南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (5): 702-709.doi: 10.12122/j.issn.1673-4254.2021.05.10

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热打击可通过调控内质网应激通路促进神经细胞凋亡

高经华，刘亚伟，吉晶晶，刘志锋   

  • 出版日期:2021-05-20 发布日期:2021-06-11

Heat stress induces neuronal apoptosis by up-regulating endoplasmic reticulum stress pathway

  • Online:2021-05-20 Published:2021-06-11

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摘要：

目的 研究内质网应激通路在热打击导致神经元细胞凋亡的作用。方法 体外培养神经元SH-SY5Y细胞，设置正常对照组和实验组。正常对照组：始终37 ℃培养，实验组：细胞培养箱设置43 ℃，热打击2 h后，置回37 ℃细胞培养箱复温0、3、6 h。光学显微镜观察细胞形态变化；CCK8法检测细胞活力；流式细胞术检测热打击后细胞凋亡情况;流式细胞术和免疫荧光共聚焦显微镜检测和观察细胞内钙离子水平；qRT-PCR检测Caspase-12、BIP和XBP-1的RNA表达水平；Western blot检测内质网应激通路相关蛋白Caspase-12、BIP、P-JNK、JNK和XBP-1的蛋白表达；利用4-苯基丁酸(4-PBA)预处理后，检测热打击后细胞凋亡水平。结果 热打击后复温0 h，细胞收缩明显，随着复温时间延长，细胞逐渐伸展；热打击后细胞活力明显下降（P=0.001）；热打击后细胞内钙离子显著上调并随复温时间延长而恢复；热打击后细胞Cleaved-caspase3蛋白水平增加，内质网应激通路相关蛋白Caspase-12（P=0.002）和BIP（P=0.008）蛋白水平和mRNA水平增加并有时间依赖性，P-JNK/JNK蛋白水平在复温0 h显著增加（P=0.003），并随着复温时间延长而逐渐减少。XBP-1蛋白和mRNA水平在热打击后逐渐减少（P=0.005，P=0.002），而4-PBA处理组，细胞中Cleaved-caspase3表达水平明显降低。结论 热打击可通过诱发内质网应激及促进胞内钙离子稳态失衡而促进神经细胞发生凋亡。

关键词: 热打击；内质网应激；钙离子；凋亡

Abstract: Objective To explore the role of endoplasmic reticulum stress in heat stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cells were incubated at 43 ℃ for 2 h followed by further culture at 37 ℃ for 0, 3 h, or 6 h. With the cells cultured at 37 ℃ as the control, the cells exposed to heat stress were examined for morphological changes under optical microscope and changes in cell viability using CCK-8 assay. Flow cytometry was performed for detecting apoptosis of the cells following heat stress, and intracellular Ca2 + level in the cells was determined using flow cytometry and immunofluorescence confocal microscopy. The mRNA expression levels of caspase-12, BIP and XBP-1 in the cells were detected using qRT-PCR, and the protein expressions of caspase-12, BIP, P- JNK, JNK and XBP-1 were examined using Western blotting. The effect of pretreatment with 4-PBA on cell apoptosis following heat stress was analyzed with Western blotting. Results SH-SY5Y cells showed obvious cell shrinkage immediately after the exposure to heat stress, followed then by gradual cell stretching over time. The cell viability decreased significantly after heat stress (P=0.001), and the intracellular Ca2+ level increased significantly at 0 h and gradually recovered the normal level at 3 and 6 h. Heat stress induced significant increase in the protein expression of cleaved caspase-3 and time-dependent increase of caspase-12 (P=0.002) and BIP (P=0.008) expression at both the protein and mRNA levels. The expression of P-JNK/JNK protein increased significantly at 0 h (P=0.003) followed by gradual decrease; the expression levels of XBP-1 protein and mRNA gradually decreased after heat stress (P=0.005, P=0.002). Pretreatment with 4-PBA significantly reduced the expression level of cleaved caspase-3 in SH-SY5Y cells following heat stress. Conclusion Heat stress induces apoptosis of SH- SY5Y cells by triggering endoplasmic reticulum stress and the imbalance of intracellular calcium ion homeostasis.

Key words: heat stress; endoplasmic reticulum stress; Ca2+; apoptosis