inhibiting CYP4A with the selective inhibitor HET0016 or a specific siRNA ameliorated steatosis-related pathology through reduced ER stress and improved insulin signaling. Conclusions We provide here a novel 3D human cell-based hepatic model that can be easily generated and reliably simulate hepatic steatosis pathology. We have experimentally validated its potential for target validation and drug evaluation by focusing on CYP4A, which may serve as a translational platform for drug development.

中文翻译：

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靶向 CYP4A 可减轻新型多细胞器官型肝脏模型中的肝脂肪变性。

背景 非酒精性脂肪性肝病 (NAFLD) 始于单纯的肝脂肪变性，但进一步发展为慢性肝病会导致严重的肝损伤和肝功能衰竭。然而，由于缺乏可靠的人类体外肝脏模型来了解疾病进展机制和开发治疗方法，治疗选择很少。结果 我们在此描述了一种使用 HepaRG 细胞、血管内皮细胞和间充质干细胞生成 3D 肝球体的新方法，这些细胞在狭窄的锥形管中的厚层软基质上培养；这种方法提高了自组织效率和功能能力。我们进一步开发了具有过量葡萄糖和棕榈酸酯的 3D 肝脂肪变性模型，准确地概括了脂肪变性表型，例如中性脂质积累，脂肪生成和糖异生标志物的表达增强，细胞内甘油三酯含量增加，葡萄糖摄取减少。在我们的体外脂肪变性模型中诱导细胞色素 P450 4A (CYP4A) 的表达和活性，这是一种肝脏葡萄糖和脂质稳态酶，在 NAFLD 患者的肝组织中高度表达，并用选择性抑制剂 HET0016 或 a特异性 siRNA 通过减少 ER 应激和改善胰岛素信号传导来改善与脂肪变性相关的病理学。结论 我们在此提供了一种新颖的基于人类细胞的 3D 肝脏模型，该模型可以轻松生成并可靠地模拟肝脏脂肪变性病理学。我们通过专注于 CYP4A 实验验证了其在靶点验证和药物评估方面的潜力，CYP4A 可作为药物开发的转化平台。