HepG2脂肪变模型中氧化应激的发生及维生素E对其的干预作用

Occurrence of oxidative stress and interventional effects of vitamin E on HepG2 steatosis model

【摘要】目的建立HepG2脂肪变模型，探讨混合脂肪酸对肝细胞氧化应激的作用，以及维生素E对脂肪变肝细胞氧化还原系统的影响。方法采用不同浓度梯度（0．125、0．250、0．500、1．000mmol／L）的混合脂肪酸（油酸：软脂酸=2：1）孵育HepG2细胞，细胞计数试剂盒细胞增殖法测细胞活力。分别采用0．5和1．0mmol／L的混合脂肪酸孵育HepG2细胞，在12、24、48h进行油红0染色和细胞内TG含量的测定。建立HepG2脂肪变模型后检测细胞内过氧化产物丙二醛含量和超氧化物歧化酶（SOD）、还原型谷胱甘肽水平，并予以维生素E干预，检测维生素E对丙二醛、SOD和还原型谷胱甘肽的影响。两组计量资料比较采用t检验。结果随着作用时间的延长和混合脂肪酸浓度的增加，光学显微镜下可见HepG2细胞内脂滴数量逐渐增加，TG含量也逐渐增加。干预后24h，混合脂肪酸0．5mmol／L组和1．0mmol／L组的HepG2细胞内丙二醛含量较对照组分别增加了56．3％（t=4．28，P=0．0129）和66．6％（t=6．87，P=0．0024），还原型谷胱甘肽水平较对照组分别降低了30．6％（t=4．24，P=0．0133）和43．7％（t=6．13，P=0．0036），差异均有统计学意义。混合脂肪酸1．0mmol／L组细胞内SOD水平在24h较对照组降低了39．2％（t=3．20，P=0．0328），0．5mmol／L组在48h较对照组降低了40．6％（t=8．27，P=0．0012），差异均有统计学意义。维生素E干预24、48h时，可使脂肪变性HepG2细胞内丙二醛含量比同时间实验组下降31．8％（t=4．00，P=0．0161）和37．5％（t=8．27，P=0．0042），差异有统计学意义；干预12、24、48h时，SOD水平分别升高1．9％、14．5％和50．5％；还原型谷胱甘肽水平分别升高4．1％、33．2％和52．0％。结论油酸和软脂酸比例为2：1、浓度为0．5mmol／L的混合脂肪酸可成功诱导HepG2细胞脂肪变模型。HepG2细胞脂肪变性时发生明显的氧化应激，抗氧化剂维生素E能在一定程度上减轻氧化应激。

Objective To establish hepatocyte steatosis model, explore the oxidative stress role of fatty acid mixtures in HepO2 cells and the effects of vitamin E on redox system of steatosis hepatocyte. Methods HepG2 cells were incubated with different gradient concentrations （0. 125, 0. 250, 0. 500 and 1. 000 retool/L） of fatty acid mixtures （the ratio of oleic acid to palmitic acid was 2 to 1）. The cell viability was detected by cell counting kit-8 assay. HepG2 cells were incubated with fatty acid mixtures at 0. 5 mmol/L and 1.0 mmol/L, at 12, 24 and 48 hour the cells were stained with oil red O staining and the content of triglyceride in the cells was tested. After the hepatocyte steatosis model established, the content of lipid peroxidation product malondialdehyde（MDA） and the level of superoxide dismutase （SOD） and reduced glutathione （GSH） were detected. Then the cells were intervened by vitamin E, and the effects of vitamin E on MDA, SOD and reduced GSH were examined, t test was used for two groups of measurement data comparison. Results With the extension of time and increased concentration o{ {arty acid mixtures, the number of lipid droplet in HepG2 cells under microscope and the content of triglyceride gradually increased. After treated for 24 hours, the content of MDA in HepG2 cells treated with fatty acid mixtures at0.5 mmol/L and 1.0 mmol/L increased 56.3% （t=4.28, P=0.012 9） and 66.6% （t= 6.87, P=0. 002 4） compared with control group, the level of reduced GSH decreased 30.6% （t=4.24, P=0. 013 3） and 43.7% （t=6.13, P=0. 003 6） compared with control group and the differences were statistically significant. The level of SOD in cells treated with 1.0 mmol/L fatty acid mixtures decreased 39.2% （t=3.20, P=0. 032 8） compared with control group at 24 hour and with 0.5 mmol/L decreased 40.6% （t=8. 27, P= 0. 001 2） at 48 hour compared with control group and the differences were statistically significant. After intervened by vitamin E for 24 hours and 48 hours, the content of MDA in steatosis HepG2 cells decreased 31.8% （t=4.00, P=0.016 1） and 37.5% （t=8.27, P=0.004 2） compared with control group, the differences were statistically significant. After intervened fQr 12, 24 and 48 hours, the level of SOD increased 1. 9%, 14. 5% and 50. 5%; the level of reduced＇GSH increased 4.1%, 33.2% and 52.0%. Conclusions The hepatocyte steatosis model successfully induced by fatty acid mixture at concentration of 0.5 mmol/L and the ration of oleic acid to palmitic acid is 2 to 1. There is remarkable oxidative stress in steatosis HepG2 ceils. Antioxidant vitamin E could reduce oxidative stress to certain degree.