油酸钠、棕榈酸钠添加剂诱导L-02脂肪肝细胞模型

油酸钠、棕榈酸钠添加剂诱导L-02脂肪肝细胞模型

                                            鲲创生物/鲲创科技（kunchuang biotechnology）整理发布

非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）是西方国家最常见的慢性肝脏疾病之一。NAFLD通过引起氧化应激和脂质过氧化，逐步进展为非酒精性脂肪性肝炎、肝纤维化甚至肝硬化，最终可能导致肝细胞癌、门脉高压和肝衰竭。此外，NAFLD患者同时也是多器官胰岛素抵抗、2型糖尿病、血脂紊乱和冠心病的高风险人群。

NAFLD患者的特征是肝脏脂肪变性和肝内甘油三酯（TG）过度积聚。肝内甘油三酯（TG）过度积聚的机制包括：（1）因脂肪分解增加和饮食中摄入过多引起的自由脂肪酸（FFAs）供应过多；（2）肝脏脂肪生成增加；（3）FFAs氧化利用减少；（4）由于极低密度脂蛋白分泌下降导致的TG清除减少。肝脏脂肪中的三分之二来自于FFAs向肝脏运输，因此，外周血脂肪酸水平和肝脏脂肪生成是NAFLD患者肝脏脂肪积聚的主要原因。

2022年2月，一篇题为Farnesoid X Receptor Deficiency Induces Hepatic Lipid and Glucose Metabolism Disorder via Regulation of Pyruvate Dehydrogenase Kinase 4发表于Oxidative Medicine and Cellular Longevity杂志（影响因子7.310），报道了胆汁酸受体FXR的缺失引起肝脏脂质沉积和葡萄糖代谢紊乱的现象，并阐明了其分子机制。

在这篇文章中，作者首先采用FXR基因敲除（FXR^-/-）小鼠和野生（WT）小鼠，分别饲喂普通饲料（SD）和高脂饲料（HFD）。发现3月后，HFD-FXR^-/-组小鼠发生肝肿大（图1a和b）和脂肪肝（图2a-c），表现为肝脏脂滴增多、TG增加、FFAs升高。

图1a.四组小鼠肝脏照片，b.小鼠肝脏/体重量比值

图2a.四组小鼠肝脏HE染色、油红O染色照片，b.肝脏甘油三酯（TG）含量，c.肝脏自由脂肪酸（FFA）含量。

接着，作者在细胞水平，利用CRISPR/Cas9基因编辑技术构建了FXR敲除（KO）L-02肝脏细胞系，联合应用油酸钠和棕榈酸钠（500μM/250μM）作为FFAs诱导L-02细胞脂滴形成，通过BODIPY染色和油红O染色检测细胞中脂滴的积聚。作者发现，与对照L-02细胞相比，FXR KO L-02细胞中的脂滴形成明显增加、细胞内TG和FFAs显著增高（图3a-d）。

图3a. L-02细胞BODIPY染色照片，其中绿色指示脂滴，蓝色指示细胞核。

            图3b. L-02细胞油红O染色照片，其中红色指示脂滴。

         图3c. L-02细胞甘油三酯（TG）含量；d. L-02细胞自由脂肪酸（FFA）含量。

小编总结：油酸高脂试剂盒（油酸钠+棕榈酸钠）主要用于诱导脂肪肝细胞模型，包括HepG2、L-02和原代肝细胞，一般采用的浓度比为油酸钠:棕榈酸钠=2:1，处理时间2-3天。本研究采用500μM油酸钠和250μM棕榈酸钠联合处理，成功诱导了L-02脂肪肝细胞模型，具有较好的参考价值。

References

Wenyi Deng, Wenjing Fan, Tingting Tang. Farnesoid X Receptor Deficiency Induces Hepatic Lipid and Glucose Metabolism Disorder via Regulation of Pyruvate Dehydrogenase Kinase 4 [J] . Oxidative Medicine and Cellular Longevity. Volume 2022, Article ID 3589525, 19 pages. https://doi.org/10.1155/2022/3589525

Reagents and Antibodies

Sodium oleate/sodium palmitate (#SYSJKJ006) was purchased from Xi’an Kun Chuang Technology Company. 

Abstract

Farnesoid X receptors (FXR) are bile acid receptors that play roles in lipid, glucose, and energy homeostasis. Synthetic FXR-specific agonists have been developed for treating nonalcoholic fatty liver disease (NAFLD) patients. However, the detailed mechanism remains unclear. To investigate the effects of FXR on NAFLD and the possible mechanism, FXR-null mice were fed either a normal or a high-fat diet. The FXR-null mice developed hepatomegaly, steatosis, accumulation of lipid droplets in liver cells, glucose metabolism disorder, and elevated serum lipid levels. Transcriptomic results showed increased expression of key lipid synthesis and glucose metabolism-related proteins. We focused on pyruvate dehydrogenase kinase 4 (PDK4), a key enzyme involved in the regulation of glucose and fatty acid (FA) metabolism and homeostasis. Subsequently, we confirmed an increase in PDK4 expression in FXR knockout cells. Moreover, inhibition of PDK4 expression alleviated lipid accumulation in hepatocytes caused by FXR deficiency in vivo and in vitro. Our results identify FXR as a nuclear transcription factor that regulates glucose and lipid metabolism balance through PDK4, providing further insights into the mechanism of FXR agonists in the treatment of metabolic diseases.