Yu Jun, Yang Ming, Jin Yun, Yang Kaijie, Yang Haibo
Orthopaedic Trauma Department, General Hospital of Ningxia Medical University, 804 Shengli South Street, Xingqing District, Yinchuan City, Ningxia, 750001, China.
Emergency Department, People's Hospital of Ningxia Hui Autonomous Region, Ningxia, China.
J Orthop Surg Res. 2025 May 29;20(1):539. doi: 10.1186/s13018-025-05898-7.
Osteomyelitis resulting from a traumatic fracture is a recurrent and difficult-to-treat bone infection. Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme, and Sirtuin-1 (SIRT1), an NAD+-dependent deacetylase, both play critical roles in regulating cellular processes, including inflammation. It has been discovered that exosomes originated from bone marrow mesenchymal stem cells (BMSCs-exo) can promote the repair and regeneration of bone fractures. In this study, we aimed to investigate the role of BMSCs-exo in osteoblast differentiation in osteomyelitis and the related molecular mechanisms. MC3T3-E1 cells induced with S. aureus were used as an in vitro model of osteomyelitis. BMSCs-exo were isolated and characterized using ultracentrifugation, transmission electron microscopy (TEM), and Western blot. RT-qPCR, Western blot, CCK-8, ALP staining, ELISA, and CO-IP were utilized to evaluate USP14 and SIRT1 levels, the osteogenic differentiation ability of MC3T3-E1 cells, and the deubiquitination level of SIRT1. Low expression of USP14 and SIRT1 was observed in the bone tissue of osteomyelitis patients. BMSCs-exo could upregulate the expression of USP14 and promote the expression of SIRT1 protein in the cell model of osteomyelitis. In addition, BMSCs-exo reduced the levels of inflammatory factors TNFα and IL-6, enhanced cell viability, promoted the expression of osteogenic differentiation markers RUNX2 and OCN in MC3T3-E1 cells, and improved cell osteogenic capacity. However, these trends were significantly reversed in MC3T3-E1 cells following treatment with BMSCs-exo transfected with si-USP14. Furthermore, knockdown of USP14 promoted SIRT1 ubiquitination and degradation, the process that was reversed by the proteasome inhibitor MG132, whereas USP14 overexpression inhibited SIRT1 ubiquitination. In MC3T3-E1 cells infected with S. aureus, BMSCs-exo delivers USP14, which may enhance SIRT1 deubiquitination and increase SIRT1 protein activity. This process inhibits inflammation and promotes osteogenesis, warranting further investigation into its mechanisms and in vivo efficacy.
创伤性骨折引起的骨髓炎是一种复发性且难以治疗的骨感染。泛素特异性蛋白酶14（USP14），一种去泛素化酶，以及沉默调节蛋白1（SIRT1），一种NAD+依赖性脱乙酰酶，在调节包括炎症在内的细胞过程中都起着关键作用。现已发现，源自骨髓间充质干细胞的外泌体（BMSCs-exo）可促进骨折的修复和再生。在本研究中，我们旨在探讨BMSCs-exo在骨髓炎中成骨细胞分化中的作用及相关分子机制。用金黄色葡萄球菌诱导的MC3T3-E1细胞作为骨髓炎的体外模型。使用超速离心、透射电子显微镜（TEM）和蛋白质印迹法分离并鉴定BMSCs-exo。利用RT-qPCR、蛋白质印迹法、CCK-8法、碱性磷酸酶染色、酶联免疫吸附测定（ELISA）和免疫共沉淀法（CO-IP）评估USP14和SIRT1水平、MC3T3-E1细胞的成骨分化能力以及SIRT1的去泛素化水平。在骨髓炎患者的骨组织中观察到USP14和SIRT1的低表达。在骨髓炎细胞模型中，BMSCs-exo可上调USP14的表达并促进SIRT1蛋白的表达。此外，BMSCs-exo降低了炎症因子TNFα和IL-6的水平，增强了细胞活力，促进了MC3T3-E1细胞中成骨分化标志物RUNX2和OCN的表达，并提高了细胞的成骨能力。然而，在用转染了si-USP14的BMSCs-exo处理后的MC3T3-E1细胞中，这些趋势被显著逆转。此外，敲低USP14促进了SIRT1的泛素化和降解，蛋白酶体抑制剂MG132可逆转这一过程，而USP14的过表达则抑制了SIRT1的泛素化。在感染金黄色葡萄球菌的MC3T3-E1细胞中，BMSCs-exo传递USP14，这可能增强SIRT1的去泛素化并增加SIRT1蛋白活性。这一过程抑制炎症并促进成骨，有必要对其机制和体内疗效进行进一步研究。
