竹节参皂苷Ⅳa对FFA诱导肝细胞脂肪沉积保护作用及机制

Protective effect and mechanism of Chikusetsusponin Ⅳa on FFA-induced fatty deposition in hepatocytes

目的研究竹节参皂苷Ⅳa(CHS)抗混合游离脂肪酸(FFA)诱导的L-02肝细胞脂肪沉积的作用及机制。方法取人L-02肝细胞,分为对照组、FFA处理组和3个CHS药物干预组,采用FFA和CHS处理分别处理FFA处理组和3个CHS药物干预组2 w。2 w后,流式细胞仪荧光法分别检测细胞总活性氧(T-ROS)和线粒体活性氧(M-ROS)水平;采用相应的试剂盒检测细胞和线粒体丙二醛(MDA)水平及甘油三酯(TG)水平;Western blot法检测细胞中LC3、AMPK、p-AMPK、ULK1和p-ULK1水平。结果 FFA处理可以明显提高L-02细胞T-ROS和M-ROS水平,以及MDA和TG水平。不同剂量CHS干预可以剂量依赖地降低FFA处理引起的上述变化,同时FFA处理可以显著降低LC3II蛋白水平、AMPK及ULK1磷酸化水平,而不同剂量CHS可以拮抗FFA对这些蛋白分子的抑制作用。结论 CHS可以通过降低细胞线粒体ROS水平,提高AMPK/ULK1途径的自噬水平,从而发挥抗FFA诱导的肝细胞脂肪沉积作用。

Objective To study the anti-lipid deposition effect and mechanism of Chikusetsusponin Ⅳa(CHS) on fatty deposition induced by mixed free fatty acids(FFA) in human liver L-02 cells. Methods Human liver L-02 were divided into a control group, a FFA treatment group and three CHS drug intervention groups. The FFA treatment group and the three CHS drug intervention group were treated with FFA and CHS respectively for two weeks. After two weeks, the total reactive oxygen species(T-ROS) and mitochondrial reactive oxygen species(M-ROS) levels were detected by flow cytometry fluorescence method;the levels of MDA and TG in cells and mitochondria were detected by corresponding kits;the levels of LC3, AMPK, p-AMPK, ULK1 and p-ULK1 in cells were detected by Western blot method. Results FFA treatment significantly increased T-ROS, M-ROS, MDA and TG levels in L-02 cells. Different doses of CHS intervention could reduce the above changes caused by FFA treatment in a dose-dependent manner, and FFA treatment could significantly reduce the levels of LC3 II protein, AMPK and ULK1 phosphorylation, while different doses of CHS could antagonize the inhibition of FFA on these protein molecules. Conclusion CHS can inhibit FFA-induced fatty deposition in hepatocytes by decreasing the level of mitochondrial ROS and increasing the level of autophagy in AMPK/ULK1 pathway.