Hypaconitine confers protection on ketamine-induced neuronal injury in neonatal rat brain via a mechanism involving PI3K/Akt/Bcl-2 pathway

Abstract

Purpose: To investigate the neuroprotective effect of hypaconitine against ketamine-induced neuronal injury in the brains of neonatal rats, and the underlying mechanism of action. Methods: Seven day-old Sprague-Dawley pups weighing 15.0 to 20.0 g (mean weight = 17.5 ± 2.5 g), and aged 7 days were used for this study. The pups were sacrificed, and their forebrains isolated and used to prepare cell suspensions. The isolated cells were treated with ketamine (100 µM) or varied concentrations of hypaconitine (0.1 – 2 µM) or LY294002 (10 µM). The cells were trypsinized and cultured at 37 °C in 10 % fetal bovine serum (FBS) supplemented Dulbecco's modified Eagle's medium (DMEM) in a humidified incubator containing 5 % CO2. Cell viability was determined using MTT assay, while TUNEL assay was used to determine the extent of apoptosis in the cells. The expressions of pAkt, Bcl-2 and caspase-3 were determined using Western blotting. Results: There were only few viable cells in the ketamine-treated group, and cell viability was significantly and dose-dependently increased in hypaconitine-treated groups (p < 0.05). The extent of apoptosis was significantly higher in ketamine-treated cells than in control cells, but treatment with hypaconitine significantly reduced the number of apoptotic cells (p < 0.05). However, in the presence of LY294002 (a PI3K-specific inhibitor), the effect of hypaconitine on neuronal cell apoptosis was significantly reversed (p < 0.05). The expressions of p-Akt and Bcl-2 were significantly down-regulated while the expression of caspase-3 was significantly upregulated in ketamine-treated neuronal cells, when compared with control group (p < 0.05). However, in cells treated with hypaconitine, the expressions of p-Akt and Bcl-2 were significantly upregulated, while the expression of caspase-3 was significantly down-regulated (p < 0.05). Treatment of neuronal cells with hypaconitine in the presence of LY294002 significantly reversed the effect of hypaconitine on the expressions of p-Akt, Bcl-2 and caspase-3 (p < 0.05). Conclusion: These results suggest that hypaconitine ameliorates ketamine-induced neuronal injury in neonatal rats via a mechanism involving the PI3K/Akt/Bcl-2 pathway.