Proteomics-Based Analysis of Mechanisma Underlying Celastrol-Induced Fibroblast Transdifferentiation

doi:10.19803/j.1672-8629.20250638

Abstract

Abstract: Objective To explore the mechanisms of celastrol-induced cardiotoxicity and its potential promotion of fibrosis via proteomic analysis. Methods Primary cardiac fibroblasts were isolated from neonatal mice. The Cell Counting Kit-8 (CCK-8) assay was used to determine the concentration and duration of celastrol treatment. Bright-field microscopy was employed to observe the morphological changes of fibroblasts before and after treatment. Seventy-two hours after treatment with 0.01 μmol·L^-1, real-time quantitative PCR (qPCR) was used to measure the expression levels of related pro-fibrotic genes. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify changes in the levels of extracellular matrix (ECM), including tissue inhibitor of metalloproteinases-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2). Immunofluorescence staining was performed to calculate the expressions of fibrosis-related proteins. Proteomic analysis was used to investigate the potential mechanisms by which celastrol promoted fibrosis. Results 72 hours after treatment with celastrol 0.01μmol·L^-1, the morphology of cells in the treatment group changed from spindle-shaped to polygonal or flattened, and the cell volume increased compared with the blank control group. The expression levels of pro-fibrotic genes and proteins were significantly elevated in the celastrol-treated group, along with increased levels of the extracellular matrix proteins TIMP-1 and MMP2. Differential protein enrichment analysis based on proteomics indicated that celastrol affected the expressions of such proteins as fibronectin 1 (Fn1), integrin alpha 11 (Itga11), integrin beta 3 (Itgb3), and transforming growth factor beta 1 induced transcript 1 (Tgfb1i1) in fibroblasts. By activating inflammatory and extracellular matrix-related pathways, celastrol subsequently promoted cardiac fibrosis. Conclusion Short-term administration of celastrol may potentially impact cardiac function, likely by influencing collagen synthesis and disrupting extracellular matrix (ECM) homeostasis, thereby promoting the transformation of cardiac fibroblasts into myofibroblasts and exacerbating the fibrosis process.

Key words: Celastrol, Fibroblast, Transdifferentiation, Fibrosis, Extracellular Matrix (ECM), Poteomics, Neonatal Mice

CLC Number:

R282
R994.11

SUN Yue, ZHANG Yunan, SUN Yuanbo, LI Gaofu, YANG Fang, WANG Ningning, ZHANG Pengfei, GAO Yue, ZHOU Wei. Proteomics-Based Analysis of Mechanisma Underlying Celastrol-Induced Fibroblast Transdifferentiation[J]. Chinese Journal of Pharmacovigilance, 2026, 23(3): 241-249.

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https://zgywjj.magtechjournal.com/EN/Y2026/V23/I3/241

