Full
Outline
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).
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, 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.
① Compared 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), reduced ΔΨM (P<0.05), up-regulated demethylated-PP2Ac (P<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 (P<0.01), up-regulation of PINK1, LC3Ⅱ and VDAC proteins, down-regulation of P62 (P<0.05), reduced total and mitochondrial ROS levels, and enhanced ΔΨM (P<0.01). The mRNA expression of Fizz1, Arg-1, and TGF-β was 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).
Fructose induces PP2Ac demethylation and then mitochondrial oxidative damage in M2-type macrophages. PP2A activation promotes mitophagy and reverses fructose-induced damage.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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