吉林大学学报(医学版)

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人参蛋白对β淀粉样蛋白1-40和H2O2损伤神经元的保护作用及其机制

李红艳1，曹阳2，白雪媛3，赵丹1，张晓丹1，杨静娴1，康廷国1   

  1. （1.辽宁中医药大学药学院药理学教研室，辽宁 大连  116600；2.辽宁省大连市华信理化检测中心有限公司检测部，辽宁 大连 116600；3.长春中医药大学中医药与生物工程研发中心，吉林 长春 130117）
  • 收稿日期:2013-04-07 发布日期:2013-11-28
  • 通讯作者: 康廷国 E-mail:（Tel：0411-87586078，E-mail：kangtg@126.com）
  • 作者简介:李红艳（1982-），女，内蒙古自治区赤峰市人，讲师，中药学博士，主要从事中药药理学研究。
  • 基金资助:

    中国博士后基金资助课题（2013M530945）；辽宁省科技厅科研项目资助课题（2008226011-1）

Protective effect of ginseng protein on neurons  damaged by amyloid-β protein1-40 and H2O2 and its mechanism

LI Hong-yan1,CAO Yang2,BAI Xue-yuan3,ZHAO Dan1,ZHANG Xiao-dan1,YANG Jing-xian1,KANG Ting-guo1   

  1. (1. Department of Pharmacology,Liaoning University of Traditional Chinese Medicine,Dalian 116600,China;2. Department of Detection,Huaxin Physical and Chemical Analysis Center CO.,Ltd,Dalian 116600，China;3. Chinese Medicine and Bioengineering Research Center,Changchun University of Traditional Chinese Medicine，Changchun 130117，China)
  • Received:2013-04-07 Published:2013-11-28

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摘要：

目的：探讨人参蛋白(GP)对β淀粉样蛋白1-40（Aβ1-40）和过氧化氢（H2O2）诱导神经元损伤的影响，阐明GP对神经元的保护作用及其机制。方法：从新生乳鼠大脑皮层中分离纯化神经元，培养12 d后，分别采用Aβ1-40（30 μmol•L-1）和H2O2 （200 μmol•L-1）诱导神经元损伤，并随机分为模型组（Aβ1-40模型组与H2O2模型组，不加药物）、GP组和GP含药血清组。采用MTT法检测各组神经元存活率；试剂盒法检测细胞培养上清液中一氧化氮合酶（NOS）活性和一氧化氮（NO）水平；Hoechst 33342/PI荧光染色检测细胞凋亡与坏死率。结果：倒置荧光显微镜，Aβ1-40和H2O2处理后，神经元数量明显减少，轴突缩短或消失，
胞体变圆、缩小，大小不等，核固缩，核染色质聚集；Heochst 33342染色呈现高蓝光；部分细胞PI染色呈现高红光。加入GP及其含药血清后，神经元数量增多，胞体增大，细胞核着色形态为圆形、淡蓝色，PI阳性细胞数量减少。MTT法，GP组细胞存活率明显高于模型组（P<0.05或P<0.01），GP含药血清组与模型组无显著差异（5％GP含药血清组除外，P>0.05）；试剂盒法，GP组细胞培养上清液中NOS活性与NO水平明显低于模型组（P<0.05　或P<0.01）
；Hoechst 33342/PI法，GP及其含药血清组细胞凋亡率低于模型组，以GP组差异更明显（P<0.05或P<0.01），GP及其含药血清组细胞坏死率与模型组比较差异无统计学意义（P>0.05）。结论：GP具有神经保护作用，作用机制与减少NOS活性、降低NO水平、抑制神经元凋亡有关。

关键词: 人参蛋白, 血清, 神经元, &beta, 淀粉样蛋白, 细胞凋亡

Abstract:

To investigate the effect of ginseng protein (GP) on neurons damaged  by beta amyloid protein1-40 (Aβ1-40) and hydrogenperoxi
de (H2O2),and to clarify the neuroprotective effect of GP and its mechanism. Methods The primary neurons were isolated and purified from cerebral cortex of suckling mouse and  cultured for 12 d,and damaged by 30  μmol•L-1Aβ1-40 and 200 μmol•L-1 H2O2. Then the damaged cells  were randomly divided into model group (Aβ1-40 model group and H2O2 model group,no drugs),GP group and  GP medicated  serum  group. MTT method was used to detect the  survival rates of cells;Qiagen was used to detect the  activities of NOS and levels of NO  in culture supernatant,and  Hoechst 33342/PI fluorescent staining method was used to detect the apoptotic  and necrotic rates of cells. Results It was observed that by the treatment of Aβ1-40  and H2O2,the  number of neuron was decreased,the axons shortened or disappeared,the karyopyknosis concentrated into a bright blue（dyed by Hoechst 33342）,and some cells were dyed into red by PI under inverted flurescence microscope; after adding GP and its medicated serum,the  number of cells was obviously increased,the cells stretched,the  nuclei　were round and light blue,and the number of cells dyed by PI into red was reduced. The MTT results showed that the  survial rate of cells in   GP group was obviously higher than that in model group (P<0.05 or P<0.01).There was no significant difference in  the  survival rate of cells between  GP medicated serum group and  model group (except 5% GP medicated serum group,P>0.05). The activity of NOS and level of NO in culture supernatant in  GP group were significantly lower than those in model group (P<0.05 or P<0.01). The apoptotic rates of cells in GP group and GP medicated serum group were obviously lower than that in model group detected by Hoechst 33342/PI method(P<0.05 or P<0.01). There was no significant differences of  the necrotic rates of neurons between GP group, GP medicated group and model group(P>0.05).Conclusion GP protein has the neuroprotective effect,and its mechanism is related to the reduction of the activity of NOS and the level of NO and the inhibition of apoptosis of neurons.

Key words: ginseng protein;serum;neuron;amyloid-&beta, protein;apoptosis

中图分类号: 

  • R961
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