References

[1] LIANG LC, YANG YX, GUO FJ.Recent Advances of Pharmacological Activities and Structural Modifications of Celastrol[J]. Chinese Journal of Medicinal Chemistry(中国药物化学杂志), 2020, 30(10): 622-635.
[2] LI JZ, HAN M, SHENG S, et al.Research Progress on the Regulatory Role and Mechanism of Tripterygium wilfordii Hook. F. in the Cardiov-ascular System[J]. Geriatric Medicine Research(老年医学研究), 2025, 6(2): 86-89.
[3] WANG C, DAI S, ZHAO X, et al.Celastrol as an Emerging Anticancer Agent: Current Status, Challenges and Therapeutic Strategies[J]. Biomed Pharmacother, 2023, 163: 114882.
[4] LI G, ZHOU L, DENG HF, et al.Targeting OPA1-Mediated Mitochondrial Fusion Contributed to Celastrol's Anti-Tumor Angiogenesis Effect[J]. Pharmaceutics, 2022, 15(1): 48.
[5] ZHENG S, YANG H, ZHENG J, et al.Unveiling the Anti-Obesity Potential of Thunder God Vine: Network Pharmacology and Compu-tational Insights into Celastrol-Like Molecules[J]. Int J Mol Sci, 2024, 25(23): 12501.
[6] ZHOU D, LI X, XIAO X, et al.Celastrol Targets the ChREBP-TXNIP Axis to Ameliorates Type 2 Diabetes Mellitus[J]. Phytomedicine, 2023, 110: 154634.
[7] CHEN Y, LIU HM, QIAN WX, et al.Exploration of Characteristics and Regularity of Cardiotoxicity Induced by Traditional Chinese Medicine Based on TCMSTD[J]. Chinese Traditional and Herbal Drugs(中草药),2024, 55(7): 2332-2342.
[8] LIU MB, HE XY, YANG XH, et al.Interpretation of Annual Report on Cardiovascular Health and Diseases in China 2024[J]. Biomed Environ Sci, 2025, 38(8): 893-917.
[9] JIANG YW, WANG YS.Research Progress on the Mechanism Linking Fibroblast Cytoskeletal Protein Changes to Myocardial Fibrosis[J]. Shanghai Medical Journal(上海医学), 2023, 46(12): 854-858.
[10] XU J.The Effect and Mechanism of miR-30c on Atrial Fibrosis and Fibrogenisis of Cardiac Fibroblasts Induced by Transforming Growth Factor-β1[D]. Shanghai: Shanghai Jiao Tong University, 2017.
[11] LIN XJ, TONG XY.Advances on Cell Types and Their Mechanisms Involved in the Development of Cardiac Fibrosis[J]. Life Science Instruments(生命科学仪器), 2019, 17(Z1): 46-54, 33.
[12] CZUBRYT MP.Cardiac Fibroblast to Myofibroblast Phenotype Conversion-An Unexploited Therapeutic Target[J]. Cardiovasc Dev Dis, 2019, 6(3): 28.
[13] KUMAR S, NAGESH D, RAMASUBBU V, et al.Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis[J]. Bio Protoc, 2023, 13(4): e4616.
[14] ZHANG CC, WANG X, CHENG NX, et al.Establishment of the Senescence Cell Model of Mouse Cardiac Fibroblast[J]. Journal of Cardiovascular and Pulmonary Diseases(心肺血管病杂志), 2022, 41(7): 821-826.
[15] ZHENG M, LI S, XU J, et al.Nonylphenol Exposure Induces Myocardial Fibrosis via the RhoA/ROCK1/MLC-Signaling Pathway[J]. Ecotoxicol Environ Saf, 2025, 299: 118423.
[16] GUO H.The Effect of miR-21 on the Gene Expression Profile of Irradiated Rat Cardiac Fibroblasts and the Mechanism of miR-21/GDF15 on Promoting Radiation-Induced Fibrosis[D]. Lanzhou: Lanzhou University, 2021.
[17] ZHANG P, WANG F, SONG J, et al.Fasudil Attenuates Myocardial Fibrosis in Rats with Diabetes Mellitus via TGF-β1/Smad Signaling Pathway[J]. Cell Mol Biol, 2023, 69(10): 233238.
[18] WISNIEWSKI JR, ZOUGMAN A, NAGARAJ N, et al.Universal Sample Preparation Method for Proteome Analysis[J]. Nature Methods, 2009, 6(5): 359-362.
