Relationship between TDAG8 and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration-induced apoptosis in rat neurons

Abstract

Objective To investigate the relationship between T-cell death-associated gene 8 (TDAG8) and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat neurons. Methods The primary cortical neurons obtained from fetal rats were seeded in 6-well plates at a density of 1×105 cells/ml and divided into 5 groups using a random number table: control group (group C, n=24), group OGD/R (n=48), TDAG8 agonist BTB09089 group (group BTB, n=24), TDAG8-siRNA group (group siRNA, n=24), and blank vehicle group (group V, n=24). The medium was replaced with glucose- and serum-free Locke′s buffer, and the neurons were exposed to 95% N2-5% CO2 in an air-tight incubator at 37 ℃ for 60 min followed by routine culture to establish the model of OGD/R.In BTB, siRNA and V groups, 20 μmol/L TDAG8 agonist BTB09089, 200 pmol/L TDAG8-siRNA, and 6 μl/200 μl transfection reagent were added, respectively, at 24 h before oxygen-glucose restoration.At 6 h of oxygen-glucose restoration, the neuronal viability and amount of lactic dehydrogenase (LDH) released were measured, and the expression of TDAG8 and caspase-3 mRNA in neurons was detected by fluorescent quantitative real-time polymerase chain reaction.In group OGD/R, the expression of TDAG8 and caspase-3 was measured by Western blot at 0, 3, 6, 12 and 24 h of oxygen-glucose restoration.In C, OGD/R, BTB, siRNA and V groups, the expression of TDAG8, caspase-3 and p-Akt was detected at 6 h of oxygen-glucose restoration. Results In group OGD/R, the expression of TDAG8 was gradually up-regulated after oxygen-glucose restoration, and the expression of caspase-3 peaked at 6 h of oxygen-glucose restoration.Compared with group C, the neuronal viability was significantly decreased, the amount of LDH released was significantly increased, and the expression of TDAG8 and caspase-3 protein and mRNA and p-Akt was significantly up-regulated in OGD/R, V and siRNA groups (P 0.05). Conclusion TDAG8 is partially involved in the endogenous neuron-protective mechanism against OGD/R-induced apoptosis in rat neurons, which may be related to activation of Akt signaling pathway. Key words: Receptors, G-protein-coupled; Apoptosis; Neurons; Reperfusion injury