青钱柳总皂苷对游离脂肪酸诱导的H4-ⅡE细胞脂肪代谢的影响及作用机制

Effect and Mechanism of CPS on Fat Metabolism in H4-ⅡE Cells Induced by Free Fatty Acids

目的:研究青钱柳总皂苷(CPS)对游离脂肪酸(FFA)诱导的H4-ⅡE肝细胞脂肪代谢的影响和作用机制。方法:以FFA诱导的H4-ⅡE大鼠肝细胞为模型,培养72 h后,采用Neutral red法检测CPS(0,0.05,0.10,0.20,0.40 g·L^(-1))对细胞活性的影响;根据细胞活性检测结果选择CPS低剂量组(CPS-L,0.10 g·L^(-1))和高剂量组(CPS-H,0.20 g·L^(-1))干预,油红O(Oile red)染色并测定细胞内脂质含量;药物干预2 h后实时荧光定量PCR(Real-time PCR)检测肝细胞相应基因表达,药物干预30 min后,利用蛋白免疫印迹法(Western blot)检测细胞相应蛋白磷酸化水平。结果:与正常组比较,0.10,0.20 g·L^(-1)CPS干预后肝细胞活性明显升高(P<0.05);干预72 h后,Oile red染色显示FFA(20μmol·L^(-1))诱导后呈现大片红染的脂滴,细胞脂质含量显著增加(P<0.01),CPS干预后细胞中红染的脂滴减少,细胞脂质含量较FFA(20μmol·L^(-1))诱导组明显减少(P<0.05,P<0.01);与模型组比较,CPS-L和CPS-H组腺苷酸活化蛋白激酶(AMPK),乙酰Co A羧化酶(ACC)蛋白磷酸化水平明显升高(P<0.05,P<0.01);胆固醇应答元件结合蛋白1c(SREBP1c),脂肪酸合成酶(FAS)mRNA表达水平明显降低(P<0.05,P<0.01)。结论:CPS能够显著降低体外FFA诱导肝细胞脂质沉积,可能是通过上调AMPK,ACC蛋白磷酸化水平,下调SREBP1c,FAS mRNA表达而发挥作用。

Objective：To explore the effect and mechanism of Cyclocarya paliurus saponin（CPS） on FFA-induced H4-ⅡE in hepatic cells.Method：FFA-induced H4-ⅡE liver cells of rat were cultivated for 72 h,and then exposed to various concentrations of CPS（0,0.05,0.10,0.20,0.40 g·L-1）,and their viability was measured by Neutral red.According to the results of CPS cell activity,these cells were treated in the low-dose group（CPS-L,0.10 g·L-1） and the high-dose group（CPS-H,0.20 g·L-1）,Oile red staining was performed to determine lipid content in the cells.After treatment of the cells for 2 h,mRNA levels were measured by Real-time PCR,while phosphorylation levels of target proteins were detected by Western blot after treatment for 30 min.Result：It was found that the cell viability indexes in the cells intervened by 0.10,0.20 g·L-1CPS increased significantly,compared with normal group（P〈0.05）.After treatment for 72 h,Oile red staining showed that FFA（20 μmol·L-1）-induced cells contained a large number of red lipid droplets,and lipid content increased significantly（P〈0.01）.However,after treatment CPS,red lipid droplets decreased,and lipid content significantly decreased（P〈0.05,P〈0.01）.After intervention with CPS,phosphorylation levels of mitogenactivated protein kinase（AMPK）,and acetylcoacarboxylase（ACC） in CPS-L and CPS-H groups were higher than those in control group（P〈0.05,P〈0.01）.And sterol regulatory element-binding protein 1c（SREBP1c） and fatty acid synthase（FAS） mRNA expressions in CPS-L and CPS-H groups increased,compared with control group（P〈0.05,P〈0.01）.Conclusion：CPS can play a significant role in reducing FFA-induced lipid deposition in liver cells in vitro;The mechanism may be correlated with up-regulation of AMPK,ACC protein and phosphorylation levels,and down-regulation of SREBP1 c,FAS mRNA expressions.