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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 investigate the effect and mechanism of Cyperus volosure total saponin (CPS) on fat metabolism of H4-ⅡE hepatocytes induced by free fatty acid (FFA). Methods: The H4-ⅡE rat hepatocytes induced by FFA were used as the model. After cultured for 72 h, Neutral red method was used to detect the effect of CPS (0,0.05,0.10,0.20,0.40 g · L -1) on cell viability (CPS-L, 0.10 g · L -1) and high dose group (CPS-H, 0.20 g · L -1) according to the results of cell activity test. O (Oile red) staining and determination of intracellular lipid content; 2 h after drug intervention real-time PCR detection of liver cell gene expression, drug intervention 30 min, Western blotting ) To detect the corresponding cell phosphorylation level. Results: Compared with the normal group, the activity of hepatocytes was significantly increased after treated with 0.10,0.20 g · L -1 CPS (P <0.05). After 72 h intervention, Oile red staining showed that FFA (20μmol·L ~ (-1) (P <0.01). Lipid droplets stained with red dye in the cells decreased after CPS intervention. Compared with FFA (20μmol·L ~ (-1)) lipid droplets, (P <0.05, P <0.01). Compared with the model group, the expressions of AMPK, ACC and CPS-1 in CPS-L and CPS- (P <0.05, P <0.01). The mRNA expressions of SREBP1c, FAS mRNA were significantly decreased (P <0.05, P <0.01). CONCLUSION: CPS can significantly reduce FFA-induced lipid deposition in hepatocytes in vitro, which may be related to up-regulation of AMPK and ACC protein phosphorylation and down-regulation of SREBP1c and FAS mRNA expression.