NaAsO2对L-02肝细胞氧化应激性损伤及SOD1、AUF1表达影响

目的 观察不同剂量亚砷酸钠（NaAsO2）对L-02肝细胞氧化应激性损伤作用及超氧化物歧化酶1（SOD1）、AU 碱基富集元件RNA 结合因子1（AUF1）表达影响。方法 用0、10、20、40μmol/L NaAsO2分别处理L-02肝细胞24小时,化学比色法检测谷胱甘肽巯基转移酶（GST）、γ-谷氨酰转肽酶（γ-GT）活力及总胆汁酸（TBA）含量和SOD1、谷胱甘肽过氧化物酶（GPx）活性;荧光探针2,7-二氯二氢荧光素二乙酸酯检测细胞内活性氧族（ROS）相对荧光密度;实时荧光定量PCR和Western Blotting分别检测SOD1和AUF1转录和蛋白表达。结果 （1）与对照组比较,20、40μmol/L NaAsO2组GST活性和TBA含量均升高（P<0.05）;10、20、40μmol/L NaAsO2组γ-GT活性也升高（P<0.05）。（2）与对照组比较,20、40 μmol/L NaAsO2组SOD1活性均下降（P<0.05）;GPx活性仅在10μmol/L升高（P<0.05）;细胞内ROS先降低后升高（P<0.05）。（3）与对照组比较,20和40μmol/L NaAsO2组AUF1和SOD1 mRNA表达均升高（P<0.05）,AUF1蛋白表达先升高后降低（P<0.05）;10、20μmol/L NaAsO2组SOD1蛋白表达升高（P<0.05）,但40μmol/L NaAsO2组SOD1蛋白表达有降低趋势。结论 NaAsO2对L-02肝细胞产生氧化应激性损伤,其机制可能是NaAsO2通过降低AUF1的蛋白表达降低,从转录后调控降低了SOD1 mRNA的稳定性而使其蛋白表达和酶活力降低。

Effects of NaAaO2 on oxidative stress injury and expression of SOD1 and AUF1 in L-02 hepatocytes

Objective To observe effects of sodium arsenite (NaAsO2) at different concentrations on oxidative stress injury and the expression of superoxide dismutase 1 (SOD1) and AU-rich element RNA-binding factor 1 (AUF1) in L-02 hepatocytes. [WTHZ]Methods The L-02 hepatocytes were exposed to 0, 10, 20, 40μmol/L NaAsO2 for 24 hours, and then the colorimetry was used to detect levels of glutathione S-transferase (GST), γ-glutamyltranspeptidase (γ- GT), total bile acid (TBA) and SOD1, glutathione peroxidase (GPx). Fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate was used to detect relative fluorescence density of reactive oxygen species (ROS) in cells. Real-time quantitative PCR and Western blotting were used to detect the transcription and protein expression of SOD1 and AUF1, respectively. [WTHZ]Results (1) With increasing of NaAsO2 concentrations, the levels of GST and TBA in 20 and 40μmol/L NaAsO2 groups increased more than those in the control group(P<0.05), the γ-GT activity in 10, 20 and 40 μmol/L NaAsO2 groups also increased(P<0.05). (2) With the increasing of NaAsO2 concentrations, compared with the control group, the SOD1 activity in the 20 and 40μmol/L NaAsO2 groups decreased(P<0.05). However, the GPx activity increased only in 10μmol/L group compared with that in the control group(P<0.05). The relative fluorescence density of ROS decreased at first and then increased(P<0.05). (3) With increasing of NaAsO2 concentrations, the mRNA expression levels of AUF1 and SOD1 in 20 and 40μmol/L NaAsO2 groups increased than those in the control group(P<0.05); the protein expression levels of AUF1 increased firstly and decreased at last(P<0.05); the protein expression levels of SOD1 in the 10 and 20μmol/L NaAsO2 groups increased than those in the control group(P<0.05), but there only had a decreasing trend in the 40μmol/L NaAsO2 group. [WTHZ]Conclusion NaAsO2 exposure could cause oxidative stress damage to L-02 hepatocytes. The mechanism may be that NaAsO2 could reduce the AUF1 protein expression. That cause the stability of SOD1 mRNA reduction by post-transcriptional regulation and then cause the protein expression and enzyme activity of SOD1 reduce.