Effect of sodium arsenite exposure on nuclear factor E2-related factor 2 signaling pathway in SVEC4-10 cells / 中华地方病学杂志

ABSTRACT

Objective:To investigate the effect of sodium arsenite (NaAsO 2) on transcriptional activity of nuclear factor E2-related factor 2 (Nrf2) signaling pathway in mouse lymph node vascular endothelial cell line (SVEC4-10). Methods:In vitro cell culture method was used to treat SVEC4-10 cells for 24 h with different doses of NaAsO 2 [0 (control), 2, 5, 10, 20, 50, 100, 150 μmol/L], and the cell viability was detected by tetrazole compound (MTS) method. The time-response relationship was studied with SVEC4-10 cells treated with 5 μmol/L NaAsO 2 for 0 (control), 2, 6 and 12 h; the dose-response relationship was studied with SVEC4-10 cells treated with 0 (control), 2, 5 and 10 μmol/L NaAsO 2 for 6 h; real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA expression of Nrf2 and its downstream genes glutamate-cysteine ligase catalytic subunit (Gclc), glutamate-cysteine ligase modifier subunit (Gclm), NAD(P)H dehydrogenase quinone 1 (Nqo1) and metallothionein 1 (Mt1). Establishment of Nrf2 gene stably silenced (Nrf2-KD) cells using SVEC4-10 cells, the interference control (scramble, SCR) cells and Nrf2-KD cells were treated with 0(control), 10 and 20 μmol/L NaAsO 2 for 16 h, and apoptosis was detected by flow cytometry. Results:MTS test results showed that the cell viability of the control, 2, 5, 10, 20, 50, 100, 150 μmol/L NaAsO 2 treatment groups was (100.00 ± 19.53)%, (98.18 ± 9.85)%, (96.09 ± 30.04)%, (90.64 ± 8.74)%, (59.75 ± 12.09)%, (35.43 ± 8.58)%, (26.35 ± 5.89)% and (17.54 ± 4.48)%, respectivily. There was statistically significant difference in cell viability between different dose groups ( F = 18.30, P < 0.05); and the cell viability of the 20, 50, 100, 150 μmol/L NaAsO 2 treatment groups was significantly lower than that of the control group ( P < 0.05). The time-response relationship results showed that there were statistically significant differences in Nrf2, Gclc, Gclm, Nqo1 and Mt1 mRNA level between control, 2, 6 and 12 h treatment groups ( F = 56.69, 85.28, 90.82, 80.46, 758.60, P < 0.05); with extension of arsenic exposure time, the mRNA level of Nrf2, Gclc, Gclm and Mt1 first increased and then decreased, the mRNA level of Nqo1 increased continually; among them, the mRNA level of Nrf2 peaked at 2 h, the mRNA levels of Gclc, Gclm and Mt1 peaked at 6 h, and the mRNA level of Nqo1 peaked at 12 h. The dose-response relationship results showed that there were statistically significant differences in Nrf2, Gclc, Gclm, Nqo1 and Mt1 mRNA levels between control, 2, 5 and 10 μmol/L NaAsO 2 treatment groups ( F = 68.39, 72.26, 30.41, 397.00, 28.88, P < 0.05); with increasing of arsenic exposure dose, the mRNA levels of Nrf2, Gclc, Gclm, Nqo1 and Mt1 increased. The mRNA level of Nrf2 peaked at a dose of 5 μmol/L, and the mRNA levels of Gclc, Gclm, Nqo1 and Mt1 peaked at a dose of 10 μmol/L. Apoptosis test results showed that there were statistically significant differences in the apoptosis rates of SCR and Nrf2-KD cells between control, 10 and 20 μmol/L NaAsO 2 treatment groups ( F = 8.18, 9.66, P < 0.05); compared with the control group, the apoptosis rates of SCR and Nrf2-KD cells in the 20 μmol/L NaAsO 2 treatment group increased ( P < 0.05); and the apoptosis rate of Nrf2-KD cells in the 20 μmol/L NaAsO 2 treatment group was higher than that of SCR cells in the same dose group ( P < 0.05). Conclusions:NaAsO 2 exposure has caused the activation of Nrf2 signaling pathway in mouse lymph node vascular endothelial cell line SVEC4-10 cells, activated the adaptive antioxidant response, and altered transcriptional activity; while silence of Nrf2 has made SVEC4-10 cells more sensitive to NaAsO 2 toxicity.