The role of neuroglobin in oxygen-glucose deprivation and reoxygenation-induced mitochondrial depolarization and reactive oxygen species production in SH-SY5Y cells

Abstract

Objective: To investigate the role of neuroglobin (NGB) in oxygen-glucose deprivation and reoxygenation (OGD/R) induced mitochondrial depolarization and reactive oxygen species (ROS)production in a human neuroblastoma cell line (SH-SY5Y). Methods: SH-SY5Y cells were transfected with lentivirus to establish a stable cell line of NGB knockdown (KD). After treated with OGD/R, cells were collected at different time points to analyze NGB mRNA and protein levels. Furthermore, cells were stained with JC-1 and DCFH-DA to evaluate mitochondrial depolarization and ROS production by inverted fluorescence microscope. Also, to determine the neurotoxicity, we measured the lactate dehydrogenase(LDH)level in the cell culture medium. Results: After the treatment of OGD/R, the NGB mRNA and protein started to elevate and peak at 4 h and 8 h (2.04±0.35 fold, 1.69±0.18 fold). Compared with the vector group, NGB KD group had much more mitochondrial depolarization [JC-1 red/green (1.10±0.10) vs (1.46±0.11), P<0.05] and ROS production [DCFH-DA fluorescence (36.30±5.32) vs (16.26±2.97), P<0.05]. Furthermore, NGB KD groups had a higher level of LDH release [(63.42±6.14)%vs (49.65±5.09)%, P<0.05]. Conclusions: NGB plays an important role in the homeostasis of mitochondria. Knockdown of NGB results in increased mitochondrial depolarization, ROS production and neurotoxicity under hypoxia circumstances.