BACKGROUND Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5-15), and old (> 15 years old). METHODS ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. RESULTS Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. CONCLUSIONS Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.

中文翻译：

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从马中分离出的脂肪干细胞的年龄依赖性损伤。


背景技术由年龄相关的细胞代谢损伤引起的细胞功能的逐渐丧失不仅涉及成熟的特化细胞，还涉及其祖细胞，这显着降低了它们的再生潜力。脂肪干细胞（ASC）最常用于兽医医学，作为韧带和软骨损伤的替代治疗选择，特别是对于高价值的运动马。因此，本研究的主要目的是确定来自三个年龄匹配马组的 ASC 的分子变异：年轻马组 (< 5)、中年马组 (5-15) 和老年马组 (> 15 岁) 。方法使用酶法从三个年龄匹配的马组中分离ASC。使用 qRT-PCR、ELISA 和蛋白质印迹方法、基于流式细胞术的系统以及共聚焦和扫描电子显微镜评估分子变化。结果我们的研究结果表明，中老年组来源的ASC表现出典型的衰老表型，如G1/G0停滞细胞百分比增加、双核、β-半乳糖苷酶活性增强、γH2AX聚集点积累，以及细胞增殖减少。此外，衰老的 ASC 的特点是促炎细胞因子和 miRNA（白细胞介素 8 (IL-8)、IL-1β、肿瘤坏死因子 α (TNF-α)、miR-203b-5p 和 miR-16）的基因表达增加-5p)，以及凋亡标记物（p21、p53、caspase-3、caspase-9）。此外，我们的研究表明，线粒体融合蛋白 1 (MFN1) 的蛋白水平随着年龄的增长而显着降低。 衰老的 ASC 还表现出参与干细胞稳态和归巢的基因的 mRNA 水平降低，例如 TET-3、TET-3（TET 家族）和 CXC 趋化因子受体 4 型 (CXCR4)，以及 DNA 甲基转移酶的蛋白质表达(DNMT1) 和八聚体转录因子 3/4 (10 月 3/4)。此外，我们观察到 XBP1（X-box 结合蛋白 1）mRNA 的剪接率较高，表明肌醇需求酶 1 (IRE-1) 活性升高，从而增加内质网 (ER) 应激。我们还观察到葡萄糖转运蛋白 4 (GLUT-4) 和胰岛素受体 (INSR) 水平降低，这表明胰岛素敏感性受损。结论 获得的数据表明，源自 5 岁以上马的 ASC 表现出多种分子改变，这明显限制了它们的再生能力。这些结果提供了有价值的信息，可以更好地了解 ASC 在衰老过程中发生的分子事件，并可能有助于识别新的潜在药物靶点，以恢复其再生潜力。