目的：非酒精性脂肪肝病（NAFLD）及其进展为非酒精性脂肪性肝炎（NASH）和肝癌是一个严重且日益严重的问题。然而，由于缺乏高通量药物检测方法，新疗法的开发受到严重阻碍。方法：我们开发了一种简单的 Transwell 检测，由 HepG2 肝细胞、肝 LX-2 星状细胞和分化的 THP-1 细胞组成。将细胞与含有 NASH 相关危险因素、葡萄糖、胰岛素、游离脂肪酸 (FFA) 和脂多糖 (LPS) 的激活混合物一起孵育 72 小时。我们比较了不同的培养条件组合，以获得一个模型系统，该系统概括了 NAFLD/NASH 的主要特征，即脂肪变性增加、活性氧 (ROS)、促炎细胞因子/趋化因子的分泌以及纤维化的存在。为了确认优化模型系统的有用性，我们筛选了抑制肝细胞脂肪变性的化合物，并评估了三培养模型系统中最有效的化合物。结果：与单一培养物中的 HepG2 细胞相比，活化混合物刺激三培养物中的 HepG2 细胞积累更多脂肪并产生更高水平的活性氧 (ROS)。此外，与单一培养物相比，这种三培养物产生了更高水平的炎症细胞因子和趋化因子（IL-8、IL-6、MIP-1α等）。此外，在所有 LX-2 单一培养物和共培养物中，暴露于活化混合物会增加纤维化标志物。我们的三培养系统的主要优势在于，它可以同时监测 NASH 的 4 个主要特征，即脂肪变性、氧化应激、炎症和纤维化。筛选可能减少 HepG2 细胞脂肪变性的潜在调节剂揭示了异生物碱小檗碱的保护作用。使用这种新颖的三培养测定进行测试，用 5 µM 小檗碱治疗可减少 HepG2 肝细胞中的脂肪变性和 ROS，减少炎症细胞因子的产生并抑制 LX-2 细胞的胶原蛋白产生。结论：这种简单的三培养模型概括了 NAFLD/NASH 的主要特征，对于高通量临床前药物发现应该有用。在此模型中，小檗碱在减少脂肪变性和 ROS 以及防止纤维化方面显示出有希望的结果。版权所有 © 2023 Rafiei、Yeung、Kowalski、Krystal 和 Elisia。

Objectives: Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing. Methods: We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72 h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system. Results: The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1α, etc.) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5 µM berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells. Conclusion: This simple triculture model recapitulates the main features of NAFLD/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.Copyright © 2023 Rafiei, Yeung, Kowalski, Krystal and Elisia.