Local Health Unit Treviso, Department of Pediatric Surgery, 31100 Treviso, Italy.
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy.
Int J Mol Sci. 2023 Jun 20;24(12):10397. doi: 10.3390/ijms241210397.
Glucosamine (GlcN) is a glycosaminoglycan (GAGs) constituent in connective tissues. It is naturally produced by our body or consumed from diets. In the last decade, in vitro and in vivo trials have demonstrated that the administration of GlcN or its derivates has a protective effect on cartilage when the balance between catabolic and anabolic processes is disrupted and cells are no longer able to fully compensate for the loss of collagen and proteoglycans. To date, these benefits are still controversial because the mechanism of action of GlcN is not yet well clarified. In this study, we have characterized the biological activities of an amino acid (AA) derivate of GlcN, called DCF001, in the growth and chondrogenic induction of circulating multipotent stem cells (CMCs) after priming with tumor necrosis factor-alpha (TNFα), a pleiotropic cytokine commonly expressed in chronic inflammatory joint diseases. In the present work, stem cells were isolated from the human peripheral blood of healthy donors. After priming with TNFα (10 ng/mL) for 3 h, cultures were treated for 24 h with DCF001 (1 μg/mL) dissolved in a proliferative (PM) or chondrogenic (CM) medium. Cell proliferation was analyzed using a Corning Cell Counter and trypan blue exclusion technique. To evaluate the potentialities of DCF001 in counteracting the inflammatory response to TNFα, we measured the amount of extracellular ATP (eATP) and the expression of adenosine-generating enzymes CD39/CD73, TNFα receptors, and NF-κB inhibitor IκBα using flow cytometry. Finally, total RNA was extracted to perform a gene expression study of some chondrogenic differentiation markers (COL2A1, RUNX2, and MMP13). Our analysis has shed light on the ability of DCF001 to (a) regulate the expression of CD39, CD73, and TNF receptors; (b) modulate eATP under differentiative induction; (c) enhance the inhibitory activity of IκBα, reducing its phosphorylation after TNFα stimulation; and (d) preserve the chondrogenic potentialities of stem cells. Although preliminary, these results suggest that DCF001 could be a valuable supplement for ameliorating the outcome of cartilage repair interventions, enhancing the efficacy of endogenous stem cells under inflammatory stimuli.
氨基葡萄糖（GlcN）是连接组织中糖胺聚糖（GAGs）的成分。它是由我们的身体自然产生的，也可以从饮食中摄取。在过去的十年中，体外和体内试验表明，当分解代谢和合成代谢过程之间的平衡被破坏，细胞不再能够完全补偿胶原蛋白和蛋白聚糖的损失时，GlcN 或其衍生物的给药对软骨具有保护作用。迄今为止，这些益处仍然存在争议，因为 GlcN 的作用机制尚不清楚。在这项研究中，我们研究了 GlcN 的一种氨基酸（AA）衍生物 DCF001 对经过肿瘤坏死因子-α（TNFα）预刺激的循环多能干细胞（CMCs）生长和软骨诱导的生物学活性，TNFα 是一种在慢性炎症性关节疾病中普遍表达的多效细胞因子。在本研究中，我们从健康供体的人外周血中分离出干细胞。用 TNFα（10ng/ml）预刺激 3 小时后，用 DCF001（1μg/ml）在增殖（PM）或软骨形成（CM）培养基中处理 24 小时。用康宁细胞计数器和台盼蓝排除技术分析细胞增殖。为了评估 DCF001 对抗 TNFα 炎症反应的潜力，我们使用流式细胞术测量了细胞外三磷酸腺苷（eATP）的含量和产生腺苷的酶 CD39/CD73、TNFα 受体以及 NF-κB 抑制剂 IκBα 的表达。最后，提取总 RNA 以进行一些软骨分化标志物（COL2A1、RUNX2 和 MMP13）的基因表达研究。我们的分析揭示了 DCF001 能够：（a）调节 CD39、CD73 和 TNF 受体的表达；（b）在分化诱导下调节 eATP；（c）增强 IκBα 的抑制活性，减少 TNFα 刺激后的磷酸化；（d）保持干细胞的软骨形成潜力。尽管这只是初步结果，但这些结果表明，DCF001 可能是改善软骨修复干预效果的有价值的补充剂，可增强炎症刺激下内源性干细胞的疗效。
