Role and mechanism of ubiquitin-specific protease 35 in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes.
- Author:
Lianghua FENG
1
;
Lirong HONG
1
;
Yujia CHEN
1
;
Xueming CAI
1
Author Information
1. Department of Rheumatology and Immunology, Xiamen Fifth Hospital, Xiamen 361101, Fujian, China.
- Publication Type:Journal Article
- Keywords:
Ferroptosis;
Glutathione peroxidase 4;
Rheumatoid arthritis;
Solute carrier family 7 member 11;
Ubiquitin-specific protease 35
- MeSH:
Ferroptosis;
Humans;
Arthritis, Rheumatoid/metabolism*;
Synoviocytes/pathology*;
Reactive Oxygen Species/metabolism*;
Ubiquitin-Specific Proteases/metabolism*;
Fibroblasts/pathology*;
Cell Survival;
Piperazines/pharmacology*;
Endopeptidases/metabolism*;
Cells, Cultured;
Cell Line;
Amino Acid Transport System y+
- From:
Journal of Peking University(Health Sciences)
2025;57(5):919-925
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To elucidate the role and underlying mechanism of ubiquitin-specific protease 35 (USP35) in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes (RA-FLS), thereby enhancing our comprehension of the pathogenesis of RA and identifying potential therapeutic targets for its treatment.
METHODS:(1) RA-FLS were cultured in vitro and transduced with lentiviral vectors to establish stable cell lines: A USP35-knockdown line (short hairpin ribonucleic acid of USP35, shUSP35) and its control (negtive control of short hairpin ribonucleic acid, shNC), as well as a overexpression of USP35 line (USP35 OE) and its control (Vector). To investigate the role of USP35 in ferroptosis regulation, a ferroptosis model was induced in RA-FLS by treatment with 1 μmol/L Erastin. The cells were divided into six groups: shNC, shNC + Erastin, shUSP35 + Erastin, Vector, Vector + Erastin, and USP35 OE + Erastin. (2) Cell viability was detected using the cell counting kit-8 (CCK-8). (3) Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG) ratios, and Ferrous ion (Fe2+) levels were measured using specific assay kits to evaluate oxidative stress, lipid peroxidation, and glutathione redox status in the cells. (4) Protein expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected using Western blotting to investigate their potential involvement in USP35-mediated ferroptosis regulation.
RESULTS:(1) Compared with the shNC +Erastin group, the cell viability of the shUSP35+Erastin group was significantly decreased (P < 0.001), while it was notably increased in the USP35 OE+Erastin group compared with the Vector+Erastin group (P < 0.001). These findings indicated that USP35 could alleviate the inhibitory effect of Erastin on RA-FLS cell viability. (2) In comparison to the shNC+Erastin group, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe2+ (P < 0.001) were significantly elevated, and the GSH/GSSG ratio was increased (P < 0.05) in the shUSP35+Erastin group. Conversely, the levels of ROS (P < 0.001), MDA (P < 0.05), and Fe2+ (P < 0.05) were significantly decreased, and the GSH/GSSG ratio was decreased (P < 0.05) in the USP35 OE+Erastin group compared with the Vector+Erastin group. These results suggested that USP35 could inhibit Erastin-induced oxidative stress and lipid peroxidation in RA-FLS. (3) In Erastin-induced RA-FLS, the expression of USP35 was positively correlated with the protein levels of SLC7A11 and GPX4, indicating a potential mechanism by which USP35 regulated ferroptosis in these cells.
CONCLUSION:USP35 inhibits ferroptosis in RA-FLS, potentially through the increased expression of SLC7A11 and GPX4.
