胡柚皮黄酮对棕榈酸诱导的脂肪变性肝细胞中线粒体膜电位以及自噬相关蛋白表达的影响

Effect of pure total flavonoids from citrus on mitochondrial membrane potential and expression of autophagy-related proteins in palmitic acid-induced hepatocytes steatosis

目的观察胡柚皮黄酮(PTEC)对棕榈酸(PA)诱导的肝细胞脂肪变性的影响。方法MTT、油红O染色联合检测,设立0.3 mmol/L PA诱导HepG2细胞建立脂肪变性模型,不同浓度(0、20、40、80、160 mg/L)PTEC干预后,采用油红O染色法观察肝细胞脂肪变性;荧光探针法观测细胞内活性氧水平及线粒体膜电位变化;免疫印迹法检测自噬效应蛋白(Beclin-1)、微管相关蛋白质1轻链3(LC3)及P62蛋白表达。结果PA(0.3 mmol/L)诱导后,HepG2细胞内脂滴[(39.13±3.15)比(1.00±0.13),P<0.01]和活性氧[(18.56±1.48)比(1.00±0.25),P<0.01]含量明显增高;与PA组比较,PA+40、80、160 mg/L PTEC组细胞内脂滴[(16.35±4.00)、(6.26±1.69)、(2.76±2.05)比(39.13±3.15),P<0.01]和活性氧[(9.13±1.12)、(7.00±0.45)、(5.03±1.00)比(18.56±1.48),P<0.01]均明显降低,并在荧光显微镜下观察到线粒体膜电位恢复;进一步通过Western blot检测发现,LC3和Beclin-1蛋白表达上调,P62蛋白表达下调。结论PTEC能改善肝细胞脂肪变性和氧化应激反应,机制可能与其调控线粒体自噬,维持线粒体功能稳定有关。

Objective To assess the effect of pure total flavonoids from citrus(PTFC)on palmitic acid(PA)-induced hepatocyte steatosis.Methods HepG2 cells were induced by PA(0.3 mmol/L)to established hepatic steatosis cell model.The protective effects of PTFC(0,20,40,80,and 160 mg/L)on hepatic steatosis were assessed using the oil red O staining.Fluorescence probe method was used to detect intracellular reactive oxygen species(ROS)levels and mitochondrial membrane potential,while Western blot was used to assay expression of Beclin-1,LC3 and P62 proteins.Results The content of lipid droplets(39.13±3.15 vs.1.00±0.13)and ROS(18.56±1.48 vs.1.00±0.25)in 0.3 mmol/L PA-treated HepG2 cells were increased(All P<0.01).However,compared to those of PA treatment,PA plus 40,80,and 160 mg/L PTFC treatments significantly decreased the lipid droplets contents(16.35±4.00,6.26±1.69,and 2.76±2.05 vs.39.13±3.15;P<0.01)and ROS level(9.13±1.12,7.00±0.45,and 5.03±1.00 vs.18.56±1.48;P<0.01),but the mitochondrial membrane potential was restored.The expression of LC3 and Beclin-1 proteins was also increased,but P62 protein was downregulated.Conclusion PTFC was able to improve hepatic steatosis and oxidative stress response by regulating mitochondrial autophagy and maintaining mitochondrial functional stability.