褪黑激素对氯胺酮诱导胎鼠海马神经元凋亡的影响

Effect of melatonin on ketamine.induced apoptosis in hippocampai neurons of fetal rats

目的探讨褪黑激素对氯胺酮诱导胎鼠海马神经元凋亡的影响。方法原代培养孕16—18dSD大鼠胎鼠海马神经元5d，采用随机数字表法，将神经元随机分为5组（n=6）：对照组（C组）、氯胺酮组（K组）和不同浓度褪黑激素组（M。组，浓度分别为1．0、2．5和5．0mmol／L），K组加入氯胺酮（终浓度为1000μmol／L）孵育3h，M1-3组给予氯胺酮前60min时加入褪黑激素，使各组褪黑激素浓度分别为1．0、2．5、5．0mmol／L，C组加入等量生理盐水。采用MTY法检测发育期神经元活性，流式细胞仪检测线粒体膜电位（ψm），Westernblot法测定cAMP反应元件结合蛋白[p-CREB（Serl33）]、Bcl．2、Bax、细胞色素C表达。结果与C组相比，K组神经元活性降低，神经元线粒体膜电位（ψm）明显降低（P〈0．05），p-CREB（Serl33）和Bcl．2表达下调，Bax和细胞色素c表达上调（P〈O．05）；与K组相比，M2和M3组神经元ψm升高，M1-3组神经元活性增加，Bcl-2表达上调，Bax和细胞色素c表达下调（P〈0．05），p-CREB（Serl33）表达差异无统计学意义（P〉0．05）。结论褪黑激素通过调节Bcl-2和Bax表达，稳定ψm，抑制线粒体内细胞色素c释放，阻止凋亡复合体的形成，进而抑制氯胺酮诱导胎鼠海马神经元凋亡。

Objective To investigate the effect of melatonin on ketamine-induced apoptosis in hippocam- pal neurons of fetal rats. Methods Sixteen tO eighteen day pregnant Sprague Dawley rats were anesthetized. The fetal rats were obtained under sterile condition and decapitated. The hippocampal neurons were isolated and prima- ry cultured for 5 days. The primary cultured neurons were randomly divided into 5 groups （ n = 6 each） ： control group （group C）, ketamine group （group K）, and 1.0, 2.5 and 5.0 mmol/L melatonin groups （groups M1-3 re- spectively）. Ketamine with the final concentration of 1 000μmol/L was added to the culture medium and the neu- rons were incubated for 3 h in group K. In groups M1-3, 1.0, 2.5 and 5.0 mmol/L melatonin were added to the culture medium, respectively, at 60 min before the addition of ketamine, and the neurons were incubated for 3 h. While the equal volume, of normal saline was added instead in group C. The neuronal viability during the develop- mental phase was assessed by MTY assay. The mitochondrial membrane potential （ψm） was measured by flow cy- tometry. The expression of cAMP response element binding protein phosphorylation （p-CREB （Ser133））, Bcl-2, Bax, and cytochrome C was detected by Western blot. Results Compared with group C, the neuronal viability and ψm were significantly decreased, and the expression of p-CREB and Bcl-2 was down-regulated, while the expres- sion of Bax and cytochrome C was up-regulated in group K （ P 〈 0.05）. Compared with group K, ψm was signifi- cantly increased in groups M2 and M3, and the neuronal viability was significantly increased, the expression of Bcl2 was up-regulated, while the expression of Bax and cytochrome C was down-regulated in groups M1.3 （P 〈 0.05） , Conclusion Melatonin can protect the hippocampal neurons of fetal rats from apoptosis triggered by ketamine via regulating the expression of Bcl-2 and Bax, stabilizing ψm, inhibiting the release of cytochrome C from mitochon- dria, and preventing apoptosome formation.