钙稳态失衡在1-甲基-4-苯基吡啶离子诱导的人神经母细胞瘤SH-SY5Y细胞凋亡中的作用

目的研究钙稳态失衡在1-甲基-4-苯基吡啶离子（MPP+）诱导SH-SY5Y细胞凋亡中的作用。方法MTT法检测细胞存活
率；Hoechst 33342 和Annexin V+PI 染色检测MPP+诱导人神经神经母细胞瘤SH-SY5Y细胞的凋亡作用；Western blot 检测
PARP蛋白表达的变化；罗丹明123 染色检测MPP+对SH-SY5Y细胞线粒体膜电位的影响；激光共聚焦显微镜检测MPP+对
SH-SY5Y细胞胞质钙、线粒体钙和内质网钙浓度的影响以及线粒体钙通道抑制剂钌红与MPP+联合应用对线粒体游离钙浓度、
胞质游离钙浓度的影响。结果MPP+导致SH-SY5Y细胞存活率下降且具有剂量-反应关系、MPP+能导致SH-SY5Y细胞凋亡、
MPP+导致SH-SY5Y细胞线粒体膜电位下降、MPP+使SH-SY5Y细胞胞质和内质网游离钙离子浓度下降而使线粒体游离钙离子
浓度上升。钌红可对抗MPP+诱导的SH-SY5Y细胞凋亡、阻止线粒体膜电位下降，减少PARP的切割片段以及阻断线粒体游离
钙浓度的上升。结论线粒体钙超载在MPP+诱导的SH-SY5Y细胞凋亡中起重要作用。MPP+诱导人神经母细胞瘤SH-SY5Y细
胞的凋亡可能与细胞质、线粒体及内质网的钙稳态失衡有关。

Role of calcium dyshomeostasis in 1-methyl-4-phenylpyridinium ion-induced apoptosis of human neuroblastoma SH-SY5Y cells

Objective To investigate the role of calcium dyshomeostasis in 1-methyl-4-phenylpyridinium ion (MPP+ )-induced
apoptosis of human neuroblastoma SH-SY5Y cells. Methods The viability of SH-SY5Y cells exposed to varying concentrations
of MPP + was assessed using MTT colorimetric assay, and MPP +-induced cell apoptosis was detected with hoechst 33342
staining and Annexin V+ PI assay. Western blotting and rhodamine 123 staining were employed to examine the changes in
cellular poly(ADP-ribose)polymerase (PARP) protein expression and mitochondrial membrane potential in response to MPP+
exposure. The effects of ruthenium red and/or MPP + on calcium concentration in the cytoplasm, mitochondria and
endoplasmic reticulum were evaluated using confocal microscopy. Results MPP+ induced apoptosis and caused reduced cell
viability and mitochondrial membrane potential in SH-SY5Y cells. The cells exposed to MPP + showed a lowered calcium
concentration in the cytoplasm and endoplasmic reticulum and an increased mitochondrial Ca2 + uptake. Ruthenium red
rescued MPP+-induced apoptosis and mitochondrial membrane potential reduction, reduced PARP cleavage, and inhibited the
increase of mitochondrial matrix free Ca2 + in the cells exposed to MPP+ . Conclusion Mitochondrial calcium overload plays an
important role in MPP+-induced apoptosis of SH-SY5Y cells, which is closely associated with dysregulation of intracellular Ca2+
homeostasis.