Xu Yingxing, Jiang Yaping, Jia Bin, Wang Yingzhen, Li Tao
Department of Joint Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266003, China; Qingdao University, Qingdao, Shandong, 266071, China; Medical Department of Qingdao University, Qingdao, Shandong, 266071, China.
Department of Oral Implantology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266003, China; Qingdao University, Qingdao, Shandong, 266071, China.
Phytomedicine. 2021 May;85:153485. doi: 10.1016/j.phymed.2021.153485. Epub 2021 Jan 29.
Icariin (ICA) is a bioactive compound isolated from epimedium-derived flavonoids that modulates bone mesenchymal stem cell osteogenesis and adipogenesis. However, its precise mechanism in this process is unknown.
The purpose of this study was to elucidate the role of ICA on human bone mesenchymal stem cell (hBMSC) osteogenesis and adipogenesis by focusing on miR-23a mediated activation of the Wnt/β-catenin signaling pathway.
After ICA treatment, hBMSC osteogenesis and adipogenesis were evaluated using alkaline phosphatase staining, an alkaline phosphatase activity assay, Oil Red O staining, and cellular triglyceride levels. Moreover, the mRNA and protein expression levels of osteogenic and adipogenic markers as well as key factors of the Wnt/β-catenin signaling pathway were measured using quantitative reverse transcription polymerase chain reaction and western blotting. Lithium chloride, an activator of the Wnt/β-catenin signaling pathway, was used as a positive control. Finally, to investigate the role of miR-23a in ICA-induced activation of the Wnt/β-catenin signaling pathway, hBMSCs were transfected with miR-23a mimics or a miR-23a inhibitor.
ICA significantly promoted hBMSC osteogenic differentiation by upregulating alkaline phosphatase activity and the expression of bone sialoprotein II (BSPII) and runt-related transcription factor-2 (Runx-2). In contrast, ICA inhibited hBMSC adipogenic differentiation by reducing lipid droplet formation and cellular triglyceride levels as well as by downregulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT enhancer-binding protein-α (C/EBP-α). ICA mediated its effects on hBMSCs by activating the Wnt/β-catenin signaling pathway. It did so by upregulating β-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and T cell factor 1 (TCF1). Notably, the up-regulation of these proteins was blocked by Dickkopf-related protein 1 (DKK1). Critically, the effects of ICA on hBMSCs were similar to that of the positive control, lithium chloride. Notably, ICA-induced activation of the Wnt/β-catenin signaling pathway was significantly attenuated following miR-23a up-regulation. Conversely, miR-23a downregulation affected hBMSCs in the same manner as ICA; i.e., it activated the Wnt/β-catenin signaling pathway.
ICA promotes and inhibits, respectively, hBMSC osteogenesis and adipogenesis via miR-23a-mediated activation of the Wnt/β-catenin signaling pathway.
淫羊藿苷（ICA）是从淫羊藿衍生的黄酮类化合物中分离出的一种生物活性化合物，可调节骨髓间充质干细胞的成骨和成脂作用。然而，其在此过程中的精确机制尚不清楚。
本研究旨在通过聚焦miR-23a介导的Wnt/β-连环蛋白信号通路激活，阐明ICA对人骨髓间充质干细胞（hBMSC）成骨和成脂的作用。
ICA处理后，使用碱性磷酸酶染色、碱性磷酸酶活性测定、油红O染色和细胞甘油三酯水平评估hBMSC的成骨和成脂情况。此外，使用定量逆转录聚合酶链反应和蛋白质印迹法测量成骨和成脂标志物以及Wnt/β-连环蛋白信号通路关键因子的mRNA和蛋白质表达水平。Wnt/β-连环蛋白信号通路激活剂氯化锂用作阳性对照。最后，为研究miR-23a在ICA诱导的Wnt/β-连环蛋白信号通路激活中的作用，用miR-23a模拟物或miR-23a抑制剂转染hBMSC。
ICA通过上调碱性磷酸酶活性以及骨唾液酸蛋白II（BSPII）和 runt相关转录因子2（Runx-2）的表达，显著促进hBMSC的成骨分化。相反，ICA通过减少脂滴形成和细胞甘油三酯水平以及下调过氧化物酶体增殖物激活受体γ（PPAR-γ）和CCAAT增强子结合蛋白α（C/EBP-α）的表达，抑制hBMSC的成脂分化。ICA通过激活Wnt/β-连环蛋白信号通路介导其对hBMSC的作用。它通过上调β-连环蛋白、低密度脂蛋白受体相关蛋白5（LRP5）和T细胞因子1（TCF1）来实现。值得注意的是，这些蛋白质的上调被Dickkopf相关蛋白1（DKK1）阻断。至关重要的是，ICA对hBMSC的作用与阳性对照氯化锂相似。值得注意的是，miR-23a上调后，ICA诱导的Wnt/β-连环蛋白信号通路激活显著减弱。相反，miR-23a下调对hBMSC的影响与ICA相同；即，它激活了Wnt/β-连环蛋白信号通路。
ICA通过miR-23a介导的Wnt/β-连环蛋白信号通路激活，分别促进和抑制hBMSC的成骨和成脂。
