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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (5): 919-925. doi: 10.19723/j.issn.1671-167X.2025.05.016

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Role and mechanism of ubiquitin-specific protease 35 in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes

Lianghua FENG*(), Lirong HONG, Yujia CHEN, Xueming CAI   

  1. Department of Rheumatology and Immunology, Xiamen Fifth Hospital, Xiamen 361101, Fujian, China
  • Received:2024-07-31 Online:2025-10-18 Published:2025-08-14
  • Contact: Lianghua FENG

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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.

Key words: Ubiquitin-specific protease 35, Rheumatoid arthritis, Ferroptosis, Solute carrier family 7 member 11, Glutathione peroxidase 4

CLC Number: 

  • R593.22

Figure 1

Influence of USP35 on the viability of RA-FLS cells (n=3) *P < 0.05, ***P < 0.001. A, qPCR detection of USP35 mRNA expression; B, CCK8 detection of RA-FLS cell viability. shNC, negtive control of short hairpin ribonucleic acid; shUSP35, short hairpin ribonucleic acid of USP35; USP35 OE, overexpression of USP35; USP35, ubiquitin-specific protease 35; qPCR, quantitative real-time PCR; CCK8, cell counting kit-8; RA-FLS, rheumatoid arthritis-fibroblast like synoviocytes; ns, no statistic."

Figure 2

USP35 regulates the Fe2+ level of RA-FLS cells (n=3) *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations as in Figure 1."

Figure 3

Detection of ROS, MDA, GSH, and GSSG levels in RA-FLS cells (n=3) *P < 0.05, **P < 0.01, ***P < 0.001. A, cellular ROS assay; B, cellular MDA assay; C, cellular GSH assays; D, cellular GSSG assays; E, cellular GSH/GSSG assays. MFI, mean fluorescent intensity; DCF, 2', 7'-dichlorofluorescein; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; GSSG, glutathione sulfide; Other abbreviations as in Figure 1."

Figure 4

USP35 influences GPX4 and SLC7A11 protein expression in RA-FLS cells A, USP35 knockdown; B, USP35 overexpressing. GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7 member 11; Other abbreviations as in Figure 1."

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