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Journal of Peking University (Health Sciences) ›› 2024, Vol. 56 ›› Issue (6): 950-955. doi: 10.19723/j.issn.1671-167X.2024.06.002

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

Kelin ZHAO1, Xue XIA1, Naixu SHI2, Han ZHOU1, Jingwen GAI1, Ping LI1,*()   

  1. 1. Department of Rheumatology and Immunology, China-Japan Union Hospital, Jilin University, Changchun 130000, China
    2. Department of Stomatology, China-Japan Union Hospital, Jilin University, Changchun 130000, China
  • Received:2024-07-31 Online:2024-12-18 Published:2024-12-18
  • Contact: Ping LI E-mail:li_ping@jlu.edu.cn
  • Supported by:
    the Jilin Provincial Department of Finance Health Research Talent Special Project(2021SCZ14)

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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.

Key words: Systemic sclerosis, Fibroblasts, Ferroptosis, Fibrosis

CLC Number: 

  • R593.25

Figure 1

The morphological effects of TGF-β1 on NIH3t3 A, representative images of NIH3t3 with and without added TGF-β1 for 24 h under the microscope (×20); B, construction pattern diagram of NIH3t3 cell model induced by TGF-β1. TGF-β1, transforming growth factor-β1."

Figure 2

Profibrotic effect of TGF-β1 on NIH3t3 * * P < 0.01. A, RT-qPCR detection of changes in expression levels of Vimentin gene in NIH3t3; B, Western blot determination of Vimentin in NIH3t3. TGF-β1, transforming growth factor-β1; RT-qPCR, reverse transcription quantitative real-time PCR."

Figure 3

Flow cytometry detection of ROS fluorescence intensity in NIH3t3 induced by TGF-β1 TGF-β1, transforming growth factor-β1; ROS, reactive oxygen species."

Figure 4

Up-regulated expression of 4-HNE in a systemic sclerosis cell model * P < 0.05. A, immunofluorescence representation of 4-HNE in NIH3t3 cells (×200). NIH3t3 cells were treated with 0.1% Triton X-100 for per-meabilization and blocked with 5% bovine serum albumin (BSA). Cells were incubated overnight at 4 ℃ with a primary antibody against 4-HNE (dilution 1 ∶ 50). The following day, cells were labeled with a FITC-conjugated secondary antibody (dilution 1 ∶ 100) and incubated at room temperature for 1 h. Nuclear staining was performed using DAPI. B, quantification of average immunofluorescence intensity of 4-HNE in NIH3t3 cells (P < 0.05). C, Western blot determination of 4-HNE in NIH3t3. 4-HNE, 4-hydroxynonenal."

