[Expression and significance of ferroptosis marker 4-HNE in in vitro model of systemic sclerosis].

Abstract

Objective: To investigate the expression and physiological significance of the ferroptosis marker 4-hydroxynonenal (4-HNE) in myofibroblasts induced by transforming growth factor-β1 (TGF-β1), providing theoretical evidence for its potential role in the diagnosis and treatment of fibrosis in systemic sclerosis (SSc).

Methods: Mouse embryonic fibroblasts (NIH3t3) were cultured and divided into two groups after 12 h of starvation: the control group (cultured in 1% serum-containing medium) and the TGF-β1 group (cultured in 10 μg/L TGF-β1 with 1% serum-containing medium). Cell morphology changes in both groups were observed under a microscope. To confirm successful establishment of the SSc cell model, fibrosis markers were analyzed using reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot. Next, flow cytometry was employed to assess the intracellular levels of reactive oxygen species (ROS) in both groups. Finally, Western blot and immunofluorescence staining were used to measure the expression of 4-HNE in the TGF-β1-treated cells.

Results: Microscopic observations revealed that TGF-β1 treatment caused the NIH3t3 cells to transition from a typical spindle shape to a flat, polygonal shape with multiple protrusions, indicating fibroblast activation. The RT-qPCR and Western blot analyses showed that the expression of the fibrosis marker Vimentin was significantly upregulated in the TGF-β1 group compared with the control group (P < 0.01), confirming that TGF-β1 effectively promoted fibrosis-related gene and protein expression. Flow cytometry results indicated that TGF-β1 significantly elevated intracellular ROS levels, suggesting the induction of oxidative stress. Furthermore, both Western blot and immuno-fluorescence staining demonstrated a significant increase in 4-HNE expression in the TGF-β1-treated cells (immunofluorescence intensity P < 0.05).

Conclusion: TGF-β1 promotes fibroblast activation and fibrosis while inducing ROS production, leading to a marked increase in 4-HNE expression. Given the role of 4-HNE as a marker of lipid peroxidation and its elevated levels in the SSc cell model, this study suggests that 4-HNE could serve as a potential biomarker for fibrosis in SSc. The findings highlight the importance of investigating the mechanisms of 4-HNE in fibrosis and suggest that targeting this pathway could offer new therapeutic opportunities for treating SSc.