Ferroptosis patterns in SH-SY5Y cells induced by sodium arsenite

Background Arsenic can cause neurotoxicity by inducing neuronal loss, but it is not clear whether it can induce ferroptosis in nerve cells.

Objective This experiment is designed to study the ferroptosis patterns in SH-SY5Y human neuroblastoma cells, and provide insights to study the mechanism underlying neurotoxicity induced by sodium arsenite (NaAsO₂).

Methods SH-SY5Y cells were divided into a blank control group (with normal medium), a positive control group (with a final concentration of 10 μmol·L^-1 ferroptosis inducer Eratin), three NaAsO₂ exposure groups (with a final concentration of 20, 40, 80 μmol·L^-1 NaAsO₂, respectively), four inhibitor groupswith a final concentration of 20 μmol·L^-1 apoptosis inhibitor Z-VAD-FMK, 10 μmol·L^-1 specific ferroptosis inhibitor Fer-1, 100 μmol·L^-1 specific ferroptosis inhibitor deferoxamine (DFO), and 20 μmol·L^-1 necrotic apoptosis inhibitor Nec-1, respectively, and sixteen intervention groups (four inhibitors were added to a group with the same positive control treatment and three groups with the same NaAsO₂ exposure treatments respectively at the same dose of the inhibitor groups). A total of 25 groups were exposed following the designed protocol for 24 h. The experiment was repeated three times. CCK-8 method was used to detect cell survival rate of each group; corresponding kits were used to detect malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione (GSH) content, glutathione peroxidase (GPXS) activity, and Fe²⁺ content; flow cytometry was used to detect lipid reactive oxygen species (ROS) level.

Results The survival rate of SH-SY5Y cells showed a decreasing trend with the increasing NaAsO₂ exposure concentration (b=-0.984, P < 0.001). The cell survival rates of the 20, 40, and 80 μmol·L^-1 NaAsO₂ exposure groups and the Erastin group were (85.15±1.32)%, (72.63±2.67)%, (65.28±1.71)%, and (74.34±2.07)%, respectively, and were lower than that of the control group (P < 0.01). The Z-VADFMK intervention increased the survival rates of the cells exposed to 20, 40, and 80 μmol·L^-1 NaAsO₂ and the Erastin-treated cells to (88.30±1.92)%, (81.72±2.43)%, (77.72±1.05)%, and (85.28±1.97)%, respectively (P < 0.05), decreased the lipid ROS level and the MDA content (P < 0.05), and elevated the SOD activity (P < 0.05), but did not change the GSH content, GPXS activity, and Fe²⁺ content (P>0.05). The Fer-1 intervention increased the cell survival rates of the 40 μmol·L^-1 and 80 μmol·L^-1 NaAsO₂ exposure groups and the Erastin group to (86.33±2.31)%, (82.24±1.24)%, and (88.76±2.87)%, respectively, and the DFO intervention increased the rates to (82.83±2.55)%, (79.66±0.67)%, and (87.38±1.23)%, respectively (P < 0.01); the two inhibitors also decreased the lipid ROS level and MDA content (P < 0.05), increased the SOD activity, GSH content, and GPXS activity (P < 0.05), and decreased the Fe²⁺ content (P < 0.05). The Nec-1 intervention did not change the cell survival rate, lipid ROS level, MDA content, SOD activity, GSH content, GPXS activity, and Fe²⁺ content in each NaAsO₂ exposure group and the Erastin treatment group (P>0.05).

Conclusion NaAsO₂ exposure can induce apoptosis of SH-SY5Y cells, and with the increase of exposure dose it can induce ferropotsis.