摘要
目的观察树鼩海马微环境中谷氨酸(Glu)及钙(Ca2+)改变对细胞色素C(CytC)释放以及caspase凋亡基因激活的影响,探讨银杏内酯B(GB)干预海马神经元线粒体应激的分子机制。方法使用单泵等速微灌流系统行树鼩海马Glu及Ca2+微灌流,24h后用免疫组化法观察海马神经元CytC蛋白表达;免疫印迹法检测线粒体及胞质的CytC含量;实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca2+溶液后6h于舌下静脉注射银杏内酯B5mg/kg,24h后观察上述指标的改变。结果树鼩海马微灌流Glu和Ca2+溶液后24h,线粒体CytC含量显著下降,而胞质部分出现CytC;海马组织caspase-3、caspase-9 mRNA明显升高。GB治疗组线粒体CytC含量增多,胞质中仍有CytC存在,而海马组织caspase-9 mRNA显著降低,但caspase-3 mRNA无差异。结论海马微环境中Glu与Ca2+的大量堆积促进线粒体CytC的释放,可能是激活caspase级联反应而导致线粒体应激、神经元继发损伤的始动环节;GB的神经保护效应可能与其特异性拮抗血小板活化因子(PAF)受体,抑制Ca2+内流,部分阻滞CytC释放入胞质,从而减少caspase-9基因转录有关。
Objective To observe the influence of the cbanges of glutamate and calcium in tree shrew＇ s hippocampal microenvironment on cytochrome C release and caspase apoptotic gene action, and to explore the molecular mechanism of Ginkgolide B intervening mitochondrial stress. Methods A lateral hippocampus was microperfused by glutamate and calcium chloride solution by a kind of single-pumped push-pull perfusion system in Tree Shrews. After 24h, the cytochreme c expression was observed by immunochemistry; the cytochrome contents in mitochondria and cytoplasmic fragment were assessed through western blot; The relative amounts of caspase-3 and caspase-9 mRNA were evaluated by real time fluorescence polymerase chain reaction. After perfusing the glutamate-calcium chloride solutions into the hippocampus, Ginkgolide B（5 mg/kg） was intravenously injected at 6h and the above-mentioned items were inspected at 24h. Results After perfusing glutamate-calcium solutions in tree shrew＇ s hippocampus, the contents of cytochrome c markedly decreased in mitochondria and appeared in cytosol; the caspase-3 and caspase-9mRNA clearly rose. In GB group, the cytochrome C increased in mitochondria but existed in cytosol, caspase-9mRNA reduced, but there was no difference in caspase-3 mRNA. Conclusion It may be an initiatory step that glutamate and calcium ions accumulate in hippocampal microenvirenment, which can activate cytochrome C release, caspase cascade activation, and finally result in mitochondria stress and neuronal secondary injury. The neuronal protection of Ginkgolide B is related to inhibiting platelet active factor （PAF） receptor and reducing the calcium ions influx, consequently partly protect from cytochrome c release and transition of caspase mRNA.
出处
《中国微循环》
北大核心
2007年第3期158-162,共5页
Journal of Chinese Microcirculation
基金
国家自然科学基金(N03066056)
教育部博士点专项基金(N020050678008)
云南省自然科学基金资助项目(N02004C0043M)
关键词
海马
线粒体
细胞色素C
银杏内酯
Hippocampus
Mitochondria
Cytochrome c
Ginkgolide B
