砷对Nrf2抑制HaCaT细胞凋亡及抗氧化水平的影响

Effect of arsenic on inhibition of apoptosis and antioxidant level of HaCaT cells by Nrf2

目的以Nrf2基因抑制的人永生化皮肤角质细胞(HaCaT)作为模型细胞,通过染砷模型细胞,观察抗氧化水平及细胞凋亡等的变化。探讨对氧化应激的影响,为探究砷致皮肤角化紊乱机制提供基础。方法以人正常HaCaT细胞为实验空白对照组。无机砷混合物暴露于砷污染组,高、中、低砷浓度为LC50浓度的1/10、1/50、1/100,即为21. 0μmol/L、4. 2μmol/L、2. 1μmol/L。每组细胞培养8、24、48和72 h。酶联免疫吸附试验(ELISA)检测各组HO-1、GSH和COX-2蛋白水平。结果与阴性对照组相比,高、中、低剂量组在8 h和24 h时早期凋亡率增加,差异有统计学意义(F=298. 696,P <0. 01),在低、中、高剂量组中HO-1蛋白水平在8 h和24 h时上调(F=279. 229,P <0. 01;F=530. 741,P <0. 01),48 h和72 h HO-1蛋白含量均下调(F=329. 470,P <0. 01;F=814. 141,P <0. 01),各组8 h、72 h GSH蛋白浓度先上调后下调(F=100 064. 804,P <0. 001,F=158 817. 814,P <0. 05),各组24 h、48 h GSH蛋白浓度上调(F=77 549. 323,P <0. 05),除8 h外,其余时间点各组COX-2蛋白浓度均下调(F=147. 203,P <0. 001;F=3 054. 025,P <0. 001;F=126 920. 724,P <0. 001)。结论砷暴露通过增加ROS生成,调控Nrf2通路,下调COX-2和HO-1蛋白,上调GSH蛋白,诱导皮肤角质形成细胞凋亡及皮肤角化紊乱。

Objective Human immortalized skin keratinocytes(HaCaT)inhibited by Nrf2 gene were used as model cells to observe the changes of antioxidant level and apoptosis by arsenic staining.To explore the effects of arsenic on oxidative stress,and to provide a basis for exploring the mechanism of arsenic-induced skin keratosis disorder.Methods Human normal HaCaT cells were used as the blank control group.The inorganic arsenic mixture was exposed to arsenic contamination groups at concentrations of 1/10,1/50,and 1/100 of LC50,namely 21.0 mol/L,4.2 mol/L,and 2.1 mol/L.Each group was cultured for 8,24,48 and 72 h.Protein levels of HO-1,GSH and COX-2 in each group were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with the negative control group,the early apoptosis rate increased in the high,medium and low dose groups at 8 h and 24 h.Difference was statistically significant(F=298.696,P<0.01),in the low dose group,medium dose group and high dose group of HO-1 protein levels in 8 and 24 hours increase(F=279.229,P<0.01;F=530.741,P<0.01),48 h and 72 h HO-1 protein content were lower(F=329.470,P<0.01;F=814.141,P<0.01),each group 8 h,72 h GSH protein concentration increase before cut(F=100 064.804,P<0.001;F=158 817.814,P<0.05),each group of 24 h,48 h increase GSH protein concentration(F=77 549.323,P<0.05),in addition to the 8 h,the rest of the time point groups of COX-2 protein concentrations were lower(F=147.203,P<0.001;F=3 054.025,P<0.001;F=126 920.724,P<0.001).Conclusion Arsenic exposure can increase ROS production,regulate Nrf2 pathway,down-regulate COX-2 and HO-1 proteins,up-regulate GSH proteins,and induce apoptosis of keratinocytes and dermal keratosis disorders.