Wen Jiaxin, Yi Lingxian, Chen Lei, Xu Jinhan, Zhang Yanmin, Cheng Qiheng, Ping Hangyu, Wang Huanyu, Shuang Feng, Chai Wei, Weng Tujun
Senior Department of Orthopaedics, The Fourth Medical Centre, Chinese PLA General Hospital, Beijing, 100048, P.R. China.
School of Medicine, Nankai University, Tianjin, 300071, P.R. China.
Stem Cell Res Ther. 2025 Jun 2;16(1):274. doi: 10.1186/s13287-025-04422-2.
Mesenchymal stem cells (MSCs) have potential for treating degenerative and immune diseases, but their clinical efficacy is limited by senescence, characterized by mitochondrial dysfunction, impaired mitophagy, and metabolic imbalance. The goal of this study was to investigate the effects of dimethyloxalylglycine (DMOG), a hypoxia-mimetic agent that stabilizes hypoxia-inducible factor 1 alpha (HIF-1α), on rejuvenating senescent MSCs by enhancing mitochondrial function, mitophagy, and metabolic reprogramming.
Two models of MSC senescence were established: oxidative stress-induced senescence using hydrogen peroxide and replicative senescence through serial passaging. Umbilical cord derived MSCs were treated with DMOG for 48 h under normoxic conditions. Mitochondrial function, mitophagy, and metabolism were assessed using assays that measured mitochondrial membrane potential, reactive oxygen species levels, ATP production, and mitophagy. Western blotting and real-time PCR were employed to analyze the expression changes of relevant molecules. RNA sequencing (RNA-seq) was performed to identify key genes and pathways regulated by DMOG. Additionally, to evaluate the therapeutic potential of rejuvenated MSCs, a co-culture system was established, where DMOG-treated senescent MSCs were co-cultured with IL-1β-treated chondrocytes.
DMOG treatment significantly reduced key senescence markers, including senescence-associated beta-galactosidase, p53, and p21, in both senescence models. DMOG treatment restored mitochondrial morphology and function, improving mitochondrial membrane potential, reducing mitochondrial reactive oxygen species, and enhancing ATP production. DMOG also promoted mitophagy, as evidenced by increased colocalization of mitochondria with lysosomes. RNA-seq analysis revealed that DMOG activated key pathways, including HIF-1 signaling, calcium signaling, and mitophagy-related gene (BNIP3 and BNIP3L). Notably, BNIP3 knockdown greatly abolished DMOG-induced mitophagy and its anti-senescence effects. Furthermore, DMOG treatment improved metabolic flexibility by enhancing both mitochondrial respiration and glycolysis in senescent MSCs. Moreover, DMOG-treated senescent MSCs partially restored their therapeutic efficacy in an osteoarthritis model by improving extracellular matrix regulation in IL-1β-stimulated chondrocytes.
Short-term DMOG treatment rejuvenates senescent MSCs by enhancing mitochondrial function, promoting mitophagy via HIF-1α/BNIP3, and improving metabolic reprogramming. DMOG-treated MSCs also showed enhanced therapeutic efficacy in co-culture with IL-1β-treated chondrocytes, suggesting its potential to improve MSC-based therapies in regenerative medicine.
间充质干细胞（MSCs）具有治疗退行性疾病和免疫疾病的潜力，但其临床疗效受到衰老的限制，衰老的特征包括线粒体功能障碍、自噬受损和代谢失衡。本研究的目的是探讨二甲基乙二酰甘氨酸（DMOG），一种能稳定缺氧诱导因子1α（HIF-1α）的缺氧模拟剂，通过增强线粒体功能、自噬和代谢重编程对衰老MSCs年轻化的影响。
建立了两种MSCs衰老模型：用过氧化氢诱导氧化应激衰老和通过连续传代诱导复制性衰老。在常氧条件下，用DMOG处理脐带来源的MSCs 48小时。使用测量线粒体膜电位、活性氧水平、ATP产生和自噬的检测方法评估线粒体功能、自噬和代谢。采用蛋白质免疫印迹法和实时定量PCR分析相关分子的表达变化。进行RNA测序（RNA-seq）以鉴定DMOG调控的关键基因和通路。此外，为了评估年轻化MSCs的治疗潜力，建立了一个共培养系统，将经DMOG处理的衰老MSCs与经IL-1β处理的软骨细胞共培养。
在两种衰老模型中，DMOG处理均显著降低了关键衰老标志物，包括衰老相关β-半乳糖苷酶、p53和p21。DMOG处理恢复了线粒体形态和功能，改善了线粒体膜电位，降低了线粒体活性氧水平，并增强了ATP产生。DMOG还促进了自噬，线粒体与溶酶体共定位增加证明了这一点。RNA-seq分析显示，DMOG激活了关键通路，包括HIF-1信号通路、钙信号通路和自噬相关基因（BNIP3和BNIP3L）。值得注意的是，BNIP3基因敲低大大消除了DMOG诱导的自噬及其抗衰老作用。此外，DMOG处理通过增强衰老MSCs的线粒体呼吸和糖酵解改善了代谢灵活性。此外，经DMOG处理的衰老MSCs通过改善IL-1β刺激的软骨细胞中的细胞外基质调节，在骨关节炎模型中部分恢复了其治疗效果。
短期DMOG处理通过增强线粒体功能、通过HIF-1α/BNIP3促进自噬和改善代谢重编程使衰老MSCs年轻化。经DMOG处理的MSCs在与经IL-1β处理的软骨细胞共培养时也显示出增强的治疗效果，表明其在改善再生医学中基于MSCs的治疗方法方面的潜力。
