使用油酸钠添加剂诱导HepG2脂肪肝细胞模型经验分享与交流

使用油酸钠添加剂诱导HepG2脂肪肝细胞模型经验分享与交流

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

高脂血症是一种以外周血总胆固醇（TC）、总甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）升高和高密度脂蛋白胆固醇（HDL-C）降低为特征的脂质代谢疾病。近年来，由于体育活动减少，食用高热量、低纤维的快餐食品增多，高脂血症的发病率持续上升。目前研究表明，高脂血症是心血管疾病的主要危险因素，降脂治疗具有重要意义。临床上，高脂血症的主要治疗方法包括贝特类药物、他汀类药物和其他降脂药物。然而，上述药物可引起许多不良反应，如胃肠道症状、肌痛和呼吸道感染。因此，有必要研究和开发更多的治疗高脂血症的方法。

传统药物虎杖（Polygonum cuspidatum）具有祛湿、祛黄、清热、解毒、祛瘀、止痛、止咳、化痰的作用。它含有多种化合物，已作为中药材广泛用于治疗炎症、高脂血症等疾病。现代药理学研究表明虎杖提取物中的白藜芦醇苷能有效降低高脂高胆固醇兔模型血清TC、TG、LDL-C水平。有报道称白藜芦醇苷能显著降低高脂血症小鼠的血脂水平。此外，虎杖乙醇提取物可能通过下调脂质积累来抑制胰脂肪酶活性和脂肪形成。此外，利用代谢组学方法，研究人员报告了在虎杖长期治疗后，特定尿代谢物的变化与虎杖在大鼠中的降血脂效应之间的关联。虎杖的抗高脂血症作用已在多项研究中得到证实。然而，其作用机制和有效成分尚不完全清楚，仍需进一步阐明。

2021年，一篇发表于Oxidative Medicine and Cellular Longevity杂志的研究论著《Polygonum cuspidatum Extract Exerts Antihyperlipidemic Effects by Regulation of PI3K/AKT/FOXO3 Signaling Pathway》，利用油酸钠（Sodium Oleate，0.6 mM）诱导的HepG2细胞建立脂肪肝细胞模型，评估虎杖减少HepG2细胞脂肪堆积的作用和机制。通过Western blot和免疫荧光实验验证了虎杖对油酸钠诱导的HepG2细胞中PI3K/AKT/FOXO3信号通路具有显著的调控作用。此外，虎杖还可以在高脂血症细胞模型中激活ERa来降低血脂。

图1. 油酸钠诱导HepG2脂肪肝细胞模型。大黄素（emodin）、青兰苷（cynaroside）、虎杖苷（polydatin）和白藜芦醇（resveratrol）等不同化合物对油酸钠诱导的HepG2中脂质沉积（油红O染色）情况的影响。

图2. 虎杖提取物减少油酸钠诱导的HepG2细胞中脂质堆积。A. HepG2细胞经油酸钠（0.6 mM）处理24小时后的油红O染色照片；B. HepG2细胞经油酸钠（0.6 mM）处理24小时后的Bosipy荧光染色照片；C. HepG2细胞经油酸钠（0.6 mM）处理24小时后的尼罗红荧光染色照片。

图3. 虎杖提取物对油酸钠诱导的HepG2细胞中PI3K-AKT通路及其下游FOXO3和ERα的影响。A. HepG2细胞经油酸钠（0.6 mM）处理24小时后的FOXO3荧光图片；B. HepG2细胞经油酸钠（0.6 mM）处理24小时后细胞中PI3K-AKT-ERα通路蛋白表达情况。

图4. 虎杖提取物对油酸钠诱导的HepG2细胞中磷酸化AKT的影响。HepG2细胞经油酸钠（0.6 mM）处理24小时后细胞中AKT、p-AKT荧光表达情况。

图5. 虎杖提取物对油酸钠诱导的HepG2细胞中ERα表达的影响。HepG2细胞经油酸钠（0.6 mM）处理24小时后细胞中ERα荧光表达情况。

总结与分享：多数科研人员联合采用棕榈酸钠0.25mM+油酸钠0.5mM诱导HepG2脂肪肝细胞模型，既可诱导脂滴形成，也能诱导细胞损伤。本研究单独使用油酸钠0.6mM诱导HepG2脂肪肝细胞模型，也可以稳定地制备脂肪肝细胞模型，进一步说明油酸钠在诱导脂肪肝细胞模型中的重要作用。

References

Ting Tao , Qing Zhang , Zibo Liu. Polygonum cuspidatum Extract Exerts Antihyperlipidemic Effects by Regulation of PI3K/AKT/FOXO3 Signaling Pathway [J] . Oxidative Medicine and Cellular Longevity. Volume 2021, Article ID 3830671, 17 pages https://doi.org/10.1155/2021/3830671

Materials and Methodss

oleic acid (OA) was purchased from Xi’an Kunchuang Technology Development Co., Ltd. (Xi’an, China)

Abstract

Polygonum cuspidatum (PC) has been reported to exert a potent antihyperlipidemic effect. However, its mechanisms of action and active ingredients remain elusive and require further research. In this study, we first conducted in vivo experiments to validate that Polygonum cuspidatum extract (PCE) could ameliorate the blood lipid level in hyperlipidemia model rats. Then, ultrahigh performance liquid chromatography coupled with Q-Exactive MS/MS (UPLC-QE-MS/MS) was applied to verify its 12 main active ingredients. The pharmacophore matching model was employed to predict the target point of the active ingredient, and 27 overlapping genes were identified via database and literature mining. String online database and Cytoscape software were utilized to construct a Protein-Protein Interaction (PPI) network, followed by function annotation analysis and pathway enrichment analysis. The results showed that the PI3K/AKT signaling pathway and its downstream FOXO3/ERα factors were significantly enriched. Furthermore, in vitro experiments were performed to determine the lipid content and oxidative stress (OS) indicators in OA-induced HepG2 cells, and immunofluorescence and western blotting analysis were carried out to analyze the effects of PCE on related proteins. Our experimental results show that the mechanism of antihyperlipidemic action of PCE is related to the activation of the PI3K/AKT signaling pathway and its downstream FOXO3/ERα factors, and polydatin and resveratrol are the main active ingredients in PCE that exert antihyperlipidemic effects.