泛素特异性蛋白酶35对类风湿关节炎成纤维样滑膜细胞铁死亡的作用及机制

doi:10.19723/j.issn.1671-167X.2025.05.016

摘要/Abstract

摘要： 目的: 研究泛素特异性蛋白酶35(ubiquitin-specific protease 35, USP35)对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis-fibroblast like synoviocytes, RA-FLS)铁死亡的作用及机制，加深对RA发病机制的理解，并为其治疗提供潜在的标靶。方法: (1) 体外培养RA-FLS，用慢病毒载体感染以构建稳定敲低USP35的细胞系(short hairpin ribonucleic acid of USP35，shUSP35)和其对照细胞系(negtive control of short hairpin ribonucleic acid，shNC)，以及稳定过表达USP35的细胞系(overexpression of USP35，USP35 OE)和其对照细胞系(Vector)。为了探究USP35在铁死亡调控中的作用，使用1 μmol/L依拉司亭(Erastin)诱导RA-FLS细胞构建铁死亡模型。将细胞分为6组，第一组是shNC细胞，第二组是用Erastin处理shNC细胞(shNC+Erastin)，第三组是用Erastin处理shUSP35细胞(shUSP35+Erastin)，第四组是Vector细胞，第五组是用Erastin处理Vector细胞(Vector+Erastin)，第六组是用Erastin处理USP35 OE细胞(USP35 OE+Erastin)。(2)采用细胞计数试剂盒-8(cell counting kit-8, CCK8)检测细胞活力；(3)分别使用活性氧(reactive oxygen species, ROS)检测试剂盒、丙二醛(malondialdehyde, MDA)检测试剂盒、谷胱甘肽(glutathione, GSH)和氧化型谷胱甘肽(glutathione sulfide，GSSG)检测试剂盒、亚铁离子含量检测试剂盒检测细胞中ROS、MDA、Fe²⁺的含量以及GSH/GSSG比值；(4)采用蛋白免疫印迹实验(Western blotting)检测溶质载体家族7成员11(solute carrier family 7 member 11，SLC7A11)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4, GPX4)蛋白的表达水平。结果: (1) 与shNC+Erastin组相比，shUSP35+Erastin组的RA-FLS细胞活力显著降低(P＜0.001)，而与Vector+Erastin组相比，USP35 OE+Erastin组的细胞活力显著升高(P＜0.001)，表明USP35可显著缓解Erastin对RA-FLS细胞活力的抑制作用。(2)与shNC+Erastin组相比，shUSP35+Erastin组细胞的ROS(P＜0.001)、MDA(P＜0.05)和Fe²⁺水平(P＜0.001)显著升高，GSH/GSSG比值显著增加(P＜0.05)；而与Vector+Erastin组相比，USP35OE+Erastin组细胞的ROS(P＜0.001)、MDA(P＜0.05)和Fe²⁺水平(P＜0.05)显著降低，GSH/GSSG比值显著下降(P＜0.05)，表明USP35可显著抑制Erastin诱导的RA-FLS细胞氧化应激和脂质过氧化。(3)在Erastin诱导的RA-FLS中，USP35的表达与SLC7A11和GPX4蛋白水平呈正相关。结论: USP35抑制RA-FLS的铁死亡，可能与增加SLC7A11和GPX4表达相关。

关键词: 泛素特异性蛋白酶35, 类风湿关节炎, 铁死亡, 溶质载体家族7成员11, 谷胱甘肽过氧化物酶4

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

中图分类号:

R593.22

冯亮华, 洪丽荣, 陈雨佳, 蔡学明. 泛素特异性蛋白酶35对类风湿关节炎成纤维样滑膜细胞铁死亡的作用及机制[J]. 北京大学学报（医学版）, 2025, 57(5): 919-925.

Lianghua FENG, Lirong HONG, Yujia CHEN, Xueming CAI. Role and mechanism of ubiquitin-specific protease 35 in ferroptosis of rheumatoid arthritis-fibroblast like synoviocytes[J]. Journal of Peking University (Health Sciences), 2025, 57(5): 919-925.

图/表 4

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