Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages

LI Xiaoman; LAN Li; LONG Yijin; LI Huilian; WANG Minghong; WANG Xinhan; LI Xiyi; TANG Shen

doi:10.16016/j.2097-0927.202505052

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Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages

Basic Medicine | 更新时间：2025-09-28

- Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages
- Journal of Army Medical University Vol. 47, Issue 18, Pages: 2186-2196(2025)
- 作者机构：
  
  1.广西医科大学：，基础医学院免疫学教研室，广西南宁
  2.广西医科大学：，广西高校基础医学研究重点实验室，广西南宁
  3.广西医科大学：，公共卫生学院营养与食品卫生学系，广西南宁
- 作者简介：
- 基金信息：
  
  the Regional Science Fund Program of National Natural Science Foundation of China(82260320;82460638)
- DOI：10.16016/j.2097-0927.202505052
  CLC： R363.2;R392.1;R392.12
- Received：22 May 2025，
  
  Revised：2025-09-11，
  
  Accepted：11 September 2025，
  
  Published：30 September 2025
- 稿件说明：
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李晓蔓，蓝利，龙以瑾，等.蛋白磷酸酶2A促进线粒体自噬减轻果糖诱导的M2型巨噬细胞线粒体氧化损伤［J］.陆军军医大学学报，2025，47（18）：2186-2196.DOI：10.16016/j.2097-0927.202505052.

LI XIAOMAN，LAN LI，LONG YIJIN，et al.Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages［J］.J Army Med Univ，2025，47（18）：2186-2196.DOI：10.16016/j.2097-0927.202505052.
李晓蔓，蓝利，龙以瑾，等.蛋白磷酸酶2A促进线粒体自噬减轻果糖诱导的M2型巨噬细胞线粒体氧化损伤［J］.陆军军医大学学报，2025，47（18）：2186-2196.DOI：10.16016/j.2097-0927.202505052. DOI：

LI XIAOMAN，LAN LI，LONG YIJIN，et al.Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages［J］.J Army Med Univ，2025，47（18）：2186-2196.DOI：10.16016/j.2097-0927.202505052. DOI：

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+1.00 μmol/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（

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（

0.05），线粒体膜电位水平下降（

0.05），同时Total-PP2Ac未见改变，而Demethylated-PP2Ac蛋白表达显著上调（

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（

0.01），线粒体自噬相关蛋白PINK1、LC3Ⅱ及VDAC表达均上调（

0.05），自噬底物P62表达下调（

0.05），总体及线粒体源ROS水平降低、线粒体膜电位上升（

0.01）；与M2组相比，M2+Fru组标志物

Fizz1

、

Arg

及

TGF

的mRNA表达下降（

0.05），ABL127干预后上述标志物表达升高（

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。

Abstract

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).

Methods

① Immortalized 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. ② Ana-1 cells were also divided into M0

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+1.00 μmol/L ABL127

24 h) to investigate PP2A-mediated mechanisms. Numbers of mitochondria and lysosomes

ROS level

and ΔΨM 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

)

and

TGF-β

were measured using RT-qPCR.

Results

① Compared with the M2 group

fructose treatme

nt 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 (

0.05). Fructose of 5.00 mmol/L resulted in significantly elevated mitochondrial ROS and mitochondrial quantity (

0.05)

reduced ΔΨM (

0.05)

up-regulated demethylated-PP2Ac (

0.05)

and no changed total-PP2Ac protein level. ② Compared with the M2 + Fru group

the addition of ABL127 led to decreased number of mitochondria but increased number of lysosomes (

0.01)

up-regulation of PINK1

LC3Ⅱ and VDAC proteins

down-regulation of P62 (

0.05)

reduced total and mitochondrial ROS levels

and enhanced ΔΨM (

0.01). The mRNA expression of

Fizz1

Arg-1

and

TGF-β

was notably decreased in the M2+Fru group than the M2 group (

0.05)

and the levels were rescued by ABL127 treatment (

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.

关键词

Keywords

references

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