1 Hinz B , Phan SH , Thannickal VJ , et al. Recent developments in myofibroblast biology: Paradigms for connective tissue remodeling[J]. Am J Pathol, 2012, 180 (4): 1340- 1355.
doi: 10.1016/j.ajpath.2012.02.004
2 Volkmann ER , Andreasson K , Smith V . Systemic sclerosis[J]. Lancet, 2023, 401 (10373): 304- 318.
doi: 10.1016/S0140-6736(22)01692-0
3 Preliminary criteria for the classification of systemic sclerosis (scleroderma) . Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee[J]. Arthritis Rheum, 1980, 23 (5): 581- 590.
doi: 10.1002/art.1780230510
4 Leroy EC , Medsger TA Jr . Criteria for the classification of early systemic sclerosis[J]. J Rheumatol, 2001, 28 (7): 1573- 1576.
5 Vona R , Giovannetti A , Gambardella L , et al. Oxidative stress in the pathogenesis of systemic scleroderma: An overview[J]. J Cell Mol Med, 2018, 22 (7): 3308- 3314.
doi: 10.1111/jcmm.13630
6 Doridot L , Jeljeli M , Chene C , et al. Implication of oxidative stress in the pathogenesis of systemic sclerosis via inflammation, autoimmunity and fibrosis[J]. Redox Biol, 2019, 25, 101122.
doi: 10.1016/j.redox.2019.101122
7 Doll S , Proneth B , Tyurina YY , et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition[J]. Nat Chem Biol, 2017, 13 (1): 91- 98.
doi: 10.1038/nchembio.2239
8 Cao D , Zheng J , Li Z , et al. ACSL4 inhibition prevents macrophage ferroptosis and alleviates fibrosis in bleomycin-induced systemic sclerosis model[J]. Arthritis Res Ther, 2023, 25 (1): 212.
doi: 10.1186/s13075-023-03190-9
9 Dalleau S , Baradat M , Gueraud F , et al. Cell death and diseases related to oxidative stress: 4-hydroxynonenal (HNE) in the balance[J]. Cell Death Differ, 2013, 20 (12): 1615- 1630.
doi: 10.1038/cdd.2013.138
10 Galam L , Failla A , Soundararajan R , et al. 4-hydroxynonenal regulates mitochondrial function in human small airway epithelial cells[J]. Oncotarget, 2015, 6 (39): 41508- 41521.
doi: 10.18632/oncotarget.6131
11 Reyes-Jimenez E , Ramirez-Hernandez AA , Santos-Alvarez JC , et al. Coadministration of 3'5-dimaleamylbenzoic acid and quercetin decrease pulmonary fibrosis in a systemic sclerosis model[J]. Int Immunopharmacol, 2023, 122, 110664.
doi: 10.1016/j.intimp.2023.110664
12 de Virgilio A , Greco A , Fabbrini G , et al. Parkinson ' s disease: Autoimmunity and neuroinflammation[J]. Autoimmun Rev, 2016, 15 (10): 1005- 1011.
doi: 10.1016/j.autrev.2016.07.022
13 Li Y , Zhao T , Li J , et al. Oxidative stress and 4-hydroxy-2-nonenal (4-HNE): Implications in the pathogenesis and treatment of aging-related diseases[J]. J Immunol Res, 2022, 2022, 2233906.
14 Rozier P , Maumus M , Bony C , et al. Extracellular vesicles are more potent than adipose mesenchymal stromal cells to exert an anti-fibrotic effect in an in vitro model of systemic sclerosis[J]. Int J Mol Sci, 2021, 22 (13): 6837.
doi: 10.3390/ijms22136837
15 Wu C , Liu J , Chen Z , et al. Comprehensive analysis of ferroptosis-related hub gene signatures as a potential pathogenesis and therapeutic target for systemic sclerosis: A bioinformatics analysis[J]. Int J Immunopathol Pharmacol, 2023, 37, 3946320231187783.
doi: 10.1177/03946320231187783
16 Pei Z , Qin Y , Fu X , et al. Inhibition of ferroptosis and iron accumulation alleviates pulmonary fibrosis in a bleomycin model[J]. Redox Biol, 2022, 57, 102509.
doi: 10.1016/j.redox.2022.102509
17 Wu J , Feng Z , Chen L , et al. TNF antagonist sensitizes synovial fibroblasts to ferroptotic cell death in collagen-induced arthritis mouse models[J]. Nat Commun, 2022, 13 (1): 676.
doi: 10.1038/s41467-021-27948-4
18 Luczaj W , Gindzienska-Sieskiewicz E , Jarocka-Karpowicz I , et al. The onset of lipid peroxidation in rheumatoid arthritis: Consequences and monitoring[J]. Free Radic Res, 2016, 50 (3): 304- 313.
doi: 10.3109/10715762.2015.1112901
19 Chen J , Chen P , Song Y , et al. STING upregulation mediates ferroptosis and inflammatory response in lupus nephritis by upregu-lating TBK1 and activating NF-kappaB signal pathway[J]. J Biosci, 2024, 49, 9.
doi: 10.1007/s12038-023-00381-z
20 Brezovec N , Perdan-Pirkmajer K , Burja B , et al. Disturbed antioxidant capacity in patients with systemic sclerosis associates with lung and gastrointestinal symptoms[J]. Biomedicines, 2023, 11 (8): 2110.
doi: 10.3390/biomedicines11082110
21 Varga J , Pasche B . Transforming growth factor beta as a therapeutic target in systemic sclerosis[J]. Nat Rev Rheumatol, 2009, 5 (4): 200- 206.
doi: 10.1038/nrrheum.2009.26
22 Piera-Velazquez S , Makul A , Jimenez SA . Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: Regulation by transforming growth factor beta[J]. Arthritis Rheumatol, 2015, 67 (10): 2749- 2758.
doi: 10.1002/art.39242
23 Jimenez SA , Gaidarova S , Saitta B , et al. Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts[J]. J Clin Invest, 2001, 108 (9): 1395- 1403.
doi: 10.1172/JCI200112347
24 Sun X , Majumder P , Shioya H , et al. Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species[J]. J Biol Chem, 2000, 275 (23): 17237- 17240.
doi: 10.1074/jbc.C000099200
25 Svegliati S , Spadoni T , Moroncini G , et al. NADPH oxidase, oxidative stress and fibrosis in systemic sclerosis[J]. Free Radic Biol Med, 2018, 125, 90- 97.
doi: 10.1016/j.freeradbiomed.2018.04.554
26 Zhou X , Trinh-Minh T , Tran-Manh C , et al. Impaired mitochondrial transcription factor A expression promotes mitochondrial damage to drive fibroblast activation and fibrosis in systemic sclerosis[J]. Arthritis Rheumatol, 2022, 74 (5): 871- 881.
doi: 10.1002/art.42033
27 Liu W , Porter NA , Schneider C , et al. Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition[J]. Free Radic Biol Med, 2011, 50 (1): 166- 178.
doi: 10.1016/j.freeradbiomed.2010.10.709
28 Yang HJ , Hu R , Sun H , et al. 4-HNE induces proinflammatory cytokines of human retinal pigment epithelial cells by promoting extracellular efflux of HSP70[J]. Exp Eye Res, 2019, 188, 107792.
doi: 10.1016/j.exer.2019.107792
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