SIRT2抑制剂AGK2对硫代乙酰胺诱导的L02细胞线粒体保护作用

Mitochondrial protection effect of sirtuin2 inhibitor AGK2 on thioacetamide-induced L02 cells

目的探讨沉默信息调节因子2(SIRT2)的选择性抑制剂AGK2对硫代乙酰胺(TAA)诱导的L02肝细胞的线粒体保护作用及相关机制。方法体外培养人源性肝细胞系L02细胞,以不同浓度SIRT2抑制剂AGK2作为干预药物,CCK8检测不同浓度的AGK2对L02细胞活性的影响,选取适宜的浓度为AGK2干预组。正常组不予以任何药物干预;造模组给予90 mmol/L TAA进行造模;低、中、高剂量AGK2组在造模2 h前分别加入1、2、4μmol/L AGK2。CCK8检测各组细胞活性。倒置光镜下观察细胞形态变化。蛋白免疫印迹法(Western blot)检测细胞内异柠檬酸脱氢酶(IDH1)、苹果酸脱氢酶(MDH1)、SIRT2和裂变蛋白1同系物(FIS1)的蛋白相对表达量。共聚焦激光扫描显微镜下观察各组细胞SIRT2的表达情况。荧光显微镜下观察各组细胞线粒体膜电位。结果当AGK2的浓度为1、2、4μmol/L时,细胞的存活率分别为98.05%、95.76%、91.65%,与正常组相比差异均无统计学意义(均P>0.05)。当AGK2浓度为8、16、32、64、128μmol/L时,细胞存活率与正常组相比均显著下降(均P<0.05)。与模型组相比,低、中、高剂量AGK2组L02细胞活性和贴壁性较好,漂浮细胞显著减少,且AGK2浓度越高细胞活性和贴壁性越好,漂浮细胞越少。与模型组相比,AGK2组的L02细胞显示红色荧光增强,而绿色荧光减弱,且AGK2浓度越高红色荧光越强,绿色荧光越弱。与模型组相比,低、中、高剂量AGK2组L02细胞内SIRT2的荧光减弱,且AGK2浓度越高,SIRT2的荧光越弱。低、中、高剂量AGK2组L02细胞内IDH1、MDH1的蛋白表达量显著高于模型组(均P<0.05),且与AGK2的浓度呈正相关(r=0.818,P<0.05;r=0.960,P<0.05);SIRT2和FIS1的蛋白表达量显著低于模型组(P<0.05),且与AGK2的浓度呈负相关(r=-0.992,P<0.05;r=-0.998,P<0.05)。结论AGK2可以降低TAA刺激的L02细胞内线粒体膜电位、增加IDH1和MDH1蛋白表达量,减少L02细胞内SIRT2和FIS1的蛋白表达量,且作用呈剂量依赖性。

Objective To explore the protective effect of AGK2,a selective inhibitor of sirtuin 2(SIRT2),on the mitochondria of L02 hepatocytes induced by thioacetamide(TAA)and its related mechanism.Methods Human-derived hepatocyte line L02 cells were cultured in vitro.Different concentrations of SIRT2 inhibitor AGK2 were used as intervention drugs.Cell counting kit-8(CCK8)was used to detect the effects of different concentrations of AGK2 on the activity of L02 cells,and the appropriate concentration was selected as the AGK2 intervention group.The normal group was not given any drug intervention.The model group was given 90 mmol/L TAA for modeling.Low,medium and high dose AGK2 groups were added with 1,2 and 4μmol/L AGK2,respectively 2 h before modeling.CCK8 was used to detect cell activity in each group.Morphological changes of cells were observed under inverted light microscope.The relative protein expression levels of isocitrate dehydrogenase(IDH1),malate dehydrogenase(MDH1),SIRT2 and fission protein 1 homologue(FIS1)were detected by Western blot.The expression of SIRT2 in cells of each group was observed by confocal laser scanning microscope.The mitochondrial membrane potential of cells in each group was observed under a fluorescence microscope.Results When AGK2 concentration was 1,2 and 4μmol/L,the survival rate of cells were 98.05%,95.76%and 91.65%,respectively,with no statistical significance compared with normal group(all P>0.05).When AGK2 concentration was 8,16,32,64,128μmol/L,the cell survival rate was significantly decreased compared with normal group(all P<0.05).Compared with the model group,the L02 cells in low,medium and high AGK2 groups had better activity and adherence,and the floating cells were significantly reduced.The higher the concentration of AGK2,the better the cell activity and adherence,and the less floating cells.Compared with the model group,the red fluorescence of L02 cells in AGK2 group was enhanced,while the green fluorescence was weakened.The higher the AGK2 concentration was,the stronger the red fluorescence was,and the weaker the green fluorescence was.Compared with the model group,the fluorescence of SIRT2 in L02 cells of low,medium and high AGK2 groups was weakened,and the higher the concentration of AGK2,the weaker the fluorescence of SIRT2.The protein expressions of IDH1 and MDH1 in L02 cells of low,medium and high AGK2 groups were significantly higher than those of model group(all P<0.05),and were positively correlated with the concentration of AGK2(r=0.818,P<0.05;r=0.960,P<0.05);the protein expressions of SIRT2 and FIS1 were significantly lower than those of the model group(all P<0.05),and were negatively correlated with the concentration of AGK2(r=-0.992,P<0.05;r=-0.998,P<0.05).Conclusions AGK2 can reduce the mitochondrial membrane potential stimulated by TAA in L02 cells,increase the protein expression of IDH1 and MDH1,and inhibit the protein expression of SIRT2 and FIS1 in L02 cells in a dose-dependent manner.