摘要
目的:探讨当归芍药散(DSS)含药血清对β淀粉样蛋白(Aβ)1-40损伤大鼠肾上腺嗜铬细胞瘤(PC12)细胞的保护作用及对泛素-蛋白酶体途径(UPP)的调控作用机制。方法:采用Aβ_(1-40)干预PC12细胞制备损伤模型。实验分为空白组、模型组、DSS含药血清高、中、低剂量组(10%、5%、2.5%)。细胞增殖与活性检测(CCK-8)法检测细胞存活率,流式细胞术检测细胞凋亡,酶联免疫吸附测定法(ELISA)检测细胞Aβ和磷酸化(p)-Tau蛋白含量,免疫荧光细胞化学(ICC)标记UPP重要靶点泛素(Ub)、泛素连接酶E3(E3)、26S蛋白酶体、泛素羧基端水解酶1(UCHL1)和UCHL3蛋白的表达,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测Ub、E3、26S、UCHL1、UCHL3 mRNA和蛋白的表达。结果:根据CCK-8实验结果,筛选5μmol·L^(-1)、48 h为PC12细胞损伤最佳造模条件。与空白组比较,模型组细胞存活率降低(P<0.05)、凋亡率升高(P<0.05),Aβ及p-Tau表达升高(P<0.05),Ub蛋白及mRNA表达升高,26S、E3、UCHL1+3蛋白及mRNA表达降低(P<0.05)。与模型组比较,DSS中剂量组细胞存活率升高(P<0.05);DSS各剂量组细胞凋亡率下降(P<0.05);DSS各剂量组Aβ含量降低(P<0.05),DSS高、中剂量组p-Tau含量降低(P<0.05);DSS各剂量组Ub mRNA表达降低(P<0.05),DSS各剂量组26S mRNA表达升高(P<0.05),DSS中剂量组UCHL1 mRNA表达升高(P<0.05),DSS高、中剂量组E3和UCHL 3mRNA表达升高(P<0.05);DSS各剂量组Ub蛋白表达降低,DSS高、中剂量组26S、E3、UCHL1+3蛋白表达升高,以DSS中剂量效果最佳。结论:DSS含药血清可提高细胞存活率、降低细胞凋亡率、清除Aβ和p-Tau蛋白沉积,对Aβ_(1-40)损伤PC12细胞有保护作用,其作用可能是通过调控UPP降低Ub表达及升高26S、E3、UCHL1及UCHL3表达来发挥。
Objective:To investigate the protective effect of Danggui Shaoyaosan(DSS)-contained serum on β-amyloid(Aβ)1-40-injured rat adrenal pheochromocytoma PC12 cells and its mechanism in regulating ubiquitin-proteasome pathway(UPP).Method:Aβ_(1-40) was used to intervene PC12 cells to prepare the cell models of Alzheimer’s disease(AD),and the experiment was divided into the blank,model,and DSScontained serum high,medium,and low-dose groups(10%,5%,and 2.5%).Cell viability and apoptosis were detected using cell counting kit-8(CCK-8)method and flow cytometry,respectively.The content of Aβ and p-Tau protein was determined by enzyme-linked immunosorbent assay(ELISA).The ubiquitin(Ub),ubiquitin ligase E3(E3),26S proteasome,ubiquitin carboxyl terminal hydrolase1(UCHL1),and UCHL3 protein expressions of UPP were displayed using immunofluorescence cytochemistry(ICC),and the mRNA and protein expression levels of Ub,E3-parkin,26S,UCHL1,and UCHL3 were determined by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)and Western blot,respectively.Result:The data of the CCK8 experiment verified that 5μmol·L^(-1) and 48 hours were the optimal conditions for modeling Aβ_(1-40)-injured PC12 cells.As compared with the blank group,the cell viability rate in the model group decreased(P<0.05)with an increased apoptosis rate(P<0.05),the content of Aβ and p-Tau contents was elevated(P<0.05),the mRNA and protein expression levels of Ub increased,and the mRNA and protein expression levels of 26S,E3,and UCHL1+3 decreased(P<0.05).As compared with the model group,the cell viability rate in the DSScontained medium-dose group increased(P<0.05),whereas the apoptosis rate in each DSS-contained group decreased(P<0.05).The content of Aβ in each DDS-contained group decreased(P<0.05),and the content of p-Tau in the DDS-contained high and medium-dose groups decreased(P<0.05).The mRNA expression level of Ub decreased,and that of 26S increased in each DDS-contained group(P<0.05).The mRNA expression level of UCHL1 in the DDS-co
作者
陈云慧
夏军
刘兴隆
淮文英
刘丹
张天娥
谢永美
游宇
岳雯
唐宋琪
彭伟
CHEN Yunhui;XIA Jun;LIU Xinglong;HUAI Wenying;LIU Dan;ZHANG Tiane;XIE Yongmei;YOU Yu;YUE Wen;TANG Songqi;PENG Wei(Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Sichuan University,Chengdu 610041,China;Hainan Medical University,Haikou 570102,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第24期17-25,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81603537,81473668)
四川省科技厅国际合作与交流项目(2017HH0004)
国家留学基金委项目(201908510279)
四川省教育厅重点项目(17ZA0163)
成都中医药大学青年学者人才项目(QNXZ2019043)
the 2019 Internationalization Incentive Fund of University of Otago(IIFUO2019)。
