论文部分内容阅读
目的研究阿托伐他汀在炎症状态下对人肝癌细胞株Hep G2细胞氧化应激和脂质积聚的影响。方法将普通Hep G2细胞分为为3组:正常组、模型组[用100 ng·m L~(-1)肿瘤坏死因子(TNF-α)处理]及实验组(用100 ng·m L~(-1)TNF-α+10μmol·L~(-1)阿托伐他汀处理),3组同时负荷100μg·m L~(-1)低密度脂蛋白(LDL),处理时间24 h。用油红O染色测定细胞内脂质含量;以荧光定量聚合酶链式反应和免疫印迹法分别检测Hep G2细胞脂肪酸合酶(FAS)、乙酰辅酶A羧化酶(ACC)和固醇调节元件结合蛋白1(SREBP1)的基因和SREBP1蛋白表达;以二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针染色检测Hep G2细胞内活性氧化产物(ROS)的含量;以比色法检测Hep G2细胞过氧化氢(H2O2)和丙二醛(MDA)的含量。结果正常组与模型组SREBP1蛋白灰度值分别为1.01±0.001,1.61±0.34,这2组的SREBP1 mRNA水平分别为1.01±0.16,3.61±0.39,这2组的FAS的mRNA水平分别是1.03±0.32,1.99±0.36,这2组的ACC的mRNA水平分别是0.95±0.29,2.37±0.52。与正常组相比,模型组的细胞FAS、ACC、SREBP1的基因和SREBP1的蛋白表达明显增加,差异均有统计学意义(均P<0.05)。与模型组相比,实验组的Hep G2FAS、ACC、SREBP1的基因和SREBP1的蛋白灰度值分别是2.95±0.92,3.99±1.16,2.85±0.91,2.94±0.65,实验组的Hep G2 FAS、ACC、SREBP1的基因和SREBP1的蛋白表达明显增加,差异均有统计学意义(均P<0.05)。表明在炎症状态下,阿托伐他汀进一步加重了Hep G2细胞内脂质的沉积。正常组、模型组与实验组的细胞ROS含量(以荧光强度代表)分别为1.00±0.20,1.77±0.25,3.20±0.53;正常组、模型组与实验组的H2O2含量分别为(2.30±0.31),(4.32±0.77),(5.31±0.75)nmol·mg~(-1);这2组的MDA含量分别为(0.78±0.22),(1.86±0.23),(3.43±1.15)nmol·mg~(-1),与正常组比较,模型组差异均有统计学意义(均P<0.05);与模型组比较,实验组差异均有统计学意义(均P<0.05)。结论在炎症状态下,阿托伐他汀诱导Hep G2细胞氧化应激,激活SREBP1/FAS/ACC通路,加重细胞内脂质积聚。
Objective To investigate the effects of atorvastatin on oxidative stress and lipid accumulation in human hepatocellular carcinoma Hep G2 cells under inflammatory conditions. Methods Normal Hep G2 cells were divided into 3 groups: normal group, model group [treated with 100 ng · m L -1 of tumor necrosis factor (TNF-α)] and experimental group (treated with 100 ng · m L ~ (-1) TNF-α + 10μmol·L -1 atorvastatin). The three groups were treated with 100 μg · m L -1 of low density lipoprotein (LDL) for 24 hours. The intracellular lipid content was determined by Oil Red O staining. The levels of fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and sterol regulatory elements in Hep G2 cells were detected by fluorescence quantitative polymerase chain reaction and Western blot, respectively (SREBP1) gene and SREBP1 protein expression in Hep G2 cells were detected by fluorescent probe dihydrofluorescein-acetoacetate (DCFH-DA) staining to detect the content of reactive oxygen species (ROS) The colorimetric method was used to detect the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in Hep G2 cells. Results The gray value of SREBP1 protein in normal and model groups was 1.01 ± 0.001 and 1.61 ± 0.34, respectively. The SREBP1 mRNA levels in these two groups were 1.01 ± 0.16 and 3.61 ± 0.39, respectively. The mRNA levels of SREBP1 in these two groups were 1.03 ± 0.32 and 1.99 ± 0.36 respectively. The mRNA levels of ACC in these two groups were 0.95 ± 0.29 and 2.37 ± 0.52, respectively. Compared with normal group, the expression of FAS, ACC, SREBP1 gene and SREBP1 protein in model group were significantly increased (all P <0.05). Compared with the model group, the protein gray value of Hep G2FAS, ACC, SREBP1 and SREBP1 in experimental group were 2.95 ± 0.92, 3.99 ± 1.16, 2.85 ± 0.91, 2.94 ± 0.65, respectively. The Hep G2 FAS, ACC , SREBP1 gene and SREBP1 protein expression was significantly increased, the differences were statistically significant (P <0.05). This indicates that atorvastatin further aggravates lipid deposition in Hep G2 cells under inflammatory conditions. The contents of ROS in normal group, model group and experimental group were 1.00 ± 0.20, 1.77 ± 0.25 and 3.20 ± 0.53, respectively. The contents of H2O2 in normal group, model group and experimental group were (2.30 ± 0.31) , (4.32 ± 0.77) and (5.31 ± 0.75) nmol · mg ~ (-1), respectively. The MDA contents of the two groups were (0.78 ± 0.22), (1.86 ± 0.23) and (3.43 ± 1.15) nmol · mg ~ (-1). Compared with the normal group, there were significant differences in the model group (all P <0.05). Compared with the model group, the difference in the experimental group was statistically significant (both P <0.05). Conclusion Atorvastatin induces oxidative stress in Hep G2 cells and activates the SREBP1 / FAS / ACC pathway in the inflammatory state and aggravates intracellular lipid accumulation.