人参蛋白对β淀粉样蛋白_(1-40)和H_2O_2损伤神经元的保护作用及其机制  

Protective effect of ginseng protein on neurons damaged by amyloid-βprotein_(1-40) and H_2O_2 and its mechanism

作　　者：李红艳[1] 曹阳 白雪媛[3] 赵丹[1] 张晓丹[1] 杨静娴[1] 康廷国[1] 

机构地区：[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

出　　处：《吉林大学学报（医学版）》2013年第6期1138-1142,1312,共6页Journal of Jilin University：Medicine Edition

基　　金：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)

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12d后,分别采用Aβ1-40(30μmol·L-1)和H2O2(200μmol·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。Objective To investigate the effect of ginseng protein （GP） on neurons damaged by beta amyloid protein1-40 （Aβ1-40） and hydrogenperoxide （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 30μmol· L-1 Aβ1-40 and 200μmol· 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 ceils were dyed into red by PI under inverted flurescenee 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

关 键 词：人参蛋白 血清 神经元 G淀粉样蛋白 细胞凋亡 

分 类 号：R961[医药卫生—药理学]