基于转基因细胞模型研究通络醒脑泡腾片对Aβ代谢的影响  

Effects of Tongluoxingnao effervescent tablet on Aβ metabolism in transgenic cell model

作　　者：付文君[1,2] 代渊[2] 魏江平[1,2] 郑航[1,2] 马涛[3] 徐世军[1,2] 王永炎[4] 

机构地区：[1]四川省中药资源系统研究与开发利用国家重点实验室培育基地,成都611137 [2]成都中医药大学,成都610075 [3]北京中医药大学东方医院,北京100078 [4]中国中医科学院,北京100700

出　　处：《中国药理学通报》2016年第11期1571-1578,共8页Chinese Pharmacological Bulletin

基　　金：国家科技部"重大新药创制"科技重大专项(No2013ZX09103002-008);四川省学术和技术带头人培养资金资助项目(No 2015058);中药药理四川省青年科技创新研究团队(No 2014TD0007)

摘　　要：目的 采用转基因细胞模型,考察通络醒脑泡腾片对Aβ生成和降解关键靶点的影响,探索其抗阿尔茨海默病的作用机制。方法 体外培养SH-SY5Y-APP和SH-SY5Y-C99细胞,不同浓度的含药血清的培养液（含通络醒脑泡腾片血清0%-40%）干预48h,采用MTT法确定无毒浓度,LDH法检测细胞活性,ELISA法检测Aβ1-40和Aβ1-42分泌量;采用RT-PCR和Westernblot检测APP基因和蛋白表达,West-ernblot法检测APP剪切片段sAPPα和sAPPβ蛋白表达;qRT-PCR、Westernblot及荧光检测试剂盒分别测定BACE1基因、蛋白水平和酶活性;Westernblot检测Aβ降解酶IDE和NEP蛋白表达;体外培养SH-SY5Y,通络醒脑泡腾片不同浓度的含药血清培养液孵育48h,MTT法检测Aβ25-35诱导后细胞存活率。结果 通络醒脑泡腾片含药血清对SH-SY5Y-APP和SH-SY5Y-C99细胞均无明显毒性作用（P〉0.05）;通络醒脑泡腾片含药血清可明显抑制SH-SY5Y-APP细胞Aβ的分泌（P〈0.05）,对SH-SY5Y-C99细胞Aβ分泌量无影响;对SH-SY5Y-APP细胞APP基因及蛋白水平、sAPPα及Aβ降解酶NEP和IDE蛋白表达均无明显影响（P〉0.05）,但对sAPPβ蛋白有明显抑制作用（P〈0.05）,且呈剂量依赖关系,对BACE1基因、蛋白水平、活性均有明显抑制作用（P〈0.01）。此外,研究发现通络醒脑泡腾片含药血清对Aβ25-35诱导的SH-SY5Y细胞凋亡具有保护作用（P〈0.01）。结论 通络醒脑泡腾片对β-分泌酶具有明显的抑制作用,并能对抗Aβ神经细胞毒性作用,提示抑制β-分泌酶和拮抗Aβ神经细胞毒性是通络醒脑泡腾片发挥抗阿尔茨海默病的主要作用机制。Aim Tongluoxingnao effervescent tablets( TLXNET),based on the ancient formula of Qiong Gui Tang,can improve cognitive dysfunction in different AD models. This research is aimed to study the effects of TLXNET on the Aβ metabolism and explore the antiAD mechanism in SH-SY5Y-APP and SH-SY5Y-C99 cells. Methods Cells were incubated for 48 h in different concentrations of medicated serum( containing0% ~ 40% of TLXNET in the serum). Firstly,the non-toxic concentration was measured by MTT assay,the activity of cells was detected by LDH methods,and ELISA was used to measure the levels of Aβ_(1- 40) and Aβ_(1- 42). Then,the gene and protein expressions of APP were detected via RT-PCR and Western blot of which the expressions of shearing fragments such as sAPPα and sAPPβ were investigated by Western blot,the same as the expressions of IDE and NEP. Additionally,the level of mRNA,protein and activity of BACE1 were measured by qRT-PCR,Western blot and fluorescence detection kits respectively. Eventually,MTT assay was performed to detect the cell viability after the SH- SY5Y cells were treated with variousconcentrations of medicated serum and Aβ_(25- 35) for48 h. Results There was no significant toxicity of TLXNET medicated serum in SH-SY5Y-APP and SHSY5Y-C99 cells( P > 0. 05). TLXNET could significantly inhibit Aβ secretion in SH-SY5Y-APP cells( P< 0. 05),but had no effect on Aβ secretion in SHSY5Y-C99 cells,the same as the level of APP mRNA and protein in SH-SY5Y-APP cells and the expressions of IDE and NEP( P > 0. 05). Additionally,TLXNET could still notably inhibit the expression of sAPPβ protein in a dose-dependent way,with statistical significance( P < 0. 05). Meanwhile,the level of mRNA,protein and activity of BACE1 were also significantlydecreased by TLXNET( P < 0. 01). Moreover,the medicated serum of TLXNET had a protective effect on SH-SY5Y apoptosis induced by Aβ_(25- 35)( P < 0. 01).Conclusion TLXNET could obviously inhibit β-secretase enzyme,and has an antagonistic effect against Aβ neurotoxicity,which

关 键 词：阿尔茨海默病 通络醒脑泡腾片 SH-SY5Y-APP细胞 SH-SY5Y-C99细胞 Aβ生成 Aβ降解 

分 类 号：R282.71[医药卫生—中药学] R329.2[医药卫生—中医学]