Expression and significance of ferroptosis marker 4-HNE in in vitro model of systemic sclerosis
10.19723/j.issn.1671-167X.2024.06.002
- VernacularTitle:铁死亡标志物4-HNE在系统性硬化症细胞模型中的表达及意义
- Author:
Kelin ZHAO
1
;
Xue XIA
;
Naixu SHI
;
Han ZHOU
;
Jingwen GAI
;
Ping LI
Author Information
1. 吉林大学中日联谊医院风湿免疫科,长春 130000
- Publication Type:Journal Article
- Keywords:
Systemic sclerosis;
Fibroblasts;
Ferroptosis;
Fibrosis
- From:
Journal of Peking University(Health Sciences)
2024;56(6):950-955
- CountryChina
- Language:Chinese
-
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 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.
