程序性坏死在铝致神经母细胞瘤细胞SH－SY5Y死亡中的作用

Role of necroptosis in aluminum induced SH-SY5Y cell death

目的探讨程序性坏死是否存在于铝诱导的神经母细胞瘤细胞SH—SY5Y的死亡途径中。方法培养SH—SY5Y细胞，并用4mmol／L AlCl3·6H2O染毒制作铝致神经细胞死亡模型，按加入Nec-1剂量不同分别设对照组（0μmol／L）和30、60、90μmol／L组，检测SH-SY5Y细胞的活力、线粒体膜电位、活性氧含量及细胞的凋亡率和坏死率等。结果研究表明，加入Nec-1可以显著改善染铝后的细胞坏死样形态学改变。流式细胞仪检测结果表明，加入不同剂量的Nec-1（30、60、90μmol／L）后其细胞活力分别为0．58±0．03、0．68±0．04、1．03±0．17，与对照组（0．28±0．05）相比，差异有统计学意义（t值分别为3．25、3．36、4．56，P值均〈0．05）。Annexin V—PI双染法测定结果显示，不同剂量Nec-1（30、60、90μmol／L）作用后坏死率分别为10．40％±0．64％、5．43％±0．68％、6．28％±0．35％，与对照组（16．46％±0．54％）相比，差异有统计学意义（t值分别为3．62、7．32、6．96，P值均〈0．05），可以明显降低SH-SY5Y细胞的坏死率，但其作用后SH—SY5Y细胞凋亡率分别为7．66％±0．53％、5．68％±0．41％、4．13％±0．41％，与对照组（8．68％±0．36％）相比，差异无统计学意义（F=6．33，P=0．11）。加入不同剂量Nec-1（30、60、90μmol／L）后，其线粒体膜电位分别为49．42±5．96、84．79±6．86、95．51±7．01，60、90μmol／L剂量组与对照组（67．54±6．36）相比，差异有统计学意义（t值分别为3．21、4．01，P值均〈0．05）。但加入不同剂量的Nec-1（30、60、90μmol／L）后其活性氧含量分别为52．79±2．36、54．68±1．91、59．23±2．96，对照组为54．07±3．32，各组间差异无统计学意义（F=5．26，P=0．19）。结论用程序性坏死的特异阻断剂Nec-1可以有效阻断铝致SH—SY5Y细胞的死亡通路，提示程序性坏死是导致铝神经毒性的原因之一。程序性坏死在铝致SH—SY5Y细胞的死亡中发挥了重要作用。

Objective To study whether necroptosis exists or not in neural cell death induced by aluminum. Methods SH-SY5Y cells were treated with 4 mmol/L AlCl3 · 6H2O. The cell viability was determined with CCK-8 kit after treated with Nec-1 at different dosages （0, 30, 60, 90 μmol/L ）. Mitochondria membrane potential （ MMP ）, content of reactive oxygen species （ ROS ）, and apoptotic rate/ necrotic rates were measured with cytometry. Results Nec-1 ameliorated the necrotic-like cell morphology, the cell viability were 0. 28 ± 0. 05,0. 58 ± 0. 03,0. 68 ± 0. 04, and 1.03 ± 0. 17, there were significant differences between the Nec-1 treated groups and that of controls （ t values were 3.25,3.36,4. 56 ; P 〈 0. 05）. After Nec-1 treatment, the necrotic rates were 16.46% ± 0. 54%, 10. 40% ± 0. 64%, 5.43% ± 0. 68% ,and 6. 28% ±0. 35% ,there were significant differences between the Nee-1 treated cells and that of controls（ t values were 3.62,7.32,6. 96;P 〈 0. 05 ）; while the apoptotic rates were 8. 68 ± 0. 36,7. 66 ± 0. 53,5.68 ± 0. 41, and 4. 13 ± 0. 41, there was no significant difference among the groups （ F = 6. 33, P = 0. 11 ）. Cytometry had shown the increased cell MMPs after Nee-1 treatment, which were 67. 54 ± 6. 36, 49.42 ±5.96,84.79±6. 86,and 95.51 ±7.01 ,there were significant differences as comparing MMPs of the middle and high dosage of Nec-1 treated cells with those of controls（t values were 3.21,4. 01 ; P 〈0. 05） ; while ROS contents in the Nec-1 treated SH-SYSY ceils were 54. 07 ± 3.32,52.79 ± 2. 36,54.68 ± 1.91, and 59. 23 ± 2.96, there was no significant difference among the groups （ F = 5.26, P = 0. 19）. Conclusion Nec-1 ,as a specific inhibitor of necroptosis, might effectively block the cell death pathway induced by aluminum,it indicates that necroptosis should be one of the major causes of the SH-SYSY cell toxicity induced by aluminum, and necroptosis also plays an important role in aluminum induced SH-SYSY cell death.