促红细胞生成素保护神经元免受氯胺酮所致的损伤

Erythropoietin protects neuron against ketamine induced injuries

目的研究促红细胞生成素（EPO）是否可以保护神经元免受氯胺酮所致损伤。方法原代培养神经元，分别加入不同浓度的氯胺酮、EPO后培养24h。噻唑蓝（M1Tr）法检测神经元存活率，TdT介导的dUTP缺口末端标记技术（TUNEL）检测凋亡神经元，荧光法测定半胱氨酸蛋白水解酶（caspase）-3活性，蛋白质印迹法检测磷酸化蛋白激酶B（pAkt）的表达。结果l、10、30μmol／L氯胺酮处理组神经元的存活率分别为（91±5）％、（42±6）％和（23±7）％，明显低于对照组（P〈0．05或0．01）；10μmol／L氯胺酮分别加0．3、l、3、10U／mlEPO处理组神经元存活率分别为（73±6）％、（86±9）％、（78±8）％和（71±10）％，明显高于10μmol／L氯胺酮组（P〈0．05或0．01）。10μmol／L氯胺酮组神经元凋亡增加，caspase-3相对活性为（280±60）％，明显高于对照组[（97±15）％，P〈0．01]，而pAkt的表达减少。10μmol／L氯胺酮＋lU／mlEPO组caspase-3相对活性为（130±30）％，明显低于10μmol／L氯胺酮组，而pAkt表达增加。磷酸肌醇3激酶（P13K）抑制剂LY294002拮抗了EPO的作用。结论EPO可以保护神经元免受氯胺酮导致的损伤，其保护作用是通过促进pAkt表达实现的。

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 μmol/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 μmol/L, EPO ＋ ketamine group undergoing the treatment of 10 μmol/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 μmol/L ketamine, 1 U/ml EPO, and 10 μmol/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 μmol/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 I.rmol/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 μmol/L ketamine group （P 〈 0.05 or P 〈 0.01 ）. The number of apoptotic neurons of the 10 μmol/L ketamine group was significantly higher than that pf the control group, the number of apoptotic neurons of the 10 μmol/L ketamine ＋ 10 U/ml EPO group was significantly lower than that of the 10 μmol/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 μmol/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 μmol/L ketamine group was significantly lower than that of the control group （P〈0.05）,the pAkt protein level of the 10 μmol/L ketamine＋10 U/ml EPO group was significantly higher than that of the 10μmol/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＋10U/ml EPO group（P〈0.01）.Conclusion EPO affords significant neuroprotection against ketamine induced injury in neurons via PI3K/Akt-mediated signaling pathway.