黄酮类化合物F01WB1695B对肝细胞脂肪变性的抑制作用及分子机制研究

Inhibitory effects of flavonoid compound F01WB1695B on steatosis in L-02 hepatocytes

目的：研究黄酮类化合物F01WB1695B对脂肪变性人胚肝细胞L-02中脂质合成的抑制作用及其分子作用机制。方法：通过体外油酸诱导L-02细胞发生脂肪化,建立肝细胞脂肪变性细胞模型,再利用不同剂量的F01WB1695B干预脂肪化L-02细胞24 h后,通过油红O染色观察细胞内脂滴量变化,并通过定量检测细胞内甘油三酯（triglyceride,TG）和总胆固醇（total cholesterol,TC）的含量,确定F01WB1695B干预肝细胞脂肪变性的效果。利用报告基因细胞检测方法评价F01WB1695B对代谢性核受体过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptors,PPARs）的转录激动作用。利用实时荧光定量PCR方法检测F01WB1695B对脂代谢相关基因硬脂酰辅酶A去饱和酶1（stearoyl-Co A desaturase 1,SCD1）、酰基辅酶A氧化酶1（acyl-Co A oxidase 1,ACOX1）和脂肪酸合成酶（fatty acid synthase,FASN）mRNA水平的影响。结果：与正常组相比,20 mg/L油酸诱导L-02细胞48h后,油红O染色观察细胞内脂滴量明显增加,模型组TG和TC的含量显著升高,差异有统计学意义（P〈0.01）;F01WB1695B在20 mg/L浓度及以下时对脂肪化肝细胞无细胞毒作用,F01WB1695B干预脂肪化L-02细胞24 h后,与模型组相比,在0.2-2 mg/L浓度下细胞内脂滴量明显减少,胞内TG和TC值显著降低（P〈0.05）。瞬时转染的细胞报告基因的转录激活效应研究结果显示,F01WB1695B对核受体PPAR有较高的转录激活效应,其EC50值为0.5-2.1 mg/L。实时荧光定量PCR结果显示,F01WB1695B可下调脂肪酸合成相关基因SCD1和FASN mRNA的表达,上调脂肪分解代谢相关基因ACOX1 mRNA的表达。结论：黄酮类化合物F01WB1695B可明显降低脂肪变性肝细胞中TG及TC含量,抑制肝细胞脂肪变性,而该作用机制可能是通过激活PPAR通路,下调SCD1和FASN mRNA的表达,上调ACOX1mRNA的表达,进而降低细胞内甘油三脂水平。

AIM： To investigate the effects of flavonoid compound F01WB1695 B on the content of triglyceride（ TG） in steatotic L-02 hepatocytes and its molecular mechanisms. METHODS： An in vitro liver cell steatosis model induced by oleic acid was established. The inhibitory effects of F01WB1695 B were measured by oil red O staining,and quantitation of the intracellular tryglyceride（ TG） and total cholesterol（ TC） in the cells was conducted. The transactivity of F01WB1695 B on peroxisome proliferator- activated receptors（ PPARs） was measured by a reporter gene assay. The mRNA expression levels of lipid-related genes,stearoyl-Co A desaturase 1（ SCD1）,acyl-Co A oxidase 1（ ACOX1） and fatty acid synthase（ FASN）,in the cells were analyzed by real-time PCR. RESULTS： After the cells were treated with oleic acid for 48 h,the oil red O staining showed that the L02 cells displayed massive lipid droplet accumulation. The TG and TC levels in the oleic acid-treated cells were significantly increased compared with the control cells（ P〈0. 01）.F01WB1695 B treatment remarkably inhibited the lipid accumulation in the cells,and the TG and TC levels were significantly reduced. F01WB1695 B effectively activated the PPAR transcriptional function by inducing the expression of luciferase with the EC50 values of 0. 5 - 2. 1 mg / L. F01WB1695 B treatment remarkably inhibited the expression of lipogenesis-related genes SCD1 and FASN,and induced the expression of lipid metabolism-related gene ACOX1. CONCLUSION：F01WB1695B significantly inhibits the development of steatosis in hepatocytes in vitro by activating the PPAR pathway.