Acta Pharmacologica Sinica (2009) 30: 913-918; doi: 10.1038/aps.2009.70

Aim: To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress. 

Methods: A stable N 2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N 2a /Ngb-siRNA) was established by neomycin screening.  Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels.  Hydrogen peroxide was used to induce oxidative stress in N 2a cells.  Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays.  Apoptotic cells were detected by Hoechst staining.

Results: Cotransfection of Ngb-siRNA with Ngb-GFP plasmids suppressed the expression of Ngb-GFP in N 2a cells.  RT-PCR and Western blot analysis showed that the expression of endogenous Ngb was successfully knocked down to about 20% in N 2a /Ngb-siRNA cells compared with control cells.  A WST-8 assay demonstrated that viability was significantly decreased in N 2a /Ngb-siRNA cells and N 2a cells transiently transfected with Ngb-siRNA plasmids compared with controls following hydrogen peroxide treatment.  An LDH assay demonstrated a time-dependent increase in the death of Ngb-siRNA-transfected N 2a cells following hydrogen peroxide treatment.  Hoechst staining demonstrated that the quantity of apoptotic cells among N 2a /Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls.  In N 2a /Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3 γ decreased compared with that of N 2a /vec cells.  Transfection of 14-3-3γ plasmids significantly enhanced the viability of N 2a /Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls.

Conclusion: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3 γ expression.

Keywords: neuroglobin; siRNA; H₂O₂; 14-3-3 γ; oxidative stress; N 2a cells