目的 研究泽泻提取物（Alisma orientale extract，AE）及其3种主要单体成分对游离脂肪酸（free fatty acid，FFA）诱导的HepG2脂肪变性细胞模型的脂质代谢及氧化应激异常的改善作用及机制。方法 利用FFA（油酸-棕榈酸2∶1）建立稳定的HepG2脂肪变性细胞模型，CCK-8法测定AE、泽泻醇A、泽泻醇A-23-乙酸酯及泽泻醇A-24-乙酸酯对HepG2细胞的安全剂量；在FFA诱导同时给予AE（25.00、12.50、6.25 mg/L）、泽泻醇A（50.0、25.0、12.5 μmol/L）、泽泻醇A-23-乙酸酯（25.00、12.50、6.25 μmol/L）、泽泻醇A-24-乙酸酯（50.0、25.0、12.5 μmol/L）处理24 h，检测细胞内脂滴的形成情况，利用生化试剂盒测定各组细胞内三酰甘油（triglyceride，TG）、总胆固醇（total cholesterol，TC）水平，DCFH-DA检测药物对细胞内活性氧（reactive oxygen species，ROS）生成的影响，采用试剂盒检测细胞内氧化应激关键指标丙二醛（malondialdehyde，MDA）、总谷胱甘肽（glutathione，GSH）水平以及超氧化物歧化酶（superoxide dismutase，SOD）活性；采用Western blotting检测过氧化物酶体增殖物激活受体α（peroxisome proliferator-activated receptor α，PPARα）、PPAR共激活因子-1α（peroxisome proliferator-activated receptor gamma coactivator-1α，PGC-1α）、下游脂肪酸氧化和胆固醇代谢相关蛋白及核因子红细胞2相关因子2/血红素加氧酶-1（nuclear factor red blood cell 2 associated factor 2/hemeoxygenase-1，Nrf2/HO-1）通路蛋白表达。结果 与对照组比较，HepG2脂肪变性细胞模型细胞内TC、TG、ROS及MDA水平显著升高（P＜0.001），SOD活性及GSH水平降低（P＜0.01、0.001）；同时，细胞中PPARα、PGC-1α、肉碱棕榈酰转移酶1A（carnitine palmitoyl transferase 1A，CPT1A）、酰基辅酶A氧化酶1（acyl coenzyme A oxidase 1，ACOX1）、Nrf2及HO-1蛋白表达水平显著降低（P＜0.05、0.01、0.001）。与模型组比较，AE、泽泻醇A及泽泻醇A-23-乙酸酯给药组细胞内脂滴、TG和TC水平显著下降（P＜0.05、0.01、0.001），AE、泽泻醇A及泽泻醇A-24-乙酸酯给药组细胞内ROS及MDA水平均显著降低（P＜0.05、0.001），SOD活性和GSH水平显著升高（P＜0.05、0.01）；各给药组细胞中PPARα、PGC-1α、CPT1A、ACOX1、细胞色素P450酶7A1（cytochrome P450 enzyme 7A1，CYP7A1）、Nrf2及HO-1蛋白表达水平显著上调（P＜0.05、0.01）。结论 AE、泽泻醇A、泽泻醇A-23-乙酸酯及泽泻醇A-24-乙酸酯可以改善FFA诱导的HepG2脂肪变性细胞模型的脂质代谢及氧化应激异常，其机制可能是通过调控PPARα和Nrf2/HO-1通路，进而促进脂肪酸氧化及胆固醇代谢，抑制氧化应激。

Objective To explore the ameliorative effect and mechanisms of Alisma orientale extract (AE) and its three primary components on lipid metabolism abnormalities and oxidative stress in free fatty acids (FFA)-induced HepG2 steatosis cell model. Methods A HepG2 steatosis cell model was established using FFA [oleic acid-palmitic acid (2∶1)]. Safe dosages of AE, alisol A, alisol A-23-acetate, and alisol A-24-acetate for HepG2 cells were determined using CCK-8 kit. Simultaneously administering AE (25.00, 12.50, 6.25 mg/L), alisol A (50.0, 25.0, 12.5 μmol/L), alisol A-23-acetate (25.00, 12.50, 6.25 μmol/L), and alisol A-24-acetate (50.0, 25.0, 12.5 μmol/L) treatment for 24 h under FFA induction, the formation of intracellular lipid droplets was detected; Levels of triglyceride (TG) and total cholesterol (TC) were assessed using biochemical kits. DCFH-DA assay was utilized to measure intracellular reactive oxygen species (ROS) generation. Malondialdehyde (MDA), glutathione (GSH) levels and superoxide dismutase (SOD) activity as indicators of oxidative stress, were also determined. Western blotting was employed to detect the expressions of peroxisome proliferator-activated receptor α (PPARα), PPAR coactivator-1α (PGC-1α), and proteins involved in fatty acid oxidation, cholesterol metabolism, and nuclear factor erythrocyte 2-associated factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway. Results Compared with control group, HepG2 steatosis cell model exhibited significantly increased intracellular TC, TG, ROS production and MDA levels (P < 0.001), alongside reduced SOD activity and GSH level (P < 0.01, 0.001). There was also a decrease in PPARα, PGC-1α, carnitine palmitoyl transferase 1A (CPT1A), acyl coenzyme A oxidase 1 (ACOX1), Nrf2 and HO-1 protein expressions (P < 0.05, 0.01, 0.001). Compared with model group, AE, alisol A and alisol A-23-acetate led to a significant reduction in intracellular lipid droplets, TG, and TC levels (P < 0.05, 0.01, 0.001); AE, alisol A and alisol A-24-acetate decreased ROS and MDA levels (P < 0.05, 0.001), increased SOD activity and GSH level (P < 0.05, 0.01). Furthermore, the expression levels of PPARα, PGC-1α, CPT1A, ACOX1, cytochrome P450 enzyme 7A1 (CYP7A1), Nrf2, and HO-1 proteins were significantly upregulated in each administration group (P < 0.05, 0.01). Conclusion AE, alisol A, alisol A-23-acetate and alisol A-24-acetate effectively mitigate lipid metabolism abnormalities and oxidative stress in FFA-induced HepG2 steatosis cell model. The mechanism maybe involve modulation of PPARα and Nrf2/HO-1 pathways, thus enhancing the fatty acid oxidation and cholesterol metabolism, and reducing oxidative stress.

R285.5

江苏省工信厅工业和信息产业转型升级专项-多组分中药研究关键技术