摘要
目的研究果糖暴露对M2型巨噬细胞线粒体氧化损伤的影响,并采用蛋白磷酸酶甲酯化酶-1(protein phosphatase methylesterase-1,PPME-1)抑制剂ABL127特异性激活蛋白磷酸酶2A(protein phosphatase 2A,PP2A)以阐明其调控作用。方法①以永生化小鼠骨髓来源巨噬细胞系(Ana-1)为研究对象,设置M0组(常规培养)、M2组(20.00 ng/mL IL-4处理24 h)及M2+梯度果糖处理组(20 ng/mL IL-4联合0.04、0.20、1.00、5.00 mmol/L果糖处理24 h)。采用CCK-8法检测细胞活性,荧光探针法检测线粒体数量、细胞总活性氧(reactive oxygen species,ROS)、线粒体源ROS及线粒体膜电位水平,Western blot法检测PP2Ac总蛋白(total-PP2Ac)及去甲基化PP2Ac(demethylated-PP2Ac)蛋白表达水平。②设置M0组、M2组、M2+Fru组(20.00 ng/mL IL-4+5.00 mmol/L果糖处理24 h)及M2+Fru+ABL127组(20.00 ng/mL IL-4+5.00 mmol/L果糖+1.00μmol/L ABL127处理24 h)探究PP2A干预机制。荧光探针法检测线粒体与溶酶体数量、ROS及线粒体膜电位水平,Western blot法检测线粒体自噬相关蛋白PTEN诱导激酶1(PTEN induced putative kinase 1,PINK1)、自噬底物P62(sequestosome 1,SQSTM1/P62)、微管相关轻链蛋白3(microtubule-associated protein light chain 3,LC3)及电压依赖性阴离子通道(voltage-dependent anion channel,VDAC)表达,RT-qPCR检测M2型巨噬细胞表型标志物炎症区域分子1(found in inflammatory zone 1,Fizz1)、精氨酸酶1(arginase-1,Arg-1)及转化生长因子-β(transforming growth factor-β,TGF-β)的mRNA表达。结果①与M2组相比,0.04~5.00 mmol/L梯度果糖处理组在不影响细胞活性前提下呈剂量依赖性诱导M2型巨噬细胞总ROS水平升高(P<0.05),果糖浓度达5.00 mmol/L时线粒体源ROS及线粒体数量增加(P<0.05),线粒体膜电位水平下降(P<0.05),同时Total-PP2Ac未见改变,而Demethylated-PP2Ac蛋白表达显著上调(P<0.05)。②与M2+Fru组相比,M2+Fru+ABL127组线粒体数量减少、溶酶体数量增加(P<0.01),线粒体自噬相关蛋白PINK1、LC3Ⅱ及VDAC表达均上调(P<0.05),自噬底物P62表达下调(P<0.05),总体及线粒体源ROS水平降低、线粒体膜电位上升(P<0.01);与M2组相比,M2+Fru组标志物Fizz1、Arg-1及TGF-β的mRNA表达下降(P<0.05),ABL127干预后上述标志物表达升高(P<0.05)。结论果糖诱导M2型巨噬细胞PP2Ac去甲基化造成线粒体氧化损伤,激活PP2A显著促进线粒体自噬以逆转该损伤。
Objective To investigate the effects of fructose exposure on mitochondrial oxidative damage in M2-type macrophages and elucidate the regulatory role of protein phosphatase 2A(PP2A)in the process using its specific activator ABL127,an inhibitor of protein phosphatase methylesterase-1(PPME-1).Methods①Immortalized mouse bone marrow-derived macrophages Ana-1 were subjected and grouped into M0(conventional culture),M2(treated with 20 ng/mL IL-4 for 24 h),and M2+Fru groups(IL-4 plus 0.04,0.20,1.00,or 5.00 mmol/L fructose).Cell viability was assessed with CCK-8 assay.Number of mitochondria,total and mitochondrial levels of reactive oxygen species(ROS),and mitochondrial membrane potential(ΔΨM)were measured using fluorescent probes.Total and demethylated PP2Ac protein levels were detected by Western blotting.②Ana-1 cells were also divided into M0,M2,M2+Fru(20 ng/mL IL-4+5.00 mmol/L fructose,24 h),and M2+Fru+ABL127(20 ng/mL IL-4+5.00 mmol/L fructose+1.00μmol/L ABL127,24 h)to investigate PP2A-mediated mechanisms.Numbers of mitochondria and lysosomes,ROS level,andΔΨM were detected via fluorescence assays.Expression of mitophagy-related proteins,PTEN induced putative kinase 1(PINK1),P62,microtubule-associated protein light chain 3(LC3),and voltage-dependent anion channel(VDAC)was evaluated by Western blotting,and the mRNA levels of M2 markers,found in inflammatory zone 1(Fizz1),arginase-1(Arg-1),andT GF-βwere measured using RT-qPCR.Results①Compared with the M2 group,fructose treatment at a concentration ranging from 0.04 to 5.00 mmol/L showed no effect on cell viability in M2 macrophages,but increased total ROS level in a dose-dependent manner(P<0.05).Fructose of 5.00 mmol/L resulted in significantly elevated mitochondrial ROS and mitochondrial quantity(P<0.05),reducedΔΨM(P<0.05),up-regulated demethylated-PP2Ac(P<0.05),and no changed total-PP2Ac protein level.②Compared with the M2+Fru group,the addition of ABL127 led to decreased number of mitochondria but increased number of lysosomes(P<0.01),up-regulation of PINK1,LC3Ⅱand VDAC proteins,down-regulation of P62(P<0.05),reduced total and mitochondrial ROS levels,and enhancedΔΨM(P<0.01).The mRNA expression of Fizz1,Arg-1,and TGF-βwas notably decreased in the M2+Fru group than the M2 group(P<0.05),and the levels were rescued by ABL127 treatment(P<0.05).Conclusion Fructose induces PP2Ac demethylation and then mitochondrial oxidative damage in M2-type macrophages.PP2A activation promotes mitophagy and reverses fructose-induced damage.
作者
李晓蔓
蓝利
龙以瑾
李慧莲
王名鸿
王新航
李习艺
唐深
LI Xiaoman;LAN Li;LONG Yijin;LI Huilian;WANG Minghong;WANG Xinhan;LI Xiyi;TANG Shen(Department of Immunology,School of Basic Medical Sciences,School of Public Health,Guangxi Medical University,Nanning,Guangxi,China;Key Laboratory of Basic Medical Sciences of Guangxi Higher Education Institutions,School of Public Health,Guangxi Medical University,Nanning,Guangxi,China;Department of Nutrition and Food Hygiene,School of Public Health,Guangxi Medical University,Nanning,Guangxi,China)
出处
《陆军军医大学学报》
2025年第18期2186-2196,共11页
Journal of Army Medical University
基金
国家自然科学基金地区科学基金项目(82260320,82460638)。
作者简介
通信作者:唐深,E-mail:958714333@qq.com。