[19] CHANG J.Study on the Related Mechanism and Intervention Effect of Extract of Radix Angelica Sinensis and Radix Hedysari on fibrosis in Myocardial Fibroblast of Neonatal Rats after X-ray Radiation[D]. Lanzhou: Gansu University of Chinese Medicine, 2022.
[20] RODRIGUEZ P, SASSI Y, TRONCONE L, et al.Deletion of Delta-Like 1 Homologue Accelerates Fibroblast-Myofibroblast Differen-tiation and Induces Myocardial Fibrosis[J]. Eur Heart J, 2019, 40(12): 967-978.
[21] YU P.SIRT6 Regulates TGF-β Signaling Pathway in Cardiac Fibrosis Induced By Ang Ⅱ[D]. Xian: Air Force Medical University, 2020.
[22] YAN JY.Study on the Mechanism of Myocardial Injury in Rats Acute Exposed in High Altitude and Anti-Acute Mountain Sickness Drugs Development[D]. Guangzhou: Guangdong Pharmaceutical University, 2022.
[23] FU CQ, HE YH, HU XL, et al.The Anti-Inflammatory Effect of Tripterygium Wilfordii Extract[J]. Dermatology Bulletin(皮肤科学通报), 2025, 42(2): 204-208, 229.
[24] LYU SX, JIAO HC.Research Progress of Cardiotoxicity Mechanism of Common Chinese Herbal Medicine[J]. Information on TCM(中医药信息), 2024, 41(6): 82-88.
[25] ZHU RH, CHEN L, CHEN Y, et al.Research Progress of Celastrol in the Treatment of Stroke[J]. China Pharmacist(中国药师), 2024, 27(4): 711-721.
[26] CHEN Z, ZHUANG Z, MENG C, et al.Induction of the ER Stress Response in NRVMs is Linked to Cardiotoxicity Caused by Celastrol[J]. Acta Biochim Biophys Sin, 2022, 54(8): 1180-1192.
[27] MARUYAMA K, IMANAK-YOSHIDA K.The Pathogenesis of Cardiac Fibrosis: a Review of Recent Progress[J]. Int J Mol Sci, 2022, 23(5):2617.
[28] FENG Y.The Role and Mechanism of MircroRNA-130a in Cardiac Fibrosis after Myocardial Infarction[D]. Wuhan: Huazhong University of Science and Technology, 2022.
[29] ZHANG X, TIAN B, CONG X, et al.SLIT3 Promotes Cardiac Fibrosis and Differentiation of Cardiac Fibroblasts by RhoA/ROCK1 Signaling Pathway[J]. Iran J Basic Med Sci, 2024, 27(7): 832-840.
[30] GUO M, XU J, LONG X, et al.Myocardial Fibrosis Induced by Nonylphenol and Its Regulatory Effect on the TGF-β1/LIMK1 Signaling Pathway[J]. Ecotoxicol Environ Saf, 2024, 272: 116110.
[31] ZHANG JJ.Effects and Mechanism of Astragaloside IV on the Expression of CTGF and FN in Cardiac Fibroblasts[D]. Jining: Jining Medical University, 2018.
[32] YANG B, QIAO Y, YAN D, et al.Targeting Interactions between Fibroblasts and Macrophages to Treat Cardiac Fibrosis[J]. Cells, 2024, 13(9): 764.
[33] CABRAL-PACHECO GA, GARZA-VELOZ I, et al.The Roles of Matrix Metalloproteinases and Their Inhibitors in Human Diseases[J]. Int J Mol Sci, 2020, 21(24): 9739.
[34] WU LJ, WANG ZF, TAN XH, et al.Effect of Extracellular Matrix Stiffness on Tumor Progression and Treatment Strategies[J]. Chinese Journal of Tissue Engineering Research(中国组织工程研究), 2025, 29(20): 4286-4294.
[35] LEGERE SA, HAIDI ID, LEGARE JF, et al.Mast Cells in Cardiac Fibrosis: New Insights Suggest Opportunities for Intervention[J]. Front Immunol, 2019, 10: 580.
[36] WERNERSSON S, PEJLER G.Mast Cell Secretory Granules: Armed for Battle[J]. Nat Rev Immunol, 2014, 14(7): 478-494.
[37] SONG X, QIU Y, SHI J, et al.Prokaryotic Expression, Purification and Evaluation of Anti-Cardiac Fibrosis Activity of Recombinant TGF-β Latency Associated Peptide[J]. PeerJ, 2022, 10: e12797.
[38] LI Z, ZHANG J, DUAN X, et al.Celastrol: a Promising Agent Fighting against Cardiovascular Diseases[J]. Antioxidants (Basel), 2022, 11(8): 1597.