Department of Orthopaedics, Fuzhou Second Hospital, No. 47, Shangteng Road, Cangshan District, Fuzhou City, 350007, Fujian Province, China.
J Orthop Surg Res. 2023 Mar 10;18(1):188. doi: 10.1186/s13018-023-03673-0.
Chondrocytes are the main cell damage type involved in the occurrence and development of osteoarthritis (OA). Ferroptosis has been confirmed to be related to many degenerative diseases. This research aimed to explore the role of Sp1 and ACSL4 in ferroptosis in the IL-1β-treated human chondrocyte cells line (HCCs).
The cell viability was detected with CCK8 assay. The ROS, MDA, GSH, and Fe levels were assessed with corresponding detecting kits. The Col2a1, Acan, Mmp13, Gpx4 and Tfr1 levels were determined by RT-qPCR assay. Western blot was conducted to evaluate the Acsl4 and Sp1 levels. PI staining was carried out to analyze the cell death. The double luciferase report was conducted to verify the interaction between Acsl4 and Sp1.
The results showed that IL-1β stimulation elevated the LDH release, cell viability, ROS, MDA and Fe levels and declined the GSH levels in the HCCs. Additionally, the mRNA levels of Col2a1, Acan, and Gpx4 were prominently decreased, while Mmp13 and Tfr1 were prominently elevated in the IL-1β stimulated HCCs. Furthermore, Acsl4 protein levels were upregulated in the IL-1β-stimulated HCCs. Both Acsl4 knockdown and ferrostatin-1 treatment neutralized the role of IL-1β in the HCCs. What's more, Acsl4 was transcriptionally regulated by Specificity protein 1 (Sp1). Sp1 overexpression enhanced the Acsl4 levels and Sp1 knockdown declined it.
Upregulation of Sp1 activates Ascl4 transcription and thus mediates the occurrence of ferroptosis. Hence, Acsl4 may be a therapeutic target for intervention of OA.
软骨细胞是骨关节炎（OA）发生和发展中主要的细胞损伤类型。铁死亡已被证实与许多退行性疾病有关。本研究旨在探讨 Sp1 和 ACSL4 在 IL-1β 处理的人软骨细胞系（HCCs）中铁死亡中的作用。
用 CCK8 法检测细胞活力。用相应的检测试剂盒评估 ROS、MDA、GSH 和 Fe 水平。通过 RT-qPCR 测定 Col2a1、Acan、Mmp13、Gpx4 和 Tfr1 水平。用 Western blot 评估 Acsl4 和 Sp1 水平。PI 染色分析细胞死亡。双荧光素酶报告验证 Acsl4 和 Sp1 之间的相互作用。
结果表明，IL-1β 刺激可提高 HCCs 中的 LDH 释放、细胞活力、ROS、MDA 和 Fe 水平，并降低 GSH 水平。此外，IL-1β 刺激的 HCCs 中 Col2a1、Acan 和 Gpx4 的 mRNA 水平显著降低，而 Mmp13 和 Tfr1 的水平显著升高。此外，IL-1β 刺激的 HCCs 中 Acsl4 蛋白水平上调。Acsl4 敲低和 ferrostatin-1 处理均可中和 IL-1β 在 HCCs 中的作用。更重要的是，ACSL4 转录受特异性蛋白 1（Sp1）调节。Sp1 过表达增强 Acsl4 水平，Sp1 敲低则降低其水平。
Sp1 的上调激活了 Ascl4 的转录，从而介导了铁死亡的发生。因此，ACSL4 可能是干预 OA 的治疗靶点。
