游离脂肪酸对肝细胞L02的脂毒性及氧化应激机制

目的 探讨游离脂肪酸(FFA)对人正常肝细胞L02的脂毒性及其机制。方法 用含FFA 0.2, 0.4和0.8 mmol·L^-1的培养液培养L02细胞48 h,通过油红O染色和甘油三酯(TG)含量检测胞内脂质蓄积,测定培养上清谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性分析肝细胞损伤,锥虫蓝拒染计数法分析细胞存活率,测定琥珀酸脱氢酶(SDH)活性分析线粒体功能,DCFH-DA法检测活性氧类(ROS)水平,比色法检测总谷胱甘肽和丙二醛(MDA)含量,FITC-AnnexinⅤ/PI法检测细胞凋亡。结果 经FFA处理L02细胞48 h后,FFA 0.4和0.8 mmol·L^-1组TG含量131±50和(267±71)μmol·g^-1蛋白较正常对照组(32±6)μmol·g^-1蛋白显著升高(P<0.05),FFA 0.2, 0.4和0.8 mmol·L^-1组GPT活性分别为2.05±0.06, 2.78±0.64和(2.43±0.55)U·L^-1,较正常对照组(1.10±0.05)U·L^-1显著升高(P<0.05)。锥虫蓝拒染法显示各FFA组细胞存活率无显著变化;FFA 0.4和0.8 mmol·L^-1组SDH活性(A_{490 nm})分别为2.10±0.11和1.95±0.11,均较正常对照组1.46±0.06显著升高(P<0.01);FFA 0.8 mmol·L^-1组ROS水平为642±58,较正常对照组559±31显著升高(P<0.01)。FFA 0.2, 0.4和0.8 mmol·L^-1组总谷胱甘肽水平分别为176±6, 180±11和(96±4)μmol·g^-1蛋白,较正常对照组(202±13)μmol·g^-1蛋白显著降低(P<0.05),FFA 0.4和0.8 mmol·L^-1组MDA水平分别为33.8±5.5和(50.4±7.4)mmol·g^-1蛋白,均较正常对照组(8.08±5.48)mmol·g^-1蛋白显著升高(P<0.01)。各组间凋亡率无显著差异。结论 FFA对肝细胞L02有一定损伤,这种损伤对细胞存活无明显影响,亦不会引起细胞凋亡,其细胞损伤机制与肝细胞氧化应激有关。

Lipotoxicity of free fatty acid and its oxidative stress mechanism in L02 cells

OBJECTIVE To investigate the mechanisms of lipotoxicity induced by free fatty acid(FFA) in L02 cells. METHODS L02 Cells were treated with FFA 0.2, 0.4 and 0.8 mmol·L^-1 for 48 h. Cellular total lipid was determined using oil red staining while triglycerides content was measured using an enzymatic kit. The level of glutamic-pyruvic transaminase(GPT) and glutamic-oxalacetic transaminase (GOT) was tested for cytotoxicity. Cell viability was evaluated by trypan blue exclusion assay and enzymatic activity of succinic dehydrogenase (SDH). Oxidative stress and apoptosis were measured by the level of reactive oxygen species (ROS), total glutathione, malondialdehyde(MDA) and AnnexinⅤ/propidium iodide staining, respectively. RESULTS Compared with normal control group, triglycerides content in FFA 0.4 and 0.8 mmol·L^-1 groups((131±50) and (267±71)μmol·g^-1 protein) was significantly higher than in normal control group((32±6)μmol·g^-1 protein)(P<0.05). GPT level in FFA 0.2, 0.4 and 0.8 mmol·L^-1 groups((2.05±0.06), (2.78±0.64) and (2.43±0.55)U·L^-1) was significantly higher than in normal control group((1.10±0.05)U·L^-1)(P<0.05). The cell viability did not change obviously. Enzymatic activity of SDH in FFA 0.4 and 0.8 mmol·L^-1 groups((2.10±0.11) and (1.95±0.11)) was significantly higher than in normal control group(1.46±0.06)(P<0.01), so was ROS level in FFA 0.8 mmol·L^-1 group (642±58), but total glutathione level in FFA 0.2, 0.4 and 0.8 mmol·L^-1 groups((176±6), (180±11) and (96±4)μmol·g^-1 protein) was significantly lower than in normal control group((202±13)μmol·g^-1 protein)(P<0.05). MDA level in FFA 0.4 and 0.8 mmol·L^-1 groups ((33.8±5.5) and (50.4±7.4)mmol·g^-1 protein) was significantly higher than in normal control group((8.1±5.5)mmol·g^-1 protein)(P<0.01), and the cell apoptosis rate did not change obviously. CONCLUSION FFA causes damage to L02 liver cells without causing apparent apoptosis and lower viability in L02 cells. The mechanism is related to oxidative stress.