苦丁冬青苷D抑制肝细胞脂质沉积作用及机制

Inhibition effect of kudinoside D on lipid deposition in hepatocytes and its mechanism

目的探讨苦丁冬青苷D(kudinoside D,KD-D)对棕榈酸(palmitic acid,PA)诱导肝细胞脂质沉积的影响。方法体外培养AML-12细胞,随机分为Control组、PA组、PA+KD-D 20μmol·L^(-1)组、PA+KD-D 40μmol·L^(-1)组和PA+KD-D 80μmol·L^(-1)组。除Control组外,其他各组细胞加入PA(0.4 mmol·L^(-1))孵育24 h,KD-D在PA加入前1 h给予。MTT法检测细胞存活率;油红O染色和透射电子显微镜检测肝细胞脂质沉积;DCFH-DA荧光探针检测细胞内活性氧族;MitoSOX线粒体超氧化物红色荧光探针检测细胞线粒体超氧化物。结果不同浓度KD-D明显改善PA诱导的肝细胞形态学改变。与Control组比较,PA组细胞内红色脂滴明显增加;与PA组比较,KD-D不同浓度干预的细胞内红色脂滴减少。透射电子显微镜结果提示,KD-D减少PA诱导的肝细胞脂肪变性并改善超微结构。此外,KD-D明显降低PA诱导的细胞活性氧水平(P<0.01),降低细胞线粒体超氧化物含量(P<0.01)。结论KD-D可抑制PA诱导的肝细胞脂质沉积,其机制可能与调控氧化应激反应有关。

Aim To investigate the effect of kudinoside D(KD-D)on palmitic acid(PA)-induced lipid deposition in hepatocytes.Methods Mouse hepatocytes AML-12 were cultured and randomly divided into the Control group,PA group,PA+KD-D 20μmol·L^(-1) group,PA+KD-D 40μmol·L^(-1) group and PA+KD-D 80μmol·L^(-1) group.AML-12 cells in PA and KD-D groups were treated with PA(0.4 mmol·L^(-1))for 24 h.AML-12 cells in KD-D groups were incubated with KD-D for 1 h before stimulation with PA.MTT assay was used to detect cell survival rate,oil red O staining and transmission electron microscopy were used to detect lipid deposition in cells,DCFH-DA fluorescence probe was used to detect intracellular reactive oxygen species(ROS)and MitoSOX mitochondrial superoxide red fluorescence probe was used to detect mitochondrial superoxide content in cells.Results KD-D at different concentrations improved PA-induced changes in cell morphology significantly.Compared with the Control group,cells in PA group showed a significant increase in intracellular lipid droplets.Compared with PA group,the red lipid droplets in KD-D groups decreased.The results of transmission electron microscopy demonstrated that KD-D reduced PA-induced hepatic steatosis and improved ultrastructure.In addition,KD-D significantly decreased PA-induced cellular ROS level(P<0.01)and reduced mitochondrial superoxide content(P<0.01).Conclusion KD-D inhibits PA-induced lipid deposition by regulating the cellular oxidative stress levels in AML-12 cells.