抑制Keap1基因对染砷HaCaT细胞Nrf2通路的影响

Effect of inhibition of Keap1 gene on Nrf2 pathway in HaCaT cells treated by arsenic

目的观察无机砷混合物对Keap1抑制HaCaT细胞的活力及形态变化,探讨Keap1抑制及染砷对Nrf2通路相关基因表达的影响,探究砷致皮肤早期氧化损伤的机制。方法培养正常与Keap1抑制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、中、高3个不同浓度组,即为2.90μmol/L、5.80μmol/L、29.00μmol/L,以0μmol/L无机砷混合受试物染毒Keap1抑制HaCaT细胞组为阴性对照组,正常细胞组为空白对照组,于4个时间点(8、24、48、72 h)采用MTT法检测细胞活力,使用实时荧光定量PCR检测Nrf2通路相关基因(Nrf2、Keap1、Bach1及CBP)mRNA表达。结果随染砷浓度增加,细胞收缩明显,边缘多不规则,脱壁明显。2.90μmol/L混合砷染毒促进细胞增殖,≥5.80μmol/L混合无机砷染毒抑制细胞增殖。与空白对照组比较,阴性组Nrf2通路相关基因(Bach1,CBP)随时间延长,基因表达持续升高,差异均有统计学意义(F=200.261,P<0.05)。与阴性组比较,2.90μmol/L染砷组24、48 h时Nrf2基因表达无差异;Bach1、CBP基因表达均上调,差异均有统计学意义(F=29.015、43.964,P<0.01)。5.80μmol/L染砷组24 h时Nrf2基因表达无差异,Bach1、CBP基因表达均上调;48、72 h时Nrf2、Bach1、CBP基因表达均下调,差异均有统计学意义(F=5.289、29.015、43.964,P<0.01)。29.00μmol/L染砷组24、48 h时Nrf2、Bach1、CBP基因表达均上调;72 h时Nrf2、Bach1、CBP基因表达均下调,差异有统计学意义(F=34.275,P<0.01)。经两两比较,72 h时随染砷浓度逐渐增加,Nrf2、Bach1、CBP基因表达逐渐下调,差异均有统计学意义(P<0.05)。结论抑制Keap1基因会引起Nrf2持续激活。Bach1和CBP协同调控Nrf2通路可能在砷致氧化应激反应中起调节作用。

Objective To observe the viability and morphology in Keapl inhibiting HaCaT cell treated by arsenic; to investigate the impact of Nrf2 pathway gene expression of Keapl inhibiting and Arsenic ; to explore the mechanism of arsenic - induced skin damage early oxidation. Methods Culturing normal and Keapl inhibiting HaCaT cells; inorganic arsenic was divided into low, medium and high dose groups according to the test LC50 1/100, 1/50, 1/10, namely 2. 90 μmol/L, 5.80 μmol/L, 29.00 μmol/L; using 0 0μmol/L inorganic arsenic exposure and Keapl inhibiting HaCaT cell group as a negative control group, the normal cell group as a blank control group ; using the MTT to test cell viability and using real - time quantitative PCR to detect the mRNA expression of Nrf2 pathway related genes （Nrf2, Keapl, Bachl and CBP）at four time points （8, 24, 48, 72 h）. Results With Arsenic concentration increases, the shrinkage of cell is more obvious, ragged edges and cell wall off significant- ly. 2. 90 μmol/L mixed arsenic exposure promotes cell proliferation, ≥5.80 μmol/L mixed arsenic exposure inhibits cell prolif- eration. Compared with the blank control group, gene expression of negative group Nrf2 pathway related genes （Bachl, CBP） continues to rise with time passing, the differences were statistically significant （ F = 200. 261, P 〈 0. 05 ）. Compared with nega- tive group, the expression of Nrf2 is no difference but it of Bachl and CBP genes is up - regulated at 24 h and 48 h in 2. 90 μmoL/L as group, and the difference was statistically significant （ F = 29. 015, 43. 964, P 〈 0. 01 ）. The expression of Nrf2 is no difference but it of Bachl and CBP genes is up - regulated at 24 h in 5.80μmol/L as group; the expression of Nrf2, Bachl and CBP genes were down- regulated at 48 h and 72 h, and the difference was statistically significant （F = 5. 289, 29. 015, 43. 964, P 〈0. 01 ）. The expression of Nrf2, Bachl and CBP gene were up - regulated at 24 h and 48 h but were down - regula- ted at 72 h in 29. 00 μmol/L as group, and the difference was statistically significant （ F = 34. 275, P 〈 0. 01 ）. By pairwise comparison, with increased exposure concentration, Nrf2, Bachl and CBP gene expression decreased gradually at 72 h, and the difference was statistically significant （ P 〈 0. 05 ）. Conclusion Inhibiting Keapl gene causes persistent activation of Nrf2. Bachl and CBP Regulating the Nrf2 pathway may play a regulatory role in arsenic -induced oxidative stress.