摘要
目的观察中药复方-还脑益聪方提取物(Huannao Yicong decoction,HYD)含药血清对β-淀粉样前蛋白swe基因(APPswe)人类早老素(PSEN1d E9,PS1)和早老蛋白1d E9基因(PSEN1d E9)人鼠淀粉样前蛋白融合体(APPswe,APP)双基因转染人胚肾293(human embryonic kidney 293,HEK293)细胞γ分泌酶活性及相关蛋白的影响,分析含药血清影响γ分泌酶活性相关蛋白的作用机制。方法以人APP/PS1双基因转染(HEK293)人胚肾细胞株为载体,将其分为5组,分别为空白组,7.5%HYD含药血清组,15%HYD含药血清组,HYD直接给药组,γ分泌酶抑制剂-DAPT组,复苏传代后采用细胞计数试剂盒CCK-8(cell counting kit-8,CCK-8)法检测HYD含药血清(大鼠灌胃10 d,取血清并进行血药成分检测)对人APP/PS1双基因转染细胞的生长活力和超微结构的影响;透射电镜观察各组细胞超微结构;双荧光素酶基因检测γ分泌酶活性;蛋白质印迹法(Western blot)检测γ分泌酶组件及相关蛋白早老素1(Presenilin 1,PS1)、鸟苷酸结合蛋白(GTP binding protein,CDC42)、热休克蛋白70羧基端作用蛋白(carboxyl terminus of Hsc70-interacting protein,CHIP)、前咽缺陷蛋白-1α(anterior pharynx defective-1α,Aph-1α)和低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的表达。结果 15%HYD含药血清组对细胞株的增殖作用高于其余各血清浓度组(P<0.05,P<0.01);透射电镜显示模型组线粒体膜不完整,有轻微肿胀,线粒体嵴模糊,胞质内有较多的脂褐素沉积,其余各组数量均有一定程度的好转和减轻。γ分泌酶活性结果,给药后24 h和48 h,与模型组比较,直接给药组、DAPT组差异有统计学意义(P<0.05或P<0.01);与DAPT组比较,15%HYD含药血清组差异有统计学意义(P<0.05)。γ分泌酶结果与模型组比较,各给药组的PS1蛋白、CDC42蛋白表达差异有统计学意义(P<0.05,P<0.01),CHIP蛋白表达和Aph-1α蛋白表达无统计学意义(P>0.05),而HYD直接给药组HIF-1α蛋白差异有统计学意义
Aim Toobservetheeffectofconcisepre-scriptions of Chinese medicine Huannao Yicong Decoc-tion（HYD）on regulatory pathway of secretase in APP/PS1 double transgenic cell line（HEK293）,and to in-vestgateitsmechanism.Methods Theproliferationof AD cell model and the toxicity of each investigational drugs were ebserved by CCK-8;the changes in micro-scopic structure of each group were observed by（Trans-mission electron microscope,TEM）;the activities of gamma-secretes was observed by Dual Luciferase Re-porter Gene Assay Kit ,and then the expression of pre-senilin 1（PS1）,carboxyl terminus of Hsc70-interacting protein（CHIP）,GTP binding protein （CDC42 ）,ante-rior pharynx defective-1α（APH-1α）,Hypoxia induc-ible factor-1α（HIF-1α） were detected by Western blot.Results 15%HYDserumincreasedthecellac-tivity compared to blank serum （P 〈0. 01 ）.Under TEM,human APP/PS1 Double Transgenic cell line’s mitochondria showed swelling and degenerat,and lipo-fuscin was deposited;after 48 h of medication,theswelling of mitochondria was reduced and crista was clear,and the number of lipofuscin was reduced.For the activity ofγsecretase,after 24 h medication,com-pared to control group,HYD directly group and DAPT group obviously inhibited the activity ofγsecretase （P〈0. 05 or P〈0. 01 ）,compared to DAPT group,only 15% serum containing HYD group had significant difference （P 〈0. 05 ）;after 48 h medication,com-pared to control group,the other group all showed sig-nificant difference（P〈0. 05 or P〈0. 01 ）.For the in-fluence of PS pathway,after 48 h medication,com-pared to control group,the other group all inhibited the PS1protein expression（P〈0. 05 or P〈0. 01）,HYD directly group could activate the CDC42 protein expres-sion（P〈0. 05 ）;compared to 0 h midicaiton,DAPT group inhibited the CHIP protein expression at the point of 48 h（P〈0. 05 ）and activated the CDC42 pro-tein expression at the point of 24 h.For the influence of Aph-1 pathway,there was no significant differenc
出处
《中国药理学通报》
CAS
CSCD
北大核心
2017年第3期417-426,共10页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81573819)
北京市科技计划课题"十病十药"研发项目资助课题(No Z141100002214003)
北京市自然科学基金项目(No 7162169)
关键词
阿尔茨海默病
Γ分泌酶
还脑益聪方提取物
含药血清
人APP/PS1双转基因细胞
中药复方
Alzheimer＇s disease（AD）
γ secretase
extract of Huannao Yicong Decoction
serum containing drug
human APP/PS1 double transgenic cell line
Chinese medicine compound
