头顶一颗珠对AD模型大鼠脑海马组织Tau蛋白磷酸化及突触发育的影响  

Trillium Tschonoskii Maxim improves cognitive dysfunction in AD rats by inhibiting GSK-3β activity,attenuating tau pathologies and promoting synaptic development

作　　者：陈玮[1] 罗洪斌[1,2,3,4] 鲁文杰 牟南樵[1] 黄胜 陈娟 樊莎莎 谢文执 商楠 杨晨宇[1] 谢枫枫[1] 谌勤 CHEN Wei;LUO Hong-bin;LU Wen-jie;MOU Nan-qiao;HUANG Sheng;CHEN Juan;FAN Sha-sha;XIE Wen-zhi;SHANG Nan;YANG Chen-yu;XIE Feng-feng;CHEN Qin(Dept of Biochemistry and Molecular Biology,Medical College,Hubei University for Nationalities;Dept of Biochemistry and Molecular Biology,Medical Faculty,Science and Technology College of Hubei University for Nationalities;Key Lab of Biologic Resources Protection and Utilization of Hubei Province,Hubei University for Nationalities;Institute of Neurological and Psychiatric Comorbidity,Hubei University for Nationalities;Dept of Neurology,Affiliated Hospital,Hubei University for Nationalities, Enshi Hubei 445000, China)

机构地区：[1]湖北民族学院医学院生物化学与分子生物学教研室,湖北恩施445000 [2]湖北民族学院科技学院医学系生物化学与分子生物学教研室,湖北恩施445000 [3]湖北民族学院生物资源保护与利用湖北省重点实验室,湖北恩施445000 [4]湖北民族学院神经精神共患病研究所,湖北恩施445000 [5]湖北民族学院附属民大医院神经内科,湖北恩施445000

出　　处：《中国药理学通报》2018年第6期775-784,共10页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81260172;81660223);湖北省自然科学基金面上项目(No 2017CFB451);湖北民族学院科技创新团队项目(No MY2011T005);湖北民族学院博士基金启动项目(No MY2012B015);生物资源保护与利用湖北省重点实验室开放基金项目(No PKLHB1318);国家级和湖北省省级大学生创新创业训练计划项目(No201610517015)

摘　　要：目的 探索头顶一颗珠(TTM)水煎液对激活糖原合酶激酶-3β(GSK-3β)诱导的阿尔茨海默病(AD)模型大鼠脑海马Tau蛋白磷酸化和突触发育的影响。方法 将♂4月龄50只SD大鼠随机均分为5组,分别为假手术组、AD模型组、TTM治疗组(低、中、高剂量组),TTM水煎液大鼠灌胃7d,假手术组和模型组灌饮用水7d,灌胃d2开始Morris水迷宫训练,5d后找出所有d5中能在15s内找到隐藏平台的模型组和治疗组大鼠,侧脑室注射各5μL的GF-109203X(GFX,PKC特异性抑制剂)和渥曼青霉素(wortmannin,WT,PI3K特异性抑制剂),浓度各为100μmol·L^(-1),假手术组同位置注射2%DMSO 10μL。24h后行水迷宫测试。免疫印迹和免疫组化技术检测脑海马中Tau蛋白磷酸化水平,及其相关GSK-3β激酶信号通路各蛋白活性和突触相关蛋白表达水平;尼氏染色法检测海马神经元尼氏小体变化情况;高尔基染色法检测海马神经突触发育、树突棘数量及形态变化。结果 水迷宫检测发现,TTM能改善大鼠空间学习记忆障碍;免疫印迹检测显示,模型组脑海马GSK-3β活性上调,进而升高Tau蛋白多个位点磷酸化水平;而低、中剂量TTM增加PKC和Akt活性,从而抑制下游激酶GSK-3β活性,导致Tau蛋白磷酸化水平下降。免疫组化结果显示,TTM能降低Tau蛋白磷酸化水平。免疫印迹结果还显示,TTM能提高模型大鼠脑海马突触相关蛋白Synapsin-1、Synaptophysin、GluR-1表达水平;尼氏染色提示AD模型鼠脑海马神经元尼氏小体数量减少,用药后明显增多。中、高剂量TTM增加模型组海马神经元树突数量、复杂性及树突棘密度。结论 TTM可有效改善GSK-3β活性升高所引起的大鼠空间认知功能障碍,其机制可能是通过作用于GSK-3β信号通路,下调GSK-3β活性,进而抑制Tau蛋白过度磷酸化,同时促进神经元和突触发育起到保护作用。Aim To assess the effects of Trillium Tschonoskii Maxim（TTM） decoction on Tau protein phosphorylation and synaptic development in AD model rats induced by high activity GSK-3β. Methods The SD rats were divided into five groups of ten animals,named sham-operated group（blank group）,AD model group,TTM group（0.5,0.25,0.125 g · kg^（-1）·d^（-1））. Treatment group received gavage once a day for seven days with TTM decoction,while other groups by gavage once a day for seven days with drinking water.On 2 nd day by gavage,Morris water maze test was used to assess the spatial learning and memory ability of the rats. After five days＇ training,rats in the treatment groups and AD model group were injected wortmannin（WT, PI3 K specific inhibitor） and GF-109203 X（GFX,PKC specific inhibitor）（100 μmol·L（-1） of each,total volume of 10 μL） into the right lateral ventricle. Western blot was used to detect the levels of phosphorylation Tau protein at multiple sites and the expression level of PI3 K,Akt,PKC,GSK-3β（S9,T216） and synapse-associated proteins. Immunohistochemical method was used to detect the hyperphosphorylation of Tau protein in hippocampus of rats.Golgi staining was applied to detect the number and morphological changes of synaptic development and dendritic spines. Nissl＇s staining was employed to observe the development of neonatal neurons in hippocampus and cortex. Results Western blot showed that the phosphorylation level of Tau in hippocampus increased in model group,and the activity of GSK-3βwas up-regulated. Among them,however,in middle dose TTM group,the phosphorylation level of Tau in hippocampus decreased and the activity of GSK-3β decreased. The expression levels of p-PKC and p-Akt in low and middle dose treatment group were higher than those in model group,thus increasing the activity of PKC and Akt to inhibit the activity of GSK-3β kinase.Immunohistochemistry also indicated that TTM could decrease the biological effects of Tau phosphorylation in hippocamp

关 键 词：阿尔茨海默病 头顶一颗珠 GSK-3Β TAU磷酸化 突触相关蛋白 突触发育 学习记忆能力 

分 类 号：R-332[医药卫生] R282.71