复智散通过细胞周期依赖性蛋白激酶5通路减轻皮层神经元Tau蛋白过度磷酸化  

Role of Fuzhisan in reducing Tau protein hyperphosphorylation in cortical neurons through a cyclin-dependent kinase 5 pathway

作　　者：张兆旭[1] 王德生[2] 

机构地区：[1]山东省千佛山医院神经内科,山东济南250000 [2]哈尔滨医科大学附属第一医院神经内科,黑龙江哈尔滨150001

出　　处：《中国药理学通报》2016年第3期422-426,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81303013)

摘　　要：目的探讨复方中药复智散(FZS)能否通过抑制细胞周期依赖性蛋白激酶5(CDK5)通路,减轻Aβ_(25-351)诱导的新生鼠皮层神经元Tau蛋白过度磷酸化。方法选用24 h内新生Wistar大鼠,分离纯化皮层神经元,进行体外培养。皮层神经元在体外培养7 d后,应用20μmol·L-1Aβ_(25-351)作用于皮层神经元24 h。药物治疗组则应用FZS(20 mg·L^(-1))、CDK5抑制剂Roscovitine(15μmol·L^(-1))、钙蛋白酶(calpain)制剂Calpeptin(20μmol·L^(-1))预处理24 h,然后用20μmol·L^(-1)Aβ_(25-351)作用24 h。用Western blot检测Tau蛋白Ser396、Ser202和Thr231位点磷酸化水平和CDK5的激活蛋白p25/p35的蛋白水平;荧光酶标仪测定荧光强度来反映calpain活性;免疫沉淀法检测CDK5激酶活性。结果 20μmol·L^(-1)Aβ_(25-351)作用于皮层神经元24 h后,Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,CDK5激酶活性升高,CDK5激活蛋白p25水平升高,calpain活性升高。20 mg·L-1FZS治疗组则明显抑制了Aβ_(25-351)导致的Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,抑制了CDK5激酶活性、p25蛋白水平以及calpain活性升高。CDK5抑制剂roscovitine和calpain抑制剂calpeptin作为阳性对照药物也显示了抑制Tau蛋白过度磷酸化的作用。结论复智散可能通过calpain-p25/CDK5通路抑制Aβ_(25-351)导致的皮层神经元Tau蛋白过度磷酸化。Aim Fuzhisan( FZS),a Chinese herbal complex prescription that has been used for the treatment of AD for over 15 years,is known to enhance the cognitive ability in AD patients.In this study,to investigate whether FZS reduces Aβ_(25-351)-induced Tau protein hyperphosphorylation in neonatal rat cortical neurons by suppressing the cyclin-dependent kinase 5(CDK5) pathway.Methods Neonatal Wistar rats born within 24 h were selected to separate and purify their cortical neurons for culture in vitro.After 7-day culture of cortical neurons in vitro,20 μmol·L^(-1)Aβ_(25-351) was used to act on them for 24 h.Medication groups were pretreated with FZS(20 mg·L^(-1)),CDK5 inhibitor roscovitine(15 μmol·L^(-1)) and calpain preparation calpeptin( 20 μmol·L^(-1)) for 24 h,followed by reaction with 20 μmol·L^(-1)Aβ_(25-351) for 24h.Tau protein phosphorylation levels at Ser396,Ser202 and Thr231 and the protein level of CDK5 activator proteins p25 / p35 were assayed by Western blot.Fluorescence intensity was measured with a fluorescence microplate reader to reflect calpain activity.CDK5 kinase activity was assayed by immunoprecipitation.Results After 20 μmol·L^(-1)Aβ_(25-351) acting on cortical neurons for 24 h,there were increments in the following: Tau protein phosphorylation levels at Ser396,Ser202 and Thr231,CDK5 kinase activity,CDK5 activator protein p25 level,and calpain activity.In the 20 mg·L^(-1) FZS treatment group,Aβ_(25-351) was suppressed markedly,resulting in increments in Tau protein phosphorylation levels at Ser396,Ser202 and Thr231,suppression of CDK5 kinase activity and p25 protein level,and elevation in calpain activity.Both CDK5 inhibitor roscovitine and calpain preparation calpeptin,as positive control drugs,also played a role in suppressing Tau protein hyperphosphorylation.Conclusion FZS can suppress Aβ_(25-351)-induced Tau protein hyperphosphorylation in cortical neurons through the calpain/CDK5 pathway.

关 键 词：阿尔茨海默病 CDK5 TAU蛋白 复智散 钙蛋白酶 p25 

分 类 号：R-332[医药卫生] R282.71