亚砷酸钠诱导SH-SY5Y细胞铁死亡的规律
Ferroptosis patterns in SH-SY5Y cells induced by sodium arsenite
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摘要:
背景 砷可通过诱导神经元丢失导致神经毒性，但其是否可诱导神经细胞铁死亡尚不清楚。
目的 探讨亚砷酸钠（NaAsO2）诱导人神经母细胞瘤细胞SH-SY5Y铁死亡发生规律，为研究NaAsO2神经毒性发生机制提供依据。
方法 选用SH-SY5Y细胞，将实验分为空白对照组（加入正常培养基）、阳性对照组（加入终浓度为10 μmol·L-1的铁死亡诱导剂Erastin）、NaAsO2染毒组（NaAsO2终浓度分别为20、40、80 μmol·L-1）、抑制剂组终浓度20 μmol·L-1凋亡抑制剂Z-VAD-FMK，10 μmol·L-1特异性铁死亡抑制剂Fer-1，100 μmol·L-1特异性铁死亡抑制剂去铁胺（DFO），20 μmol·L-1坏死性凋亡抑制剂Nec-1、干预组（在阳性对照组及NaAsO2各染毒组基础上分别加入4种抑制剂，剂量同抑制剂组），共25组，染毒24 h。实验重复3次。采用CCK-8法检测各组细胞存活率；试剂盒检测细胞内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、谷胱甘肽（GSH）含量、谷胱甘肽过氧化物酶（GPXS）活性及Fe2+含量；流式细胞仪检测脂质活性氧（ROS）水平。
结果 随NaAsO2染毒剂量增加，SH-SY5Y细胞存活率呈下降趋势（
b =-0.984，P < 0.001）；20、40、80 μmol·L-1 NaAsO2染毒组及Erastin组细胞存活率（85.15±1.32）%、（72.63±2.67）%、（65.28±1.71）%、（74.34±2.07）%均明显低于对照组存活率（P < 0.01）。Z-VAD-FMK干预使20、40、80 μmol·L-1 NaAsO2染毒组及Erastin处理组细胞存活率上升至（88.30±1.92）%、（81.72±2.43）%、（77.72±1.05）%、（85.28±1.97）%（P < 0.05），脂质ROS、MDA含量下降（P < 0.05），SOD活性上升（P < 0.05），但GSH含量、GPXS活性、Fe2+含量均无明显变化（P >0.05）。Fer-1、DFO干预使40 μmol·L-1和80 μmol·L-1 NaAsO2染毒组、Erastin处理组细胞存活率分别上升至（86.33±2.31）%、（82.24±1.24）%、（88.76±2.87）%和（82.83±2.55）%、（79.66±0.67）%、（87.38±1.23）%（P < 0.01），脂质ROS水平、MDA下降（P < 0.05），SOD活性上升（P < 0.05），同时GSH含量、GPXS活性上升（P < 0.05），Fe2+含量降低（P < 0.05）。Nec-1干预NaAsO2各染毒组及Erastin处理组后，细胞存活率、脂质ROS水平、MDA含量、SOD活性、GSH含量、GPXS活性及Fe2+含量均无明显变化（P >0.05）。结论 NaAsO2暴露可诱导SH-SY5Y细胞凋亡，随着暴露剂量的增加可诱导细胞铁死亡。
Abstract: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.
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