Objective: To investigate whether erythropoietin (EPO) protects neuron against ketamine induced injuries.
Methods: Neurons were obtained from SD rat brain, cultured, and treated with ketamine of the concentrations of 0.1, 1, 10, and 30 micromol/L respectively. Neurons not treated by any agent were used as control group. Another neurons were divided into 3 groups undergoing the treatment of ketamine of the terminal concentration of 10 micromol/L, EPO + ketamine group undergoing the treatment of 10 micromol/L ketamine and EPO of the terminal concentrations of 0.3, 1, 3, and 10 U/ml, and ketamine + EPO + LY294002 group undergoing the treatment of 10 micromol/L ketamine, 1 U/ml EPO, and 10 micromol/L LY294002, a P13k inhibitor. Twenty-four hours after the co-inoculation the survival rates of the neurons were detected by MTT method. The apoptotic rate was detected by TUNEL assay. The neuron vitality was measured by MTT assay. Apoptotic neurons were measured by TUNEL assay. The activity of caspase-3 was detected with the caspase-3 fluorometric assay kit. The level of pAkt protein was analyzed by Western blotting.
Results: The survival rates of the neurons exposed to ketamine of the concentrations of 1, 10, and 30 micromol/L were (91 +/- 5)%, (42 +/- 6)%, and (23 +/- 7)% respectively, significantly lower than that of the control group (P < 0.05 or P < 0.01). The survival rates of the neurons treated by 10 micromol/L ketamine and EPO of the concentrations of 0.3, 1, 3, and 10 U/ml were (73 +/- 6)%, (86 +/- 9)%, (78 +/- 8)%, and (71 +/- 10)% respectively, all significantly high than that of the 10 micromol/L ketamine group (P < 0.05 or P < 0.01). The number of apoptotic neurons of the 10 micromol/L ketamine group was significantly higher than that pf the control group, the number of apoptotic neurons of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly lower than that of the 10 micromol/L ketamine, and the number of apoptotic neurons of the ketamine + EPO + LY294002 group was (130 +/- 30)%, remarkably lower than that of the ketamine group. (P < 0.01), and the relative activity of caspase-3 of the 10 micromol/L ketamine group was (280 +/- 60)%, significantly higher than that of the control group, (P < 0.01). The relative activity of caspase-3 of the ketamine + EPO + LY294002 group was (220 +/- 34)%, significantly higher than that of the ketamine + EPO (P < 0.01). The pAkt protein level of the 10 micromol/L ketamine group was significantly lower than that of the control group (P < 0.05), the pAkt protein level of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly higher than that of the 10 micromol/L ketamine group (P < 0.01), and the pAkt protein level of the ketamine + EPO + LY294002 group was significantly lower than that of the ketamine + 10 U/ml EPO group (P < 0.01).
Conclusion: EPO affords significant neuroprotection against ketamine induced injury in neurons via PI3K/Akt-mediated signaling pathway.