The role and mechanism of iron overload and ferroptosis in keloid fibroblasts
10.3760/cma.j.cn114453-20230224-00041
- VernacularTitle:铁过载及铁死亡在瘢痕疙瘩成纤维细胞中的作用及机制
- Author:
Ling GONG
1
;
Yu LI
;
Mingxuan LI
;
Juan MA
;
Hongyu CHI
;
Xianglin DONG
Author Information
1. 新疆医科大学第一附属医院整形科，乌鲁木齐 830011
- Keywords:
Keloid;
Fibroblasts;
Ferroptosis;
Iron overload;
Reactive oxygen species;
Lipid peroxidation
- From:
Chinese Journal of Plastic Surgery
2023;39(12):1299-1310
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the iron content and transferrin receptor 1 (TfR1) expression levels in keloid and normal skin tissues. Erastin induced ferroptosis model of keloid fibroblasts (KFB) is constructed in vitro, and the effects of Erastin and Ferrostatin-1 (Fer-1) on cell viability, ferrous ion (Fe 2+ ) content and lipid peroxidation, ferroptosis and fibrosis-related regulatory factors are examined. Methods:Six keloid tissues and six prepuces were collected from the First Affiliated Hospital of Xinjiang Medical University from March to June 2022. The tissue iron content kit was used to determine the iron content in the dermis, and TfR1 protein expression level was detected by Western blotting. Primary KFB and normal skin fibroblasts (NFB) were obtained by tissue cultivation, Erastin-induced KFB ferroptosis model and CCK-8 assay were used to detect the effects of different concentrations of Erastin and Fer-1 on cell viability, and to screen the appropriate drug concentration. The subsequent experiments were divided into five groups: NFB group, control group, Erastin (0.6 μmol/L) group, Fer-1 (1 μmol/L) group, and Erastin (0.6 μmol/L) + Fer-1 (1 μmol/L) group. KFB was used in the last 4 groups. Cell migration ability was detected by scratch assay. The contents of malondialdehyde (MDA), reactive oxygen species (ROS) and Fe 2+ were detected by fluorescence probe and kits; the protein expression levels of TfR1, glutathione peroxidase 4 (GPx4), solute carrier family 7 member 11 (SLC7A11), α-smooth muscle actin (α-SMA) and type I collagen (COL-1) in each group of cells were detected by Western blotting; the protein expression and localization of TfR1 and Gpx4 in KFB were detected by immunofluorescence staining. GraphPad Prism 9.0 statistical software was used in the statistical analyses, and the measurement data were expressed as Mean±SD. Independent samples t-test was used for comparison between 2 groups, and one-way ANOVA was used for comparison between multiple groups, LSD- t test was used for pairwise comparison between groups. P<0.05 indicated statistical significance. Results:The iron content and TfR1 protein expression level were significantly higher in keloids compared with normal skin tissue ( P<0.01). The proliferation rate of KFB decreased as the Erastin concentration increased, the IC 50 was 0.61 μmol/L, and Fer-1 had no obvious toxicity to KFB in the range of 0.1-20 μmol/L. Scratch test showed that the migration rate of control group was significantly higher than that of NFB group ( P<0.01); compared with the control group, KFB migration rate decreased significantly after Erastin intervention ( P<0.01); compared with the Erastin group, KFB migration was significantly accelerated in the Erastin+ Fer-1 group ( P<0.01). Compared with the NFB group, ROS, MDA levels were significantly increased in the control group ( P<0.01); compared with the control group, ROS, MDA levels and Fe 2+ content were significantly higher in the Erastin group ( P<0.01), while ROS, MDA levels and Fe 2+ content were significantly lower in the Fer-1 group ( P<0.05); compared with the Erastin group, MDA, ROS levels and Fe 2+ content in the Erastin+ Fer-1 group were significantly decreased ( P<0.01). Western blotting results showed that, compared with the NFB group, ferroptosis indexes of SLC7A11 and GPx4 protein expression levels were significantly reduced ( P<0.01), TfR1 protein expression was increased ( P<0.01), and protein expression of fibrosis indexes, α-SMA and COL-1 were significantly increased ( P<0.01) in the control group; compared with the control group, Erastin group had reduced SLC7A11 expression ( P<0.01) and increased TfR1, COL-1 expression ( P<0.01), while SLC7A11, GPx4 expression increased ( P<0.01) and TfR1, α-SMA, COL-1 decreased ( P<0.01) in the Fer-1 group; compared with the Erastin group, the GPx4 and SLC7A11 expression levels were increased ( P<0.01) and TfR1, α-SMA, COL-1 expression levels were significantly decreased ( P<0.01) in the Erastin+ Fer-1 group, suggesting that Fer-1 was able to reverse the Erastin-induced ferroptosis and pro-fibrotic effects in KFB. Immunofluorescence staining showed that GPx4 was expressed in both the nucleus and the cytoplasm. Compared with the control group, Fer-1 increased the fluorescence intensity of GPx4 in KFB ( P<0.01). Compared with the Erastin group, the fluorescence intensity of GPx4 in Erastin+ Fer-1 group was significantly increased ( P<0.01). TfR1 was mainly expressed in the cytoplasm. Compared with the control group, Erastin increased the fluorescence intensity of TfR1 in KFB ( P<0.05), while Fer-1 group significantly decreased it ( P<0.01). Compared with Erastin group, the fluorescence intensity of TfR1 in Erastin+ Fer-1 group was significantly reduced ( P<0.01). Conclusion:Iron overload is present in keloids and the free iron level is increased. Erastin is able to induce ferroptosis in KFB and aggravate keloid fibrosis. Fer-1 can reverse the oxidative damage and iron accumulation induced by Erastin, and is able to effectively inhibit ferroptosis and keloid fibrosis in KFB.
