摘要
目的细胞内钙超载被认为是诱发神经元变性和死亡的关键因素。文中研究蕨麻正丁醇部位(n-butanol extract of Potentilla anserina L,NP)对大鼠海马神经元急性缺氧所致钙超载的抑制作用。方法采用体外原代培养的海马神经元建立缺氧所致钙超载的实验模型,分为正常对照组、缺氧模型组、尼莫地平干预组以及NP高、中、低剂量干预组。通过免疫荧光双染观察蕨麻正丁醇部位对海马神经元急性缺氧所致微管相关蛋白2(microtuble-associated protein2,MAP2)的保护作用。利用荧光分光光度计检测各组细胞内游离钙离子浓度([Ca2+]i)。通过半定量RT-PCR、免疫细胞化学染色及Western blot技术检测各组钙依赖中性蛋白酶1(Calpain 1)在基因及蛋白质水平的表达差异。结果缺氧3 h模型组与正常组相比较,神经元特异性细胞骨架蛋白MAP2被水解,细胞骨架形态的被破坏,[Ca2+]i显著升高,Calpain 1在基因和蛋白水平的表达量明显增加,细胞骨架发生破坏。NP各剂量干预明显抑制神经元特异性细胞骨架蛋白MAP2水解,减轻细胞骨架形态的破坏,且显著降低缺氧神经元[Ca2+]i(P<0.05);并且下调了Calpain 1在基因和蛋白的表达水平(P<0.05)。结论急性缺氧3 h可使神经元细胞内发生钙超载,NP可有效抑制缺氧所致神经元细胞钙超载,而发挥神经元作用。
Objective Intracellular calcium overload is considered to be the key factor in neuronal degeneration and death. The aim of this study is to investigate the inhibitory effects of n-butanol fraction of Potentilla anserine L. （NP） on hypoxia-induced calcium overload on hippocampus neuron in rats. Methods Hipocampus neurons of Sprague-Dawley neonatal rats were cultured in vitro to establish the hypoxia-induced calcium overload model. The cultured hippocampus neurons were randomized into control group, hypoxia injury model group, Nimodipine intervention group, and NP （high, middle and low doses） intervention group. The protective effects of NP on microtuble-associated protein2 （MAP2） in acute hypoxia injury were observed through immunofluorescent double stai- ning. The intracellular calcium ion concentration （ [ Ca^2 ＋ ] i ） in hippocampus neurons were detected through fluorescence spectropho-tometer. Changes in expression of calcium-activated neutral protease 1 （ Calpain 1 ） mRNA were detected by RT-PCR and Calpain 1 protein were detected by immuno-histochemistry stain and Western blot. Results Compared with the control group, MAP2 was hydroiyzed, eytoskeleton morphology was destroyed, [ Ca^2＋ ]i was increased, and the Calpain 1 mRNA and protein were increased in hypoxia 3 h injury model group. NP at different dosage inhibited the hydrolyses of MAP2, decreased [ Ca^2 ＋ ] i and the expression of Cal-pain 1 mRNA and protein on hypoxia injury neuron compared with model group （ P 〈 0.05 ）. Conclusion NP can attenuate the cal- cium overload induced by hypoxia 3 h on hippocampal neurons in rats.
出处
《医学研究生学报》
CAS
北大核心
2014年第2期133-137,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81073152)
天津市重点项目(10JCZDJC21100
12JCZDJC34700)
关键词
蕨麻正丁醇部位
海马神经元
缺氧
钙超载
钙依赖中性蛋白酶1
Potentilla anserina L. n-butanol extraction
Hippocampal neurons
Hypoxia
Calcium overload
Ca]pain 1
