金雀异黄酮通过调控Ca^(2+)-CaMKIV通路对Aβ_(25-35)诱导海马神经元损伤的保护作用  

Protective Effect of Genistein on Aβ_(25-35)-Induced Hippocampal Neuron Injury by Regulating Ca^(2+)-CaMKIV Pathway

作　　者：高华武[1,2] 王艳 周鹏[1,2] 叶树 宋航[1] 汪光云 蔡标[1,2] GAO Huawu;WANG Yan;ZHOU Peng;YE Shu;SONG Hang;WANG Guangyun;CAI Biao(College of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Province Key Laboratory of Chinese Medicinal Formula,Institute of Integrated Chinese and Western Medicine,Anhui Academy of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学中西医结合学院,安徽合肥230012 [2]安徽省中医药科学院中西医结合研究所,中药复方安徽省重点实验室,安徽合肥230012

出　　处：《食品科学》2021年第13期121-126,共6页Food Science

基　　金：国家自然科学基金面上项目(81574040);安徽省自然科学基金杰出青年科学基金项目(1908085J27);安徽省高校优秀青年人才支持计划项目(gxyqZD2018053);安徽中医药大学自然科学基金重点项目(2019zrzd01)。

摘　　要：目的:观察金雀异黄酮通过Ca^(2+)-钙调蛋白依赖性蛋白激酶IV(calmodulin-dependent protein kinase IV,CaMKIV)通路对Aβ_(25-35)诱导海马神经元损伤的保护作用。方法:取24 h内新生SD乳鼠的海马组织,进行神经元的分离纯化和培养,并用免疫荧光染色进行鉴定。神经元细胞随机分为空白对照组、模型组、金雀异黄酮组(50μmol/L)和阳性对照戊酸雌二醇组(10μmol/L),金雀异黄酮组和戊酸雌二醇组预处理3 h后,除空白对照组外,其他各组采用Aβ_(25-35)诱导海马神经元构建细胞损伤模型。利用噻唑蓝法检测细胞存活率,荧光探针检测神经元细胞内Ca^(2+)荧光强度,Western blot检测钙调蛋白(calmodulin,CaM)、钙调蛋白依赖性蛋白激酶激酶(calcium/calmodulin dependent protein kinase kinase,CaMKK)、磷酸化钙调蛋白依赖性蛋白激酶IV(p-calmodulin-dependent protein kinase,p-CaMKIV)和p-Tau蛋白的相对表达量。结果:免疫荧光分析结果显示大鼠海马神经元分离成功。与空白对照组比较,模型组海马神经元细胞存活率极显著下降(P<0.01),细胞Ca^(2+)荧光强度极显著升高(P<0.01),CaM、CaMKK、p-CaMKIV和p-Tau蛋白相对表达量极显著提高(P<0.01);与模型组比较,金雀异黄酮极显著提高了Aβ_(25-35)所致海马神经元损伤模型中细胞的存活率(P<0.01),降低了细胞Ca^(2+)荧光强度(P<0.01),下调了CaM、CaMKK、p-CaMKIV和p-Tau蛋白相对表达量(P<0.01)。结论:金雀异黄酮对Aβ25-35诱导的海马神经元损伤具有明显的神经保护作用,其作用可能是通过Ca^(2+)-CaMKIV通路介导的。Objective:To investigate if and how genistein can protect hippocampal neurons against Aβ_(25-35)-induced injury by regulating the Ca^(2+)-calmodulin-dependent protein kinase IV(CaMKIV)pathway.Methods:The hippocampal tissue was taken from neonatal SD rats within 24 h after birth for isolation,purification and culture of neurons as well as identification by immunofluorescence staining.The neuronal cells were randomly divided into four groups:blank control,model,and genistein treatment(50μmol/L)and estradiol and valerate treatment(10μmol/L).After being pretreated for 3 h,the hippocampal neurons in all groups except the blank control group were treated with Aβ_(25-35) to establish a cell injury model.The cell survival rate was detected by methylthiazolyl tetrazolium,the intracellular Ca^(2+)concentration was detected using a fluorescent probe,and the expression of calmodulin(CaM),calcium/calmodulin dependent protein kinase kinase(CaMKK),phosphorylation calmodulin-dependent protein kinase(p-CaMKIV)and p-Tau were detected by Western blot.Results:Immunofluorescence showed that the hippocampal neurons were successfully isolated.Compared with the blank control group,the survival rate of hippocampal neurons in the model group was significantly decreased(P<0.01),and the intracellular concentration of Ca^(2+)was significantly increased as well as the protein expression of CaM,CaMKK,p-CaMKIV and p-Tau(P<0.01).Compared with the model group,genistein significantly increased the survival rate of hippocampal neurons(P<0.01),decreased the intracellular concentration of Ca^(2+)(P<0.01),and down-regulated CaM,CaMKK,p-CaMKIV and p-Tau protein expression(P<0.01).Conclusion:Genistein has an obvious protective effect on hippocampal neuron damage induced by Aβ25-35,which may be mediated by the Ca^(2+)-CaMKIV signaling pathway.

关 键 词：阿尔茨海默病 金雀异黄酮 Ca^(2+)-CaMKIV信号通路 海马神经元 

分 类 号：R285.5[医药卫生—中药学]