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 sys-temic 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 re-vealed 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 pro-moted fibrosis-related gene and protein expression.Flow cytometry results indicated that TGF-β1 signifi-cantly 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.