Effects of sodium arsenate on apoptosis and autophagy of human hepatic stellate cells

Background Hepatic stellate cells are the main effector cells of the liver, and their activation is considered to be one of the main mechanisms that promote the development of liver fibrosis. Autophagy and apoptosis have been reported to play a key role in the activation of hepatic stellate cells.

Objective This experiment explores the effects of sodium arsenate on the apoptosis and autophagy of human hepatic stellate cells (LX-2 cells).

Methods A LX-2 cell line was cultured in vitro, and stably infected with the red fluorescent protein (RFP)-green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3) lentivirus. Flow cytometry was used to screen and determine the infection rate. LX-2 cells infected with lentivirus were treated with different concentrations of Na₂HAsO₄ (0, 0.6, 6, and 60 μmol·L^-1), and LX-2 cells without lentivirus infection were used as blank control group. CCK-8 method was used to detect the activity of LX-2 cells, flow cytometry for the apoptosis of LX-2 cells, and real-time fluorescence quantitative PCR (RT-PCR) and Western blotting for autophagy indicatorsLC3, recombinant human autophagy effector protein (Beclin-1), and sequestosome 1/P62 (SQSTM-1/P62) and apoptosis indicatorB-cell lymphoma-2 (BCL-2).

Results After the RFP-GFP-LC3 lentivirus infection, the lentivirus infection rate of the LX-2 cells was 70%. The fluorescence expression intensity was not different between RFP and GFP under laser confocal microscope. After designed Na₂HAsO₄ treatment for 24, 48, and 72 h, compared with the Na₂HAsO₄ (0 μmol·L^-1) group, the cell inhibition rate of the other Na₂HAsO₄ dose groups all increased (P < 0.05), except the LC3+Na₂HAsO₄ (0 μmol·L^-1) group. With the increase of Na₂HAsO₄ dose, the apoptosis rate showed an upward trend, and was significantly higher than that of the Na₂HAsO₄ (0 μmol·L^-1) group (P < 0.05). The mRNA and protein expression levels of LC3 (F=5.64, 340.66), Beclin-1 (F=20.59, 87.70), SQSTM-1/P62 (F=15.91, 107.24), and BCL-2 (F=113.29, 9.41) were different among the designed groups (P < 0.05).

Conclusion Na₂HAsO₄ can induce apoptosis and autophagy in LX-2 cells, and there may be an antagonistic effect between apoptosis and autophagy.