摘要
目的探讨模拟缺血再灌注引起神经元凋亡的途径和褪黑素(melatonin,MT)抗凋亡的作用机制。方法用原代培养的SD乳鼠小脑颗粒细胞建立以缺氧缺糖模拟缺血再灌注模型,并加不同浓度的MT(10-5、10-7、10-9mol/L)孵育。采用荧光比色法检测其线粒体跨膜电位及细胞内的活性氧;用Westernblot及ELISA分别检测线粒体和胞质中的细胞色素C;比色法检测胞质中Caspase3的活性。结果在模拟缺血再灌注小脑颗粒细胞模型中,细胞内的活性氧在缺氧缺糖后明显增加;线粒体跨膜电位降低并随再灌注时间延长而加重;线粒体释放入胞质的细胞色素C增加,胞质的Caspase3活性增加;MT可显著减少活性氧和抑制线粒体释放细胞色素C,抑制线粒体跨膜电位的降低,并呈一定剂量依赖性。结论①缺血再灌注引起的神经元凋亡部分是通过线粒体凋亡途径;②MT可通过抗氧化作用和阻止线粒体凋亡而抑制模拟缺血再灌注诱导的小脑颗粒细胞凋亡。
Objective To study whether melatonin can protect rat cerebellar granule neurons from apoptosis induced by oxygen-glucose deprivation and its mechanism.Methods SD rat cerebellar granule cells were isolated and cultured, and mimic ischemia-reperfusion model was established by oxygen-glucose-deprivation (OGD) in vitro. After the neuron cells were cultured with or without melatonin and at different time points after OGD for 90 min, the mitochondrial membrane potential and generation of reactive oxygen species (ROS) were measured by fluorescent spectrophotometer. Cytochrome C was assayed by Western blotting and ELISA. The activity of Caspase-3 was assessed by colorimetry.Results In the mimic ischemia-reperfusion model of cerebellar granule cells, the intracellular ROS after OGD, cytochrome C releasing from mitochondria to cytosol and activity of cytoplasmic Caspase-3 were significantly increased. Melatonin could decrease the release of ROS and cytochrome C releasing from mitochondria to cytosol in a dose-dependent manner. The decrease of the mitochondrial membrane potential was inhibited.Conclusion The neuronal injury induced by OGD is partly mediated by the mitochondrial apoptosis pathway; Melatonin can suppress the apoptosis of the cerebellar granule neurons by its anti-oxidation, prevention of the release of cytochrome C and inhibition of the decrease of mitochondrial membrane potential.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2005年第3期262-265,273,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
湖北省卫生厅第五个三年医药卫生科研计划资助项目(No.WJ01510)
关键词
小脑颗粒细胞
缺血再灌注
活性氧
细胞色素C
褪黑素
cerebellar granule neuron
ischemia/reperfusion
reactive oxygen species
cytochrome C
melatonin
