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(8):857-868
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To understand the iron content and transferrin receptor 1 (TfR1) expression levels in keloid and normal skin tissues, an in vitro model of keloid fibroblasts (KFB) ferroptosis induced by Erastin was constructed, 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 were examined. Methods:Six keloid tissues and six normal skin tissues were collected from the First Affiliated Hospital of Xinjiang Medical University from March to June 2022, and the tissue iron content kit was used to determine the iron content in the dermis of the two tissues, and TfR1 protein expression was detected in the two tissues by Western blot. Primary KFB and normal skin fibroblasts (NFB) were obtained by tissue block culture method, and the effect of different concentrations of Erastin and Fer-1 on cell activity was detected by using Erastin-induced KFB ferroptosis model and CCK-8 method to screen the appropriate drug concentration. The follow-up 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, while KFB was used as the experimental object in the last 4 groups. Cell migration ability was detected by scratch assay, and malondialdehyde (MDA), reactive oxygen species (ROS) and Fe 2+ content in each group of cells were detected by fluorescent probe method and kits; the protein expression 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 blot, and the protein expression and localization of TfR1, Gpx4 in KFB were detected by immunofluorescence. GraphPad Prism 9.0 statistical software was used, and the measurement data were expressed as Mean±SD. Independent sample t-test was used for comparison between 2 groups, and one-way ANOVA was used for comparison between multiple groups, then LSD-t test was used for pairwise comparison between groups. P< 0.05 indicated that the differences were statistically significant. Results:The iron content and TfR1 protein expression were significantly higher in keloid compared with normal skin tissue ( P<0.01). The proliferation rate of KFB with different Erastin concentrations decreased gradually, 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<0.01) ; 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 Blot showed that, compared with the NFB group, iron death indexes of SLC7A11 and GPx4 protein expression were significantly reduced ( P<0.01 or P<0.05), TfR1 protein expression was increased ( P<0.01), and fibrosis indexes of α-SMA and COL-1 protein expression were significantly increased ( P<0.01) in the control group; compared with the control group, SLC7A11 expression was reduced ( P<0.01) and TfR1, COL-1 expression increased ( P<0.01), while SLC7A11, GPx4 expression increased ( P< 0.01) and TfR1, α-SMA, COL-1 expression significantly decreased ( P<0.01) in the Fer-1 group; compared with the Erastin group, the GPx4, SLC7A11 expression was increased ( P< 0.01) and TfR1, α-SMA, COL-1 expression was significantly decreased ( P< 0.01) in the Erastin+Fer-1 group, suggesting that Fer-1 was able to reverse Erastin-induced ferroptosis and pro-fibrotic effects in KFB. Immunofluorescence showed that GPx4 was expressed in both nucleus and cytoplasm. Compared with control group, Fer-1 increased the fluorescence intensity of GPx4 in KFB ( P<0.01). Compared with Erastin group, the fluorescence intensity of GPx4 in Erastin+ Fer-1 group was significantly increased ( P<0.01). TfR1 was mainly expressed in cytoplasm. Compared with 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). Conclusions:Iron overload and free iron increase in keloids. Erastin induces ferroptosis in KFB and aggravates keloid fibrosis. Fer-1 reverses the oxidative damage and iron accumulation induced by Erastin and effectively inhibits ferroptosis and keloid fibrosis in KFB.
