Icariin stimulates osteogenesis and suppresses adipogenesis of human bone mesenchymal stem cells via miR-23a-mediated activation of the Wnt/β-catenin signaling pathway

Abstract

Background: 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.

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: ICA promotes and inhibits, respectively, hBMSC osteogenesis and adipogenesis via miR-23a-mediated activation of the Wnt/β-catenin signaling pathway.

Keywords: Adipogenesis; Human bone mesenchymal stem cells; Icariin; MicroRNA-23a; Osteogenesis; Wnt/β-catenin signaling pathway.