Related Experiment Video
Updated: Jul 12, 2025
Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis
Published on: May 9, 2025
ACSL4 inhibition prevents macrophage ferroptosis and alleviates fibrosis in bleomycin-induced systemic sclerosis
Dianyu Cao1, Jina Zheng1, Zheng Li2
1Department of Dermatology, Zhongshan Hospital, Fudan University, No.180 Fenglin Road, Xuhui District, Shanghai, 200032, P.R. China.
Background:
Systemic sclerosis (SSc), with unclear pathophysiology, is a paradigmatic rheumatic disease of immunity dysfunction-driven multi-organ inflammation and ultimate fibrosis. Pathogenesis breakthroughs are urgently needed for available treatments halting its unremitting stiffness. This study aims to investigate whether ferroptosis can regulate the progressive SSc fibrosis.
Methods:
In vivo, bleomycin (BLM)-induced mice model was subjected to ferroptosis detection using western blotting, malondialdehyde (MDA), and glutathione (GSH) assays. Pharmacological inhibitor of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was utilized to explore its potential therapeutic effects for fibrosis, from histological, biochemical, and molecular analyses. In vitro, bone marrow-derived macrophages (BMDM) were activated into inflammatory phenotype and then the relationship was evaluated between activation level and ferroptosis sensitivity in lipopolysaccharide (LPS) incubation with gradient concentrations. The potential calpain/ACSL4 axis was analyzed after calpain knockdown or over-expression in Raw264.7.
Results:
Both skin and lung tissue ferroptosis were present in SSc mice with enhanced ACSL4 expression, while ACSL4 inhibition effectively halted fibrosis progressing and provides protection from inflammatory milieu. Meanwhile, a positive regulation relationship between LPS-induced macrophage activity and ferroptosis sensitivity can be observed. After calpain knockdown, both inflammatory macrophage ferroptosis sensitivity and ACSL4 expression decreased, while its over-expression renders ACSL4-envoking condition. Also, calpain pharmacological inhibition reduced both ferroptosis and fibrosis aptitude in mice.
Conclusions:
ACSL4 induces inflammatory macrophage ferroptosis to aggravate fibrosis progressing. ACSL4 and its upregulators of calpains may be potential therapeutic targets for BLM model of SSc.
Insights
Systemic sclerosis fibrosis is worsened by ferroptosis, a cell death process involving ACSL4. Inhibiting ACSL4 and its regulator, calpain, may offer new treatments for this rheumatic disease.
Area of Science:
- Immunology
- Pathophysiology
- Fibrosis Research
Background:
- Systemic sclerosis (SSc) is a rheumatic disease characterized by immune dysfunction, inflammation, and fibrosis with unclear pathophysiology.
- Urgent need for therapeutic breakthroughs to halt the progressive stiffness and multi-organ damage in SSc.
Purpose of the Study:
- To investigate the role of ferroptosis in regulating progressive fibrosis in Systemic Sclerosis.
- To explore potential therapeutic strategies targeting ferroptosis in SSc.
Main Methods:
- In vivo: Utilized a bleomycin (BLM)-induced mouse model for ferroptosis detection (Western blotting, MDA, GSH assays). Assessed therapeutic effects of an ACSL4 inhibitor on fibrosis.
- In vitro: Evaluated ferroptosis sensitivity in activated bone marrow-derived macrophages (BMDM) and the calpain/ACSL4 axis in Raw264.7 cells.
Main Results:
- Ferroptosis and elevated ACSL4 expression were observed in SSc mouse skin and lung tissues.
- ACSL4 inhibition significantly reduced fibrosis progression and inflammation in the BLM-induced SSc model.
- Macrophage activation correlated positively with ferroptosis sensitivity; calpain modulation affected ACSL4 expression and ferroptosis.
Conclusions:
- ACSL4-induced ferroptosis in inflammatory macrophages exacerbates SSc fibrosis.
- ACSL4 and its upregulators, such as calpains, represent potential therapeutic targets for SSc treatment.
