Objective: To investigate the protective effects of lentivirus mediated Bcl-2-associated athanogene 1L (BAG-1L) over-expression on human neuroblastoma cells (SH-SY5Y) induced by hypoxia/re-oxygenation, and to study its effect on the phosphoinositide 3 kinase serine/threonine protein kinase (PI3K/AKT) pathway.
Methods: SH-SY5Y cells were cultured in vitro, and the cells at logarithmic phase were collected, and they were divided into recombined lentiviral infection group [infected by lentivirus containing BAG-1L and green fluorescent protein (GFP) gene], vector control group (infected by lentivirus containing GFP without BAG-1L gene) and cell control group (non-infection). Western Blot was used to detect the expression of BAG-1L in target cells after infection for 48 hours. SH-SY5Y cells were subjected to hypoxia for 8 hours and re-oxygenation for 24 hours, then the cell counting kit-8 (CCK-8) was used to detect the cell activity, and the apoptosis was detected by flow cytometry after allophycocyanin labeled annexin V/7-amino actinomycin D (Annexin V-APC/7-AAD) staining. Western Blot was used to detect the protein expressions of BAG-1L, heat shock protein 70 (HSP70), AKT and phosphorylated AKT (p-AKT).
Results: After infection for 48 hours, exogenous BAG-1L protein bands were observed in recombined lentiviral infection group, but not observed in cell control group and vector control group. After hypoxia/re-oxygenation treatment, the cell viability in recombined lentiviral infection group was significantly higher than that in cell control group and vector control group (A value: 0.689±0.036 vs. 0.425±0.013, 0.400±0.012), apoptosis was significantly decreased [apoptosis rate: (26.97±1.82)% vs. (36.60±1.45)%, (35.77±3.74)%], the protein levels of BAG-1L, HSP70 and p-AKT were significantly increased [BAG-1L protein (gray value): 2.405±0.167 vs. 0.529±0.141, 0.601±0.099; HSP70 protein (gray value): 0.997±0.123 vs. 0.634±0.091, 0.584±0.106; p-AKT protein (gray value): 1.234±0.118 vs. 0.661±0.210, 0.712±0.199, all P < 0.01], but the protein level of AKT was slightly increased (gray value: 1.103±0.269 vs. 0.646±0.188, 0.791±0.326) without statistically significant differences (both P > 0.05). There was no significant difference in all parameters between cell control group and vector control group (all P > 0.05).
Conclusions: Lentivirus mediated BAG-1L gene over-expression can protect nerve cells against hypoxic injury and apoptosis, and the protective effect may be related to the activation increase of pathway on PI3K/AKT.