银杏酮酯抗H_2O_2诱导衰老海马神经元氧化DNA损伤的作用研究  

The effects of Ginkgo biloba extract on anti oxidantive DNA damaging in H_2O_2-induced senescence-like hippocampal neurons and its mechanisms

作　　者：郝莉[1] 徐玉英[1] 郭春霞[2] 张志雄[2] 高崎 顾慧芬 

机构地区：[1]河南中医学院基础医学院,郑州450052 [2]上海中医药大学基础医学院,上海201203 [3]上海杏灵科技药业有限公司,上海200127

出　　处：《中药药理与临床》2015年第3期83-88,共6页Pharmacology and Clinics of Chinese Materia Medica

基　　金：上海市科委科研计划项目资助(项目批准号09ZR1432100);河南中医学院;科研苗圃项目资助(项目批准号MP2012-03)

摘　　要：目的：探讨银杏酮酯（Ginkgo biloba extract,GBE50）抗过氧化氢（H2O2）诱导大鼠衰老海马神经元氧化DNA损伤的作用及人类长寿基因（Silent information regulator 1,SIRT1）在其中的作用机制。方法：培养、鉴定大鼠原代海马神经元。将细胞随机分为4组,正常对照组,H2O2模型组,银杏酮酯组和阳性对照银杏叶提取物（EGB761）组。正常对照组用培养基24小时,模型组用H2O2（200μM）诱导18小时建立细胞衰老样模型,后两组分别用200μg/ml的银杏酮酯和银杏叶提取物预处理6小时后再加入H2O2诱导18小时。细胞免疫荧光方法观察各组神经元中8-羟基脱氧鸟苷（8-OHd G）和SIRT1的表达强度;免疫印迹方法检测SIRTl、p53和p21的蛋白水平。结果：光镜下观察培养6~7天时海马神经元逐渐成熟,免疫荧光鉴定神经元纯度达到90%以上。细胞免疫荧光结果显示：与正常对照组相比,H2O2诱导的大鼠原代衰老海马神经元中8-OHDG表达增加（P〈0.01）,SIRT1的表达降低（P〈0.01）。200μg/ml银杏酮酯和阳性对照银杏叶提取物预处理后8-OHDG表达减少（P〈0.05）,SIRT1的表达增加（P〈0.05）。免疫印迹结果显示：与正常对照组相比,H2O2诱导的大鼠原代衰老海马神经元中SIRTl蛋白表达下降,p21蛋白表达增加（P﹤0.05）;200μg/ml银杏酮酯和阳性对照银杏叶提取物预处理后H2O2诱导的海马神经元SIRTl蛋白表达增加（P〈0.05）,p21蛋白表达下降（P〈0.05）;而p53蛋白变化不明显（P﹥0.05）。结论：200μg/ml的银杏酮酯可降低H2O2诱导的大鼠原代衰老海马神经元内8-OHDG表达,其机制可能与SIRT1、p21蛋白表达有关。Objective： To investigate effects of Ginkgo biloba extract （GBES0）on anti oxidantive DNA damaging in H2 02-induced senescence-like hippocampal neurons and its mechanism. Method： Primary hippocampal neurons were cultured and identified. The cells were divided into four groups： control group, H2O2 model group, GBES0 group and positive control EGB761 group. Cells treated only with solvent for 24 h were used as control and ceils treated with H202 for 18 h were used as model. The cells were incubated with GBE50 or EGB761 for 6 h before being treated with H202 （200 μM） for 18 h staining was used to detect the 8-OHDG and Sirtl ; Western-blot was used to examine Sirtl, p53 and p21 protein levels. Results： After 6 ~ 7 days, the neurons became mature. Immunostaining was performed to visualize and determine the neuronal purity in primary hippocampal neurons, and evaluation of staining indicated the purity was up to 90%. Immuno- fluorescent staining confirmed that 200 μM H202 increased and decreased the level of 8-OHDG and SIRT1 （ P 〈 0.01 ）. Pretreatment of neu- rons with 200μg/ml GBES0 and EGB761, however, significantly decreased the level of 8-OHDG and SIRT1 （P 〈 0.05 ）, compared with cells treated with H2O2 alone. Western blot displayed that 200 μM H2O2 decreased and increased SIRT1 and p21, respectively, compared with control group （ P 〈 0.01 ）. Pretreatment of neurons with 2001μg/ml GBES0 and EGB761, however, the expression of SIRT1 showed a in- creased tendency （P 〈 0.05）, and there was significant difference in p21 protein level （P 〈 0.05）. However, the expression change of p53 was not obvious （P 〉 0.05 ）. Conclusion： GBE50 （2001xg/ml） might decrease 8-OHDG in H2 O2-induced hippocampal neurons, and its mechanism might be associated with SIRT1 and p21.

关 键 词：银杏酮酯 H2O2 海马神经元 氧化DNA损伤 SIRT1 

分 类 号：R285.5[医药卫生—中药学]