Background Arsenic can cause cell damage, autophagy, and apoptosis, but there is no clear boundary between autophagy and apoptosis, and their relatonship is unclear.
Objective This study is designed to investigate the relationship between autophagy and apoptosis in human embryonic lung fbroblasts (HELF) treated with sodium arsenite.
Methods HELF cells were cultured for three days and then randomly divided into a control group, three sodium arsenite treatment groups (5, 10, and 20 μmol/L), an autophagy inhibiton group (20 μmol/L NaAsO2+5 mmol/L 3-methyladenine), and an apoptosis inhibition group (20 μmol/L NaAsO2+20 μmol/L Caspase inhibitor z-VAD-FMK). After 48 hours of the designed exposure protocol, the cell survival rate was detected by MTT assay, and the morphological changes were observed under inverted microscope. Proteins were extracted from supernatants to determine the contents of interleukin (IL)-1β, IL-6, interferon (IFN)-α in supernatants by ELISA. The expression levels of autophagy related proteins p62 and LC3 were detected by Western blot and apoptosis related protein caspase3/7 by Caspase kit.
Results The cell survival rates of the sodium arsenite treatment groups, the autophagy inhibition group, and the apoptosis inhibition group were signifcantly lower than that of the control group at 12, 24, and 48 h afer treatment (P < 0.05). With the increase of exposure concentraton, the morphology changed from tghtly arranged spindle cells to loosely arranged round cells. The expressions levels of IL-1β in the supernatant of HELF cells in the 10 and 20 μmol/L arsenic exposure groups were increased (P < 0.05). The expression levels of proinflammatory cytokins IL-6 and IFN-α in the supernatant of arsenic treated groups were signifcantly higher than those of the control group (P < 0.05). Compared with the 20 μmol/L sodium arsenite treatment group, the expression levels of pro-inflammatory cytokines in the autophagy inhibiton group were higher, and the expression levels in the apoptosis inhibiton group were lower. Compared with the control group, the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins showed an upward trend, while the expression levels of p62 protein showed a downward trend in the arsenic exposure groups. Compared with the 20 μmol/L sodium arsenite treatment group, the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins were decreased (P < 0.05), and the expression level of p62 protein showed an increasing trend in the autophagy inhibiton group; the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins shouled an upward trend, while the expression level of p62 protein showed a downward trend in the apoptosis inhibiton group. With the increase of arsenite exposure concentraton, compared with the control group, the expression of apoptosis-related protein caspase3/7 in the 10 and 20 μmol/L arsenic exposure groups increased (P < 0.05). Compared with the 20μmol/L sodium arsenite treatment group, the expression of caspase3/7 in the autophagy inhibiton group was increased (P < 0.05), and the expression of caspase3/7 in the apoptosis inhibiton group was decreased (P < 0.05).
Conclusion Sodium arsenite can induce inflammatory reacton, autophagy, and apoptosis of HELF cells, and there may be an antagonistc relatonship between autophagy and apoptosis.
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