Ferrostatin-1 inhibits fibroblast fibrosis in keloid by inhibiting ferroptosis

Abstract

Background: Keloid is a chronic proliferative fibrotic disease caused by abnormal fibroblasts proliferation and excessive extracellular matrix (ECM) production. Numerous fibrotic disorders are significantly influenced by ferroptosis, and targeting ferroptosis can effectively mitigate fibrosis development. This study aimed to investigate the role and mechanism of ferroptosis in keloid development.

Methods: Keloid tissues from keloid patients and normal skin tissues from healthy controls were collected. Iron content, lipid peroxidation (LPO) level, and the mRNA and protein expression of ferroptosis-related genes including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), transferrin receptor (TFRC), and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined. Mitochondrial morphology was observed using transmission electron microscopy (TEM). Keloid fibroblasts (KFs) were isolated from keloid tissues, and treated with ferroptosis inhibitor ferrostatin-1 (fer-1) or ferroptosis activator erastin. Iron content, ferroptosis-related marker levels, LPO level, mitochondrial membrane potential, ATP content, and mitochondrial morphology in KFs were detected. Furthermore, the protein levels of α-smooth muscle actin (α-SMA), collagen I, and collagen III were measured to investigate whether ferroptosis affect fibrosis in KFs.

Results: We found that iron content and LPO level were substantially elevated in keloid tissues and KFs. SLC7A11, GPX4, and Nrf2 were downregulated and TFRC was upregulated in keloid tissues and KFs. Mitochondria in keloid tissues and KFs exhibited ferroptosis-related pathology. Fer-1 treatment reduced iron content, restrained ferroptosis and mitochondrial dysfunction in KFs, Moreover, ferrostatin-1 restrained the protein expression of α-SMA, collagen I, and collagen III in KFs. Whereas erastin treatment showed the opposite results.

Conclusion: Ferroptosis exists in keloid. Ferrostatin-1 restrained ECM deposition and fibrosis in keloid through inhibiting ferroptosis, and erastin induced ECM deposition and fibrosis through intensifying ferroptosis.

Keywords: Erastin; Ferroptosis; Ferrostatin-1; Fibroblasts; Fibrosis; Keloid.

Figure 1. Iron content was elevated and ferroptosis is present in keloid tissues.

We collected keloid tissues (n = 70) from keloid patients and normal skin tissues from healthy controls (n = 40). (A) Iron content was gauged using the iron assay kit. (B–E) SLC7A11, GPX4, TFRC, and Nrf2 mRNA levels were determined with RT-qPCR analysis. (F–J) SLC7A11, GPX4, TFRC, and Nrf2 protein levels were assessed with Western blot analysis. (K–L) LPO level was detected using C11-BODIPY staining. (M) Mitochondrial morphology was assessed using TEM. Data were presented as mean ± SD. **P <0.01.

Figure 2. Iron content was elevated and ferroptosis is present in KFs.

(A) Iron content was gauged using the iron assay kit. (B–E) The of SLC7A11, GPX4, TFRC, and Nrf2 mRNA levels were determined with RT-qPCR analysis. (F–J) The protein levels SLC7A11, GPX4, TFRC, and Nrf2 were assessed with Western blot analysis. (K–L) LPO level was detected using C11-BODIPY staining. (M) Mitochondrial morphology was assessed using TEM. Data were presented as mean ± SD. **P <0.01. (keloid fibroblasts KFs), NFs (normal skin fibroblasts).

Figure 3. Ferrostatin-1 restrained ferroptosis and mitochondrial dysfunction in KFs.

(A) The viability of KFs was assessed using CCK-8 assay after treatment with 0, 0.5, 1, 5, 10, and 20 µM fer-1. (B) Iron content in KFs was gauged using the iron assay kit. (C–G) SLC7A11, GPX4, TFRC, and Nrf2 protein levels were assessed with Western blot analysis. (H–I) ROS level in KFs was evaluated with immunofluorescence. (J) MDA level was tested with an ELISA kit. (K–L) LPO level was detected using C11-BODIPY staining. (M) GSH level was examined using an EKISA kit. (N) Mitochondrial membrane potential of KFs was determined with JC-1 staining. (O) ATP content was gauged with commercial kits. (P) Mitochondrial morphology was assessed using TEM. Data were presented as mean ± SD. *P <0.05, **P <0.01.

Figure 4. Ferrostatin-1 restrained ECM deposition and fibrosis in KFs.

KFs were treated with 1 and 5 µM fer-1 for 16 h, respectively. (A–D) α-SMA, collagen I, and collagen III protein levels in KFs were tested with Western blot analysis. (E–H) Immunofluorescence staining was used to evaluate α-SMA, collagen I, and collagen III protein levels in KFs Data were presented as mean ± SD. **P <0.01.

Figure 5. Erastin accelerated ferroptosis and mitochondrial dysfunction in KFs.

(A) The viability of KFs was assessed using CCK-8 assay after treatment with 0, 0.5, 1, 5, 10 µM erastin. (B) Iron content in KFs was gauged using the iron assay kit. (C–G) SLC7A11, GPX4, TFRC, and Nrf2 protein levels were assessed with Western blot analysis. (H–I) ROS level in KFs was evaluated with immunofluorescence. (J) MDA level was examined using an ELISA kit. (K–L) LPO level was detected using C11-BODIPY staining. (M) GSH level in KFs was tested with an ELISA kit. (N) Mitochondrial membrane potential of KFs was determined with JC-1 staining. (O) ATP content was gauged with commercial kits. (P) Mitochondrial morphology was assessed using TEM. Data were presented as mean ± SD. *P <0.05, **P <0.01.

Figure 6. Erastin induced ECM deposition and fibrosis in KFs.

KFs were treated with 5 and 10 µM erastin for 24 h, respectively. (A–D) α-SMA, collagen I, and collagen III protein levels in KFs were tested with Western blot analysis. (E-H) Immunofluorescence staining was used to evaluate α-SMA, collagen I, and collagen III protein levels in KFs Data were presented as mean ± SD. **P <0.01.