氯胺酮和咪达唑仑对谷氨酸致大鼠神经元样PC12细胞损伤的作用

Effects of ketamine and midazolam on glutamic acid-induced injury to rat neuronal PC12 cells

目的 探讨氯胺酮和咪达唑仑对谷氨酸致大鼠神经元样PC12细胞损伤的作用。方法 培养6-7d的神经元样PC12细胞接种于培养板培养18h后随机分为正常对照组（C组），谷氨酸组（G组），0．1、0．5、1．0mmol／L氯胺酮组（K1组-K3组），1、5、10、25、50μmol／L咪达唑仑组（M1组～M5组），0．1、0．5、1．0mmol／L氯胺酮混合10μmol／L咪达唑仑组（KM1组-KM3组）。应用MTT比色法和乳酸脱氢酶（LDH）活性检测评估细胞活力及细胞损伤程度；RT-PCR法测定c-fos mRNA表达水平；细胞免疫组化法测定Fos蛋白表达水平。结果 （1）与C组比较，除K3组外，其余各组细胞活力均降低（P〈0．05）；与G组比较，K1组。K3组、M1组-15组细胞活力均增强（P〈0．05或0．01），LDH漏出率均降低（P〈0．01）；与K1组比较，KM1组细胞活力增加，LDH漏出率降低（P〈0．05）；KM2组与K2组比较，细胞活力、LDH漏出率无明显差异（P〉0．05）。与K3组比较，KM3组细胞活力降低，LDH漏出率升高（P〈0．01）。（2）与C组比较，除K3组外，其余各组c-fos mRNA的表达均上调（P〈0．05）。与G组比较，K1组-K3组、M3组、KM2组c-fos mRNA的表达均下调（P〈0．05）；与K2组、M3组比较，KM2组c-fos mRNA的表达上调（P〈0．05）。（3）与C组比较，除K3组外，其余各组Fos染色阳性胞核数目增加，平均光密度值升高（P〈0．01）。与G组比较，K1组-K3组、M3组、KM2组Fos染色阳性胞核数减少，平均光密度值降低（P〈0．05或0．01）。与K2组、M3组比较，KM2组Fos染色阳性胞核数增加（P〈0．05），平均光密度值升高（P〈0．05）。结论 氯胺酮对谷氨酸损伤的神经元样PC12细胞具有剂量依赖性的保护作用，咪达唑仑也有神经保护作用；低剂量氯胺酮和咪达唑仑混合对神经的保护作用增强，而高剂量氯胺酮和咪达唑仑有对抗作用。

Objective To investigate the effects of ketamine and midazolam on glutamic acid-induced injury to neuronal PC12 ceils. Methods The differentiated PC12 cells purchased from Chinese Academy of Neuropharmacology Research Institute were cultured in RPMI 1640 culture medium in CO2 incubator at 37℃ for 6- 7 days and then inoculated on 96-well plates （5 ×10^4 cells/well） and randomly allocated into 5 groups： group Ⅰ control （C）; in group Ⅱ-Ⅴ the PC12 cells were incubated with glutomic acid 10 mmol/L （group Ⅱ , G）; with ketamine0.1/0.5/1.0 mmol/L ＋ glutamic acid 10 mmol/L （group m K1-K3）; with midazolam 1/5/10/25/50 μmol/L＋ glutamic acid 10 mmol/L （group Ⅳ M1-5 ） and with ketamine 0.1/0.5/1.0 mmol/L ＋ midazolam 10 μmol/L ＋ glutamic acid 10 mmol/L （groupV KM1-KM3） for another 18 h. The ceil viability was determined by MTI＂ assay （Sigma Co, USA）. LDH activity in supernatant of culture medium and ceils was measured using LDH detection kit （Nanjing Jian-Cheng Bioengineering Co）. c-fos mRNA expression was determined by RT-PCR and c-fos protein expression by immuno-histochemical method. Results （ 1 ） The viability of PC12 ceils was significantly reduced by the presence of glutamic acid in group Ⅱ- V except in group K3 as compared with control group （ Ⅰ ） Ketamine 0.1-1.0 mmol/L in group K1-K3 and midazolam 1-50 μmol/L in group M1-M5 attenuated glutamic acid-induced ceil injury in a dose-dependent manner （P 〈 0.05, 0.01 ） and significantly reduced LDH release as compared with group G （ Ⅱ ） （ P 〈 0.01 ）. The ceil viability was significantly increased and LDH release was significantly decreased in group KM1 as compared with group K1, but the cell viability was significantly decreased and LDH release was significantly increased in group KM3 as compared with group K3. There was no significant difference in cell viability and LDH release between group KM2 and K2. （2） The expression of c-fos mRNA and the number of the cells with c-fos staining positive nucleus were significantly increased in group G （ Ⅱ ）, group K1, K2, group M3 and KM2 as compared with control group and were significantly lower in group K1-K3, M3 and KM2 than in group G （ Ⅱ ）. Conclusion Ketamine as a non-competitive NMDA receptor antagonist can protect PC12 cells from glutamic acid-induced injury. Midazolam is also neuroprotective. Low dose ketatnine and midazolam can interact to enhance the neuroprotective effect, while high dose ketamine and midazolam may be antagonistic.