褪黑素和6-羟褪黑素保护神经细胞抗缺血再灌注损伤比较研究

Protective Effect of Melatonin and 6-Hydroxylmelatonin on Neurons After Exposure to Oxygen-Glucose Serum Deprivation

目的研究褪黑素(Mel)和6-羟褪黑素(6-OHMel)神经保护作用及作用机理。方法体外培养N2a细胞,模拟缺血再灌注(OGSD),加入Mel和6-OHMel,检测以下指标:①细胞生存能力:MTT法、乳酸脱氢酶释放;②细胞凋亡分析:DNA片断化,细胞色素C,Caspase3活性;③活性氧(ROS)和线粒体跨膜电位。结果①Mel和6-OHMel都能减轻OGSD诱导的N2a细胞损伤,Mel的作用强于6-OHMel。②Mel和6-OHMel均能抑制细胞色素C释放,但6-OHMel强于Mel。③Mel和6-OHMel都能稳定线粒体跨膜电位,但Mel作用时间比6-OHMel长。④Mel和6-OHMel能清除ROS,6-OHMel表现为直接作用,Mel表现为间接作用。⑤Mel和6-OHMel均能抑制caspase3的活性,但是作用时间不同。6-OHMel表现在OGSD后12h,Mel在OGSD后24h。结论Mel和6-OHMel的神经保护作用与其抗氧化、稳定线粒体功能相关,Mel的作用机制更复杂。

Objectives To study the protective effect of exogenous melatonin （Mel） and 6-hydroxylmelatonin （6-OHMel） on neurons following exposure to oxygen-glucose-serum deprivation （OGSD） insults. Methods After N2a cells cultured in vitro were deprived of glucose-oxygen serum for 90 min, Mel or 6-OHMel were added to the medium. Then, the treated cells were cultured for different time. At the end of the culturing, 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 cell ability, DNA fragmentation, reactive oxygen species （ROS） production, mitochondrial transmembrane potential, cytochrome C and caspase 3 activity. Results The Mel and 6-OHMel reduced N2a cell apoptosis induced by OGSD. The inhibitory effect of Mel on the cell apoptosis was stronger than that of 6-OHMel. Both of them could inhibit LDH and cytochrome C release and caspase 3 activity. Although 6-OHMel could no longer maintain mitochondrial transmembrane potential 6 hours after OGSD, its inhibitory effect on cytochrome C release from mitochondria and the scavenging effect on ROS were stronger than those of Mel. Condusions The protective effect of Mel and 6-OHMel on the neurons is related to their antioxidation and stabilizing mitochondrial function. The mechanism of protective effect of Mel on the neurons is more complex than that of 6-OHMel.