靶向沉默CaMKK-AMPK通路对NEFAs介导肝脂损伤的调控机制

为探究Ca²⁺/钙调素依赖性蛋白激酶(CaMKK)-AMP依赖蛋白激酶(AMPK)对非酯化脂肪酸(NEFAs)介导的AML12细胞肝脂损伤的调控机制,本试验通过添加高浓度的NEFAs刺激AML12细胞,RNA靶向沉默CaMKK,检测不同分组细胞内AMPK及固醇调节原件结合蛋白(SREBP1)的蛋白表达水平以及利用流式细胞术检测活性氧(ROS)的产生水平,同时利用激光共聚焦检测细胞内脂滴变化情况。结果显示,siCaMKK+NEFAs组AMPK蛋白表达水平较空白对照组及NEFAs组均显著降低,而SREBP1蛋白表达水平、ROS水平及脂滴数量均显著高于其他各组。结果表明,CaMKK通过AMPK信号通路调控SREBP1及线粒体脂质氧化可调节NEFAs诱导的肝脂损伤过程。

Regulatory mechanism of targeted silencing of CaMKK-AMPK pathway on NEFAs mediated liver lipid injury

To explore the regulatory mechanism of Ca²⁺/calmodulin-dependent protein kinase(CaMKK)and AMP-dependent protein kinase(AMPK)on non-esterified fatty acids(NEFAs)mediated liver lipid injury in AML12 cells,AML12 cells were firstly treated with high concentrations of NEFAs,and then the expression of CaMKK was silenced by RNAi,followed by detection of protein expression level of AMPK and sterol regulatory element binding protein 1(SREBP1)in different groups of cells.The expression level of ROS was measured by flow cytometry assay,and the change of lipid droplets in cells were detected by laser confocal microscopy.The results showed that the expression level of AMPK protein in siCaMKK+NEFAs group was significantly lower than that in control group and NEFAs group,while the expression level of SREBP1 protein,ROS level and the number of lipid droplets were significantly higher than those in other groups.The results showed that CaMKK regulates SREBP1 and mitochondrial lipid oxidation via AMPK signaling pathway,which can regulate NEFAs induced liver lipid injury.