吗啡对原代培养大鼠皮质神经元神经甾体水平的影响

目的探讨吗啡慢性处理对大鼠大脑皮质神经元合成释放神经甾体水平的影响及其机制。方法建立原代培养大鼠大脑皮质神经元吗啡依赖样模型,固相萃取结合高效液相色谱-质谱联用法测定细胞培养液中神经甾体水平;免疫印迹法检测神经元中p-CREB水平。结果与盐水对照组相比,吗啡(1μmol.L-1)处理使PREG和DS的水平降低(P<0.01);μ-受体激动剂DAMGO使PREG、DS和PS水平下降,AP水平增高;与吗啡单独处理组相比,μ-阿片受体拮抗剂CTAP与吗啡联合处理使PREG增加(P<0.01)。与盐水对照组相比,吗啡或DAMGO慢性处理均可增加神经元内p-CREB的水平(P<0.01);与吗啡处理组相比,CTAP抑制吗啡诱导的p-CREB的增加。结论μ-阿片受体参与了介导吗啡慢性处理对皮质神经元神经甾体合成释放的影响;神经元内p-CREB水平的变化反映了吗啡引起的神经元适应性改变,提示神经甾体水平的变化可能与吗啡依赖有关。

Effects of morphine on neurosteroidogenesis of primary cultured rat cerebral cortical neurons

Aim To investigate the effect and mechanism of action of chronic morphine treatment on the neurosteroidogensis of primary cultured rat cerebral cortical neurons(CCNs).Methods The effect of morphine on the production of pregnenolone(PREG),dehydroepiandrosterone(DHEA),allopregnanolone(AP),pregnenlolone sulfate(PS)and dehydroepiandrosterone sulfate(DS)in cell culture media was measured by solid-phase extraction and LC-MS,with methyltestosterone(MT)or estrogen sulfate(ES)as internal standards.The dependence-like changes of CCNs were determined by testing the p-CREB level in the nuclear lysates using western blot.Results Compared with the control group,morphine treatment significantly reduced levels of PREG and DS respectively;opioid agonist DAMGO significantly reduced levels of PREG,DS and PS respectively,and increased the level of AP significantly.Compared with the morphine group,μ-opioid-antagonist CTAP concomitant with morphine increased the level of PREG significantly.Compared with the control group,chronic morphine treatment or DAMGO treatment significantly increased the level of p-CREB in the CCNs.Compared with the morphine treatment group,μ-antagonsit CTAP significantly reduced the level of p-CREB.Conclusion μ-opioid receptor mediated the inhibitory effect of morphine on levels of neurosteroids,and changes of neurosteroid levels might be related to morphine dependence.