目的 探讨Keap1沉默及染砷对Nrf2通路相关基因表达的影响及砷致皮肤早期氧化损伤的机制.方法 培养人源性正常HaCaT细胞(正常细胞)和Keap1沉默HaCaT细胞,将无机砷混合受试物按照LC50的1/100、1/50、1/10分为低(2.90 μmol/L)、中(5.80 μmol/L)、高(29.00 μmol/L)3个不同浓度组,以0 μmol/L无机砷混合受试物染毒Keap1沉默HaCaT细胞组为阴性对照组,正常细胞组为空白对照组,于3个时间点(24、48、72 h)采用实时荧光定量PCR检测Nrf2通路相关基因(Nrf2和Bach1)mRNA表达,使用ELISA试剂盒检测GSH、HO-1蛋白表达.结果 以阴性对照组作参照,Nrf2基因24 h 2.90、29.00 μmol/L染砷组表达上调,48 h和72 h 时2.90 μmol/L染砷组表达上调,而5.80 μmol/L及29.00 μmol/L染砷组表达均下调,差异有统计学意义 (P <0.012 5);Bach1基因24 h 2.90、5.80及29.00 μmol/L染砷组表达上调,48 h 2.90、5.80 μmol/L染砷组表达上调,而72 h 2.90、5.80及29.00 μmol/L染砷组表达均下调,差异有统计学意义 (P <0.012 5).随染毒浓度的增加,GSH蛋白和HO-1蛋白表达总体呈持续增长趋势.其中GSH蛋白24 h 2.90、5.80及29.00 μmol/L染砷组,48 h 5.80 μmol/L和29.00 μmol/L染砷组以及72 h 29.00 μmol/L染砷组表达差异有统计学意义 (P <0.012 5);HO-1蛋白24、48、72 h 2.90、5.80及29.00 μmol/L染砷组差异均有统计学意义 (P <0.012 5);空白对照组GSH蛋白和HO-1蛋白表达总体上低于阴性对照组,24 h差异均有统计学意义 (P <0.012 5).结论 Keap1基因沉默会引起Nrf2持续激活.Keap1、Nrf2、Bach1、GSH和 HO-1均参与砷诱导的抗氧化反应,并在砷致皮肤氧化应激损伤中发挥作用.%Objective To investigate the effects of silencing of Keap1 and arsenic exposure on Nrf2 pathway and to explore the mechanism underlying the arsenic-induced skin early oxidative damage.Methods boththe normal human HaCaT and HaCaT whose endogenous Keap1 was silenced were cultured.The inorganic mixture arsenic was divided into three groups: low (2.90 μmol/L), middle (5.80 μmol/L) and high (29.00 μmol/L) concentration groups according to LC50 1/100, 1/50, 1/10.Using 0 μmol/L inorganic arsenic exposure and Keap1 silencing HaCaT cell group as a negative control group, the normal cell group as blank control group, the expression of Nrf2 and Bach1 mRNA was detected by real-time quantitative PCR and the expression of GSH and HO-1 was detected by ELISA kit at three time points (24, 48, 72 h).Results Compared with negative control group, the Nrf2 gene expression at 24 h was 2.90 μmol/L, 29.00 μmol/L arsenic group was up-regulated;at 48 h and 72 h, 2.90 μmol/L arsenic group was up-regulated, and 5.80 μmol/L and 29.00 μmol/L arsenic group was significantly lower, the difference was statistically significant (P <0.012 5);Bach1 gene 24 h 2.90 μmol/L, 5.80 μmol/L and 29.00 μmol/L arsenic groups up-regulated and 48 h 2.90 μmol/L, 5.80 μmol/L arsenic group was up-regulated and 72 h 2.90, 5.80 and 29.00 μmol/L arsenic group was significantly lower, the difference was statistically significant (P <0.012 5).The expression of GSH protein and HO-1 protein increased with the increase of arsenic concentration.The expression of GSH protein in arsenic treated groups was significantly higher than that in the negative control group in 2.90, 5.80 and 29.00 μmol/L at 24 h, and in 5.80, 29.00 μmol/L at 48 h and in 29.00 μmol/L at 72 h (P <0.05).The expression of HO-1 protein was significantly different between the arsenic treated groups and the negative control group in 2.90 μmol/L, 5.80 μmol/L and 29.00 μmol/L at 24 h, 48 h and 72 h.The expression of GSH protein and HO-1 protein in the blank control group was lower than that in the negative control group at 24 h (P <0.012 5).Conclusion Keap1 gene silencing can cause sustained activation of Nrf2, indicating that Keap1, Nrf2, Bach1, GSH and HO-1 were involved in the arsenic-induced antioxidant reaction and play a role in arsenic-induced skin oxidative stress injury.
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