Duan Qiuhong, Wang Zhongqiang, Lu Tao, Chen Juan, Wang Ximing
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Pineal Res. 2006 Nov;41(4):351-7. doi: 10.1111/j.1600-079X.2006.00374.x.
The protective effect of exogenous melatonin or 6-hydroxylmelatonin on neurons was examined in N2a cells following exposure to oxygen-glucose-serum deprivation insults. After N2a cells cultured in vitro were deprived of glucose, serum and oxygen for 90 min, the different concentrations of melatonin or 6-hydroxylmelatonin were added to the medium. Then, treated cells were cultured for different intervals. At the end of the treatment, the collected culture solution was used for the analysis of the activity of lactate dehydrogenase (LDH) and the cells were used for the examination of the following parameters: cell viability (MTT), DNA fragmentation, reactive oxygen species (ROS) production, mitochondrial transmembrane potential, cytochrome C and caspase 3 activity. The results show that melatonin and 6-hydroxylmelatonin both reduced oxygen-glucose-serum deprivation-mediated N2a cell apoptosis, but they could not completely inhibit the apoptosis of the cells and the inhibitory effect of melatonin was stronger than that of 6-hydroxylmelatonin. Both of them could inhibit LDH and cytochrome C release and caspase 3 activity. Although 6-hydroxylmelatonin could no longer maintain mitochondrial transmembrane potential 6 h after reperfusion, its inhibitory effect on cytochrome C release from mitochondria and the scavenging role of ROS were stronger than those of melatonin. Moreover, melatonin promoted ROS production at the 15th min of the reperfusion, and then it began to remove ROS from cells. Our study showed that melatonin and 6-hydroxylmelatonin can be used as supplements in the treatment of neurological disorders involving oxidative stress. Melatonin serves as more than a ROS scavenger and its other roles await further study.
在N2a细胞暴露于氧-葡萄糖-血清剥夺损伤后，检测外源性褪黑素或6-羟基褪黑素对神经元的保护作用。体外培养的N2a细胞在无糖、无血清和无氧条件下培养90分钟后，向培养基中加入不同浓度的褪黑素或6-羟基褪黑素。然后，对处理后的细胞进行不同时间的培养。在处理结束时，收集培养液用于分析乳酸脱氢酶（LDH）活性，细胞用于检测以下参数：细胞活力（MTT）、DNA片段化、活性氧（ROS）产生、线粒体跨膜电位、细胞色素C和半胱天冬酶3活性。结果表明，褪黑素和6-羟基褪黑素均能减少氧-葡萄糖-血清剥夺介导的N2a细胞凋亡，但不能完全抑制细胞凋亡，且褪黑素的抑制作用强于6-羟基褪黑素。二者均能抑制LDH和细胞色素C释放以及半胱天冬酶3活性。虽然6-羟基褪黑素在再灌注6小时后不能再维持线粒体跨膜电位，但其对线粒体细胞色素C释放的抑制作用和对ROS的清除作用强于褪黑素。此外，褪黑素在再灌注第15分钟时促进ROS产生，然后开始从细胞中清除ROS。我们的研究表明，褪黑素和6-羟基褪黑素可作为涉及氧化应激的神经疾病治疗的补充剂。褪黑素不仅仅是一种ROS清除剂，其其他作用有待进一步研究。
