Department of Orthopedic Surgery, Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Nangang District, Harbin City, Heilongjiang Province, 150010, China.
J Bioenerg Biomembr. 2024 Feb;56(1):31-44. doi: 10.1007/s10863-023-09991-6. Epub 2023 Nov 28.
Chondrocyte ferroptosis constitutes a major cause of the development of osteoarthritis (OA). Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have a protective role against ferroptosis in various diseases. Hence, we aimed to determine whether BMSC-Exos alleviated chondrocyte ferroptosis and its effect on OA, and to dissect out the possible mechanisms. An OA rat chondrocyte model was established by interleukin-1β (IL-1β) exposure, and treated with BMSC-Exos/ferroptosis inhibitor Ferrostatin-1. Cell viability/ferroptosis-related index levels [reactive oxygen species (ROS)/malondialdehyde (MDA)/glutathione (GSH)]/cell death/ACSL4 mRNA and protein levels and METTL3 levels were assessed by MTT/kits/immunohistochemical method and TUNEL staining/RT-qPCR and Western blot. METTL3/ACSL4 were overexpressed in rat chondrocytes to evaluate their role in BMSC-Exo-produced repression on chondrocyte ferroptosis. Bioinformatics website predicted the presence of m6A modification sites on ACSL4 mRNA, with the m6A level enriched on it assessed by MeRIP/RT-qPCR. ACSL4 mRNA stability was detected by actinomycin D assay. A surgical destabilized medial meniscus rat OA model was also established, followed by injection with BMSC-Exos to verify their function. IL-1β stimulation in rat chondrocytes inhibited cell viability, elevated Fe/ROS/MDA levels, declined GSH levels and increased TUNEL positive cell number and ACSL4 level, which were neutralized by BMSC-Exos. BMSC-Exos limited chondrocyte ferroptosis by down-regulating METTL3, with the effect abrogated by METTL3 overexpression. METTL3 regulated the m6A modification of ACSL4 mRNA, increasing ACSL4 mRNA stability and ACSL4 expression. BMSC-Exos reduced chondrocyte ferroptosis and prevented OA progression via disruption of the METTL3-m6A-ACSL4 axis. BMSC-Exos might exert a chondroprotective effect by attenuating chondrocyte ferroptosis and alleviate OA progression.
软骨细胞铁死亡是骨关节炎 (OA) 发展的主要原因。骨髓间充质干细胞来源的外泌体 (BMSC-Exos) 在各种疾病的铁死亡中具有保护作用。因此，我们旨在确定 BMSC-Exos 是否缓解了软骨细胞铁死亡及其对 OA 的影响，并探讨可能的机制。通过白细胞介素-1β (IL-1β) 暴露建立 OA 大鼠软骨细胞模型，并与 BMSC-Exos/铁死亡抑制剂 Ferrostatin-1 联合处理。通过 MTT/试剂盒/免疫组织化学方法和 TUNEL 染色/RT-qPCR 和 Western blot 评估细胞活力/铁死亡相关指标水平[活性氧 (ROS)/丙二醛 (MDA)/谷胱甘肽 (GSH)]/细胞死亡/ACSL4 mRNA 和蛋白水平以及 METTL3 水平。在大鼠软骨细胞中转染 METTL3/ACSL4 过表达载体，评估它们在 BMSC-Exo 抑制软骨细胞铁死亡中的作用。生物信息学网站预测 ACSL4 mRNA 上存在 m6A 修饰位点，通过 MeRIP/RT-qPCR 评估其 m6A 水平丰度。通过放线菌素 D 测定检测 ACSL4 mRNA 稳定性。还建立了手术不稳定内侧半月板大鼠 OA 模型，随后注射 BMSC-Exos 以验证其功能。IL-1β 刺激大鼠软骨细胞抑制细胞活力，增加 Fe/ROS/MDA 水平，降低 GSH 水平，增加 TUNEL 阳性细胞数和 ACSL4 水平，BMSC-Exos 可中和这些变化。BMSC-Exos 通过下调 METTL3 限制软骨细胞铁死亡，METTL3 过表达可消除这种作用。METTL3 调节 ACSL4 mRNA 的 m6A 修饰，增加 ACSL4 mRNA 稳定性和 ACSL4 表达。BMSC-Exos 通过破坏 METTL3-m6A-ACSL4 轴减少软骨细胞铁死亡并预防 OA 进展。BMSC-Exos 通过减轻软骨细胞铁死亡发挥软骨保护作用并缓解 OA 进展。
