Aucubin Promotes Osteogenic Differentiation and Facilitates Bone Formation through the lncRNA-H19 Driven Wnt/ β-Catenin Signaling Regulatory Axis

Abstract

Objectives: Traditional Chinese medicine Cortex Eucommiae has been used to treat bone fracture for hundreds of years, which exerts a significant improvement in fracture healing. Aucubin, a derivative isolated from Cortex Eucommiae, has been demonstrated to possess anti-inflammatory, immunoregulatory, and antioxidative potential. In the present study, our aim was to explore its function in bone regeneration and elucidate the underlying mechanism.

Materials and methods: The effects of Aucubin on osteoblast and osteoclast were examined in mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) and RAW 264.7 cells, respectively. Moreover, the lncRNA H19 and Wnt/β-catenin signaling were detected by