糖氧剥夺再灌注对SH-SY5Y细胞焦亡的影响

The effect of OGD/R on pyroptosis in SH-SY5Y cells

目的探讨细胞焦亡在糖氧剥夺诱导的人神经母细胞瘤细胞(Human neuroblastoma cells, SH-SY5Y)损伤中的作用。方法用不同糖氧剥夺时间(0、3、6、12 h)处理SH-SY5Y细胞,然后进行再灌注24 h;Hoechst33342/碘化丙啶(Propidine iodide, PI)染色试剂盒观察细胞膜破裂情况;原位末端标记法(Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL)/天冬氨酰特异性半胱氨酰蛋白酶-1(Cysteinyl aspartate specific proteinase 1,Caspase-1)共染检测细胞焦亡;蛋白免疫印迹法检测焦亡相关指标[核苷酸结合寡聚化结构导致域样受体蛋白3(Nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)、Caspase-1、Pro-caspase-1、白介素-1β(Interleukin-1β,IL-1β),Pro-IL-1β]的表达水平;使用Caspase-1抑制剂(Belnacasan, VX-765)处理糖氧剥夺/再灌注细胞模型,观察其对细胞焦亡的影响。结果糖氧剥夺再灌注处理呈时间依赖诱导SH-SY5Y细胞损伤,并诱导细胞焦亡,焦亡相关指标(NLRP 3,Caspase-1,IL-1β)表达水平升高。VX-765可减轻糖氧剥夺再灌注诱导的SH-SY5Y细胞焦亡,降低焦亡相关指标(Caspase-1,Pro-IL-1β,IL-1β)的表达水平。结论细胞焦亡在糖氧剥夺再灌注诱导的SH-SY5Y细胞损伤中起到重要作用。

Objective To investigate the role of pyroptosis in SH-SY5 Y cell injury induced by oxygen-glucose deprivation reperfusion(OGD/R).Methods The SH-SY5 Y cells were treated with different time of OGD(0,3,6,12 h), and then were treated with reperfusion for 24 h. Then cell membrane rupture and necrosis were determined by Hochest33342/PI staining, and cell pyroptosis was detected by TUNEL/Caspase-1 co-staining, and the expressions of pyroptosis indicators(NLRP3, Pro-caspase-1, Caspase-1, Pro-IL-1β, IL-1β)were analyzed by western blot.Caspase-1 inhibitory experiment: the SH-SY5 Y cellspretreated with OGD for 6 h were treated with VX-765, which was used to determine cell pyroptosis by TUNEL/Caspase-1 co-staining, and the expressions of pyroptosis indicator(Pro-caspase-1,Caspase-1,Pro-IL-1β,IL-1β)by western blot.Results The OGD/R induced SH-SY5 Y cells injury and pyroptosis, and increased expressions of pyroptosis indicators(NLRP3, Caspase-1, IL-1β).VX-765 reduced the pyroptosis of SH-SY5 Y cells induced by OGD/R, and down-regulated the expressions of pyroptosis indicators(Caspase-1,Pro-IL-1β,IL-1β).Conclusion Pyroptosis plays an important role in SH-SY5 Y cells injury induced by OGD/R.