还脑益聪方提取物含药血清对APP/PS 1双基因转染细胞γ分泌酶活性及相关蛋白表达的影响

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(PSEN1dE9,PS1)和早老蛋白1dE9基因(PSEN1dE9)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/PS 1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-interac-ting protein,CHIP)、前咽缺陷蛋白-1α(anterior pharynx defec-tive-1α,Aph-1α)和低氧诱导因子-1α(hypoxia inducible fac-tor-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(P0.05),而HYD直接给药组HIF-1α蛋白差异有统计学意义(P0. 05 );compared to control group,HYD directly group inhibited the HIF-1αprotein ex-pression after 48h medication(P<0. 05);compared to 0h midicaiton,DAPT group inhibited the HIF-1αpro-tein expression at the point of 24 h (P<0. 05 ).Con-clusions HYDcantreatADthroughprotectingthe mitochondrial function,reducing the formation of lipo-fuscin,inhibiting the activity of γsecretase by down-regulating the activity of HIF-1α,decreasing the stabil-ity and activity of PS1 by promoting the expression of CDC42.This shows that HYD has good research and development prospect as an effective drug for preven-tion and treatment of AD.