RESUMO
Objective:To investigate the effect of sodium arsenite (NaAsO 2) on autophagy protein microtubule-associated protein light chain 3 (LC3) and Beclin-1 in human hepatic stellate cells (LX-2 cells). Methods:LX-2 cells were cultured in vitro, and LX-2 cells were stably infected with red fluorescent protein-green fluorescent protein-microtubule-LC3 (RFP-GFP-LC3) lentivirus. Flow cytometry was used for screening and infection rate determination. Using a group design, different concentrations NaAsO 2 [μmol/L: 5.00 (infection + high arsenic dose group), 0.50 (infection + medium arsenic dose group), 0.05 (infection + low arsenic dose group), and 0.00 (infection group)] were incubated to stably infect LX-2 cells, and an in vitro liver fibrosis model was constructed, and a blank group was established. The effect of NaAsO 2 on the activity of LX-2 cells was detected by CCK-8 method. The mRNA and protein expression levels of LC3, Beclin-1 and α-smooth muscle actin (α-SMA) were detected by real-time fluorescent quantitative PCR and Western blotting. Results:After stable infection of LX-2 cells by RFP-GFP-LC3 lentivirus, the fluorescence rate of RFP and GFP was determined by flow cytometry, and the infection rate was about 70%. There was no significant difference in the fluorescence intensity of RFP and GFP observed under fluorescence microscope, and the infected cells were established successfully. After treatment with NaAsO 2 for 24, 48, 72 h, compared with the blank group, the cell viability of the infection group was not significantly different statistically ( P > 0.05), and the cell viability of other dose groups decreased ( P < 0.05). There were significant differences in the expression levels of LC3, Beclin-1, α-SMA mRNA and protein in blank, infection, infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups ( F = 17.450, 11.084, 11.294, 11.745, 31.635, 12.130, P < 0.05). In infection, infection + high arsenic dose, and infection + low arsenic dose groups, the levels of LC3 mRNA (20.09 ± 6.50, 36.57 ± 9.68, 14.19 ± 6.17) were higher than that of the blank group (1.25 ± 0.21, P < 0.05), and the level of LC3 mRNA in infection + high arsenic dose group was higher than that of the infection group ( P < 0.05); in infection, infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups, the levels of Beclin-1 mRNA (22.46 ± 0.66, 13.38 ± 2.27, 20.80 ± 6.95, 24.31 ± 7.09) were higher than that of the blank group (1.10 ± 0.53, P < 0.05); in infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups, the levels of α-SMA mRNA (1.07 ± 0.27, 1.65 ± 0.17, 1.73 ± 0.26) were higher than that of the blank and infection groups (0.60 ± 0.11, 0.31 ± 0.09, P < 0.05). In infection, infection + medium arsenic dose, and infection + low arsenic dose groups, the LC3 protein expressions (0.20 ± 0.06, 0.15 ± 0.00, 0.16 ± 0.01) were significantly increased compared to that of the blank group (0.04 ± 0.01, P < 0.05); in infection + medium arsenic dose, and infection + low arsenic dose groups (0.83 ± 0.03, 1.20 ± 0.02), the Beclin-1 protein expressions were significantly increased compared to that of the blank group (0.25 ± 0.01, P < 0.05), and the Beclin-1 protein expression in infection + low arsenic dose group was increased compared to that of the infection group (0.53 ± 0.03, P < 0.05); in infection + medium arsenic dose, and infection + low arsenic dose groups (0.78 ± 0.10, 0.68 ± 0.06), the α-SMA protein expressions were significantly increased compared to that of the blank and infection groups (0.40 ± 0.07, 0.48 ± 0.04, P < 0.05). Conclusion:NaAsO 2 may affect the process of arsenic-induced liver fibrosis by promoting the autophagy level of LX-2 cells.