Aim: To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress. Methods: A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/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 N2a cells. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays. Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls. In N2a/Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3γ decreased compared with that of N2a/vec cells. Transfection of 14-3-3γ plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls. Conclusion: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3γ expression.

Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen–glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia, providing novel therapeutic targets for ischemic stroke as well as other related neurological diseases.

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Background Intracellular bacteria, especially Mycobacterium tuberculosis, are important pathogenic microorganisms that endanger human health. Purified and synthesized cecropin A‐magainin 2 (CAMA‐syn) can exhibit a higher antibacterial activity and lower cytotoxicity. To enhance such antimicrobial potential, it would be desirable to deliver CAMA‐syn expressed in lung epithelial cells by an adenovirus vector using gene therapy. Methods A549 cells in vitro and lung epithelial cells in vivo were used to express CAMA‐syn by transducing recombinant adenovirus Ad‐SPC‐CAMA/GFP, and the expression of CAMA‐syn was determined by a reverse transcriptase‐polymerase reaction (RT‐PCR) and immunofluorescence. The antimicrobial activity in cells was investigated by colony‐forming rate and growth curve. Forty Kunming mice of a Bacillus Calmette‐Guerin (BCG) infection animal model were randomly divided into three groups: adenoviruses delivery of Ad‐SPC‐CAMA/GFP, Ad‐CMV‐CAMA/GFP and empty‐virus Ad‐CMV‐GFP. The expression of CAMA‐syn in mice was confirmed by RT‐PCR and immunofluorescence. After tracheal injection of adenoviral vector for 3 days, lungs from the mouse model were extracted and homogenized for detection of colony‐forming efficiency. Results CAMA‐syn expressed in lung epithelial cells A549 conferred antimicrobial activity against a series of bacteria, including Salmonella abortusovis and BCG. The results obtained in vivo showed that the colony‐forming rate of Ad‐SPC‐CAMA/GFP (74.54%) and Ad‐CMV‐CAMA/GFP (62.31%) transduced into mice was significantly lower than that of the control group. Conclusions Lung epithelial‐specific expression of antimicrobial peptide CAMA‐syn mediated by adenovirus suppressed the growth of intracellular bacteria, providing a promising approach for the control of refractory intracellular infection.

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