Inhibition of Arginyl-tRNA Synthetase Promotes the Protection of Neuronal Ischemic Tolerance in Vitro Author Imformation
Abstract BackgroundsStudies have shown that the metabolic rate of mammals decreases during hibernation, suggesting that effective reduction of the energy consumption of ischemic cells may be the basis of the protective effect of ischemic tolerance. Anderson et al. reported after inhibition of the gene encoding arginyl-tRNA synthetase, the protein translation and mortality in C. elegans under anoxic conditions decreased significantly.PurposesWhether inhibition of arginyl-tRNA synthetase (RARS), in addition to combating hypoxic injury in C. elegans, protects rat neurons from ischemic damage remains unknown. The aim of this study is to determine whether knockdown of arginyl-tRNA synthetase improves the tolerance of primary cultured rat neurons to ischemic anoxia. Methods For the primary neuronal cutlure, cerebral cortex tissues were collected from newborn 24 hours Sprague-Dawley rats. Different viral vectors were transfected into cultured primary neurons, and the optimal viral vector and time points for gene silencing were determined by detecting the expression of RARS gene and protein. The adenovirus vector expressing shRNA-RARS with the highest silencing efficiency was transfected into rat primary cultured cortical neurons. The time point at which the viral vector exhibits optimal gene silencing efficiency was selected as the detection time point for subsequent experiments. The neurons after OGD treatment were divided into 2 groups: the experimental group and the control group. Each group was divided into 3 subgroups: the normal group, the control shRNA group and the shRNA-RARS group. After 3 hours of OGD treatment, cell survival, ATP levels and RARS protein expression were evaluated , and the data was analyzed. ResultsPrimary cultured neurons were identified and purified. The adenoviral vector expressing RARS-RNAi (27394-1) exhibited the highest silencing efficiency of the three adenoviral vectors, and 3 days after transfection of the adenoviral vector was selected as the optimal time point for gene silencing. Transfection of adenoviral vectors expressing shRNA-RARS reduced RARS protein expression, ATP consumption and neuronal death, and increased CCK8 activity in neurons after OGD insult.Conclusions Our work revealed neurons transfected with adenoviral vectors expressing shRNA-RARS exhibited stronger tolerance to ischemia and hypoxia, which was due to the inhibition of RARS activity andreduced cell energy metabolism rate. These results suggested that RARS inhibition reduced protein translation and energy consumption, and played a protective role in ischemic tolerance.