Folic acid and vitamin B 12 inhibit arsenic-induced apoptosis in SH-SY5Y cells via Bcl-2/Bax pathway / 中华地方病学杂志

ABSTRACT

Objective:To explore the arsenic trioxide (As 2O 3)-induced apoptosis of human neuroblastoma cells (SH-SY5Y cells) and the protection mechanisms of folic acid (FA) and vitamin B 12 (VB 12). Methods:SH-SY5Y cells were cultured in vitro and divided into six groups by group design: control group (normal cultured), arsenic exposed group (10.00 μmol/L As 2O 3), FA intervention group (0.30 mmol/L FA + 10.00 μmol/L As 2O 3), VB 12 intervention group (0.06 mmol/L VB 12 + 10.00 μmol/L As 2O 3), combined intervention group (0.30 mmol/L FA + 0.06 mmol/L VB 12 + 10.00 μmol/L As 2O 3) and reagent control group (0.30 mmol/L FA + 0.06 mmol/L VB 12). Cells in each group were cultured for 24 h ( n = 3). Flow cytometry was used to determine the apoptosis rate of cells in each group. Transmission electron microscopy was used to observe the ultrastructural changes of the cells. The expression levels of mRNA and protein of apoptosis-related indicator B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X (Bax) were detected by fluorescence quantitative PCR and Western blotting. The activity of cysteinyl aspartate specific proteinase (Caspase) 3 was detected by luminescent assay. The above indicators were statistically analyzed. Results:There was statistically significant difference in the apoptosis rate among different groups ( F = 213.036, P < 0.05). The apoptosis rate in arsenic exposed group [(44.43 ± 3.54)%] was higher than that in control, FA intervention, VB 12 intervention, and combined intervention groups [(1.80 ± 0.06)%, (14.37 ± 0.13)%, (19.10 ± 1.56)%, (17.11 ± 2.34)%, P < 0.05]. Under transmission electron microscope, the apoptotic bodies, mitochondria swelling and degeneration, chromatin agglutination were observed in SH-SY5Y cells exposed to arsenic. The morphological and organelle changes of SH-SY5Y cells were significantly improved after respective and combined intervention of FA and VB 12. The expression levels of Bcl-2, Bax mRNA and protein were significantly different among different groups ( F = 5.178, 7.169, 6.142, 9.194, P < 0.05). The expression level of Bcl-2 protein in arsenic exposed group was lower than that in control group ( P < 0.05), and the expression levels of Bax mRNA and protein were higher than those in control group ( P < 0.05). The expression levels of Bcl-2 mRNA and protein in FA intervention group and combined intervention group were higher than those in arsenic exposed group ( P < 0.05), and Bcl-2 mRNA expression level in VB 12 intervention group was higher than that in arsenic exposed group ( P < 0.05). The expression levels of Bax mRNA and protein in FA intervention, VB 12 intervention and combined intervention groups were lower than those in arsenic exposed group ( P < 0.05). There were statistically significant differences in Caspase 3 activity among different groups ( F = 84.604, P < 0.05). Caspase 3 activity in arsenic exposed group was significantly higher than those in control, FA intervention, VB 12 intervention, and combined intervention groups ( P < 0.05). Conclusions:Arsenic exposure can lead to apoptosis and ultrastructural changes of SH-SY5Y cells. FA and VB 12 may effectively inhibit apoptosis through regulating Bcl-2/Bax pathway and decrease Caspase 3 activity, thus playing a protective role on nerve cells.