Wang Xiaolong, Chen Jiajun, Wang Hongbo, Yu Hao, Wang Changliang, You Jiabin, Wang Pengfei, Feng Chunmei, Xu Guohui, Wu Xu, Zhao Rui, Zhang Guohua
Department of Forensic Pathology, School of Forensic Medicine, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, 110122, Liaoning Province, People's Republic of China.
J Mol Neurosci. 2017 Aug;62(3-4):402-411. doi: 10.1007/s12031-017-0948-3. Epub 2017 Jul 20.
Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.
半胱天冬酶 -3激活和细胞凋亡与多种神经退行性疾病相关。钙激活是促进细胞凋亡的一个重要因素。因此，我们评估了细胞内钙在乙醇诱导的H4人神经胶质瘤细胞半胱天冬酶 -3激活中的作用，以及N-甲基-D-天冬氨酸（NMDA）受体拮抗剂美金刚对乙醇诱导的H4细胞凋亡的保护作用。H4细胞用100 mM乙醇（在培养基中）处理2天。为了进行相互作用研究，在乙醇处理前，细胞先用美金刚（4 μM）、乙二胺四乙酸（EDTA，1 mM）或1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM，10 μM）处理。使用RNA干扰技术敲低编码NMDA受体NR1亚基的基因。通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色和流式细胞术检测细胞凋亡。使用MTS细胞增殖试剂盒检测细胞活力。采用荧光双波长分光光度法测定细胞内钙浓度。通过蛋白质印迹法检测NR1、半胱天冬酶 -3、肌醇三磷酸受体1（IP3R1）和肌浆网钙ATP酶1（SERCA1）蛋白水平。通过定量聚合酶链反应（qPCR）检测NR1、IP3R1和SERCA1 mRNA水平。我们观察到，暴露于乙醇的H4细胞中NR1、IP3R1、SERCA1的表达增加，细胞内钙离子水平升高。此外, 钙螯合剂EDTA和BAPTA以及NMDA受体的RNA干扰破坏降低了乙醇诱导的H4细胞半胱天冬酶 -3激活。美金刚处理降低了乙醇诱导的细胞内钙增加、半胱天冬酶 -3激活、细胞凋亡以及乙醇诱导的细胞活力下降。我们的结果表明，乙醇诱导的半胱天冬酶 -3激活和细胞凋亡可能依赖于胞质钙水平，并且美金刚处理可以降低它们。
