摘要
目的:观察刺五加皂苷(acanthopanax senticosus saponins,ASS)对脊髓运动神经元缺氧损伤有无保护作用,并探讨其可能机制。方法:对胚鼠运动神经元进行原代分离培养,免疫组化鉴定后建立缺氧诱导的神经元凋亡模型。取运动神经元培养在24孔板中,每组10孔,分为4组:对照组,无缺氧损伤;缺氧损伤组;ASS保护组,缺氧前24 h加入50μg/ml的ASS;神经胶质细胞源性神经营养因子(GDNF)保护组,缺氧前24 h加入0.1μg/ml的GDNF。倒置显微镜及电镜下观察细胞形态学变化;MTT法测定神经元细胞活性;检测细胞外液LDH释放量来观察ASS对神经元细胞膜稳定性的影响。提取细胞匀浆液,Western印迹法检测分析ASS对缺氧的运动神经元HIF-1α表达的影响。结果:形态学观察显示ASS、GDNF保护组神经元缺氧损伤较缺氧损伤组明显减轻。ASS保护组神经元的细胞活性(0.21±0.028)比缺氧损伤组(0.15±0.012)高(P<0.05),而LDH的释放量(28.6±1.309)比缺氧损伤组(40.7±1.885)低(P<0.01);缺氧损伤组的神经元HIF-1α的相对表达量(0.72±0.027)比对照组(0.16±0.003)高(P<0.01),但ASS保护组HIF-1α的表达(1.15±0.016)最高(P<0.01)。ASS对大鼠脊髓运动神经元缺氧损伤的保护作用与GDNF相仿。结论:ASS可以提高体外缺氧损伤的运动神经元的细胞活性,对细胞的缺氧损伤有明显的保护作用,其保护作用的发挥,可能与增强细胞膜的稳定性及提高细胞HIF-1α的表达有关。
Objective：To investigate whether acanthopanax senticosus saponins（ASS） has a protective effect on anoxia-damaged rat spinal cord motoneurons（SMNs） in vitro and to reveal the possible mechanism of this effects. Methods： SMNs were obtained from the spinal cord of embryonic day 15 rats and were cultured in vitro. The cultured cells were immunohistochemically identified and were subjected to anoxia exposure to establish apoptosis model. In this study,SMNs were divided into the following 4 groups： normal control group;anoxia-induced apoptosis group; ASS pre-treated group, SMNs were treated with ASS （50μg/ml） 24 h before anoxia exposure;and glial-cell-line-derived neurotrophic factor（GDNF） pre-treated group,SMNs were treated with GDNF（0.1μg/ml） 24 h before anoxia exposure. The morphology of SMNs was observed with phase-contrast microscope and electron microscope; the viability of SMNs was detected with MTT method;and the influence of ASS on the stablility of SMNs membrane was observed through detection of lactate dehydrogenase （LDH） level in the extracellular solution. The influence of ASS on the expression of HIF-1α in anoxia-damaged SMNs was studied by Western blot analysis. Results： The morphological damage of SMNs in ASS and GDNF pre-treated groups was slighter than that in anoxia-induced apoptosis group. The results showed that the viability of SMNs was higher in ASS pre-treated group （0.21±0. 028） compared with that in anoxia-induced apoptosis group （0. 15 ± 0. 012） （P 〈 0. 05）, while the level of LDH was lower in ASS pre-treated group （28.6±1. 309） than that in anoxia-induced apoptosis group （40.7±1. 885） （P〈0.01）. The expression of HIF-1α in ASS pretreated group was the highest （1.15±0. 016） （P〈0.01） ,and that in anoxia-induced apoptosis group （0.72±0. 027） was higher than that in the control group （0.16±0. 003） （P〈0.01）. Protective effect of ASS on anoxia-damaged SMNs in rats was similar to that of GDNF. Conclusion： ASS can increase the viability of hypoxia-damaged SMNs in vitro and has obvious protective effects on them,which may be related to the enhanced stability of cell membrane and the up-regulation of HIF-1α expression.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2006年第2期173-177,共5页
Academic Journal of Second Military Medical University
基金
无锡市社会发展基金(20040006)~~
关键词
剌五加皂苷
细胞凋亡
缺氧
运动神经元
脊髓
细胞培养
保护作用
acanthopanax senticosus saponins
apoptosis
anoxia； motor neurons； spinal cord； cells, cultured
