Activation of rat hepatic stellate cells and changes of DNA methyltransferase 1, 3a mRNA expression levels induced by sodium arsenite

Abstract

Objective To observe the activation of rat hepatic stellate cells (HSC-T6) and the changes of methyltransferase (DNMT)1, DNMT3a mRNA expression with different doses of sodium arsenite stimulation. Methods HSC-T6 cells were exposed to a final concentration of 0 (control), 5 (low dose), 15 (medium dose) and 25 (high dose) μmol/L sodium arsenite in culture medium for 24, 48 and 72 h, cells and cell culture supernatant were harvested. Enzyme-linked immunosorbent assay (ELISA) kit was used to measure fibrosis factors contents of type Ⅰ collagen (COL-1), type Ⅲ collagen (COL-3) and alpha smooth muscle actin (α-SMA) and quantitative real-time PCR was used to detect the expression levels of DNMT1 and DNMT3a mRNA. Results Different arsenic exposure time (24, 48, 72 h) had a significant effect on COL-1, COL-3 and α-SMA contents in HSC-T6 cells (F= 249.574, 328.493, 3 157.436, all P < 0.01); Different arsenic exposure content (low, medium, high dose groups) had a significant effect on COL-1, COL-3 and α-SMA contents in HSC-T6 cells (F= 3 946.521, 1 006.399, 13 025.770, all P < 0.01). After arsenic exposure for 24 and 48 h, the expression levels of DNMT1 mRNA in high dose group (4.33 ± 0.24, 2.34 ± 0.43) were higher than those of control group (1.00 ± 0.00, 1.00 ± 0.00, all P < 0.05). At the same arsenic exposure levels (low, medium or high dose), the expression level of DNMT1 mRNA was declined with prolongation of sodium arsenite stimulation time (all P < 0.05). After arsenic exposure for 48 and 72 h, the expression levels of DNMT3a mRNA in high dose group (2.23 ± 0.50, 5.02 ± 0.23) were higher than those of control group (1.00 ± 0.00, 1.00 ± 0.00, all P < 0.05). The expression levels of DNMT3a mRNA in medium and high dose groups at 72 h (3.80 ± 0.14, 5.02 ± 0.23) were higher than those of 24 h (3.03 ± 0.12, 0.42 ± 0.15, all P < 0.05). Conclusion HSC-T6 cells are obviously activated with pro-fibrotic effect; the expression levels of DNMT1 and DNMT3a mRNA are both up regulated in HSC-T6 cells after being exposed to sodium arsenite. Key words: Arsenites; Fibrosis; Astrocytes; Methyltransferases