Objective To investigate the effects of mixture of arsenic on Keap1/Nrf2-ARE and MAPK/ERK signaling pathways and NF-κB gene expression in human immortalized keratinocytes (HaCaT cells) inhibited by Keap1.
Methods HaCaT cells were cultured for 72 h and then divided into a control group (normal HaCaT cells wihtout exposure to arsenic), a negative control group (suppressed by Keap1 without exposure to arsenic), and three mixed arsenic exposure groups (suppressed by Keap1 and exposed to arsenic at 2.9, 5.8, and 29.0 μmol/L, respectively). Cell growth was measured by MTT method. The mRNA expression levels of Keap1, Nrf2, ERK, and NF-κB in HaCaT cells were determined by real-time fluorescence quantitative PCR.
Results Compared with the negative control group, the cell activities of the mixed arsenic exposure groups were statistically different (F=483.9, P < 0.05). The cell activities of the medium dose and high dose arsenic exposure groups were inhibited, and the difference was statistically different (P < 0.05). The mRNA expression levels of Keap1 and Nrf2 were different between the mixed arsenic exposure groups and the negative control group (F=5.73, P=0.012; F=318.56, P < 0.05). The expression of Nrf2 mRNA was in creased at low dose (P=0.038), and inhibited at middle and high doses (P=0.014, P=0.016). The differences of ERK and NF-κB mRNA expression levels between the mixed arsenic exposure groups and the negative control group were statistically significant (F=39.88, P < 0.05; F=2 619.41, P < 0.05). Low-dose arsenic exposure promoted ERK gene expression (P=0.020), while high-dose arsenic exposure inhibited the expression (P=0.003), with significant differences. Compared with the negative control group, the expression of NF-κB gene was promoted by low-dose and middle-dose arsenic exposure (P=0.030, P=0.032), and inhibited by high-dose arsenic exposure (P=0.013), with significant differences.
Conclusion Under Keap1 inhibition, arsenic mixture could promote the expression of Nrf2, ERK, and NF-κB genes at low dose, and inhibit expression at high dose.
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