二甲双胍通过增强M 146 L细胞IDE表达促进胞内Aβ降解  

Metformin Facilitates Intracellular Amyloid β Degradation by Increasing Insulin-degrading Enzyme Expression in M146L Cell Line

作　　者：谢婷 徐采利 唐姣玲 郭开华[1] XIE Ting;XU Cai-li;TANG Jiao-ling;GUO Kai-hua(Department of Anatomy and Physiology,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou 510080,China)

机构地区：[1]中山大学中山医学院解剖生理学系,人体解剖学教研室,广东广州510080

出　　处：《中山大学学报（医学科学版）》2022年第4期522-529,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广州市科技计划项目(202002030441);广东省自然科学基金(2019A1515011184,2020A1515010012)。

摘　　要：【目的】探讨二甲双胍(Met)作用AD细胞模型M146L的淀粉样蛋白-β(Aβ)病理改善作用及其潜在机制。【方法】MTT比色法检测不同浓度Met对细胞活力的影响;Western Blotting检测Met处理后Aβ_(42)、生成Aβ相关的淀粉样蛋白前体(APP)、β分泌酶(BACE),以及Aβ降解相关的胰岛素降解酶(IDE)、脑啡肽酶(NEP)、轻链蛋白3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)的表达量变化;免疫荧光检测Met处理后Aβ_(42)、IDE的表达;Western Blotting检测IDE的特异性抑制剂杆菌肽(Bac)和Met联合处理后IDE和Aβ_(42)的表达情况。【结果】Met处理可显著下调M146L细胞Aβ_(42)蛋白表达水平(P<0.05),但Met对M146L细胞内APP、BACE表达无显著影响(P>0.05)。另外,M146L细胞内IDE、NEP及LC3Ⅱ/Ⅰ水平显著低于正常对照组(中国仓鼠卵巢细胞,CHO细胞)(P<0.05)。当Met处理24 h后,IDE蛋白表达和免疫荧光检测均明显升高(P<0.05)。一旦Met和IDE的特异性抑制剂Bac联合处理M146L细胞24 h,观察到IDE表达下降(P<0.05),而细胞内Aβ_(42)水平则明显升高(P<0.05)。【结论】本研究明确了Met对M146L细胞Aβ_(42)异常积累的改善作用,并且初步阐明其潜在机制是通过促进Aβ降解途径中IDE表达增加介导的,为T2DM药物二甲双胍作为AD潜在治疗药物提供实验基础。【Objective】To explore the effects of metformin(Met)on amyloid-β(Aβ)in AD cell model M146L and the underlying mechanism.【Methods】MTT assay was performed to determine the optimal concentration of Met.Western blotting was performed to measure the protein levels of Aβ_(42),amyloid precursor protein(APP),β-site APP-cleaving en⁃zyme(BACE)and proteolytic enzymes including insulin-degrading enzyme(IDE),neprilysin(NEP),and light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ);Immunofluorescence was performed to visualize how Aβ_(42) peptides and IDE were affected by Met.Western blotting was performed to test levels of IDE and Aβ_(42) after treatment of Met and Bacitracin(Bac),a special inhibi⁃tor of IDE.【Results】In this study,we showed that the levels of Aβ_(42) were down-regulated by the Met treatment(P<0.05).No effect was observed on the expression of APP and BACE,both of which are related to the production of Aβ,after Met treatment in M146L cells(P>0.05).Furthermore,levels of proteolytic enzymes including IDE,NEP,and LC3Ⅱ/Ⅰlevels were markedly decreased in the M146L cells compared to the CHO cells(P<0.05),and only the level of IDE was re⁃versed after the Met treatment for 24 h(P<0.05).Importantly,Met-mediated Aβ_(42) degradation in M146L cells was com pletely blocked by the Bac(P<0.05).【Conclusions】This study clarifies the ameliorating effect of Met on the abnormal ac⁃cumulation of Aβ_(42) in M146L cells,and that the underlying mechanism is mediated by the increased expression of IDE in the Aβdegradation pathway,providing an experimental basis for the T2DM drug metformin as a potential therapeutic target for AD.

关 键 词：二甲双胍 胰岛素降解酶 淀粉样蛋白-β 阿尔兹海默症 M146L细胞系 

分 类 号：R34[医药卫生—基础医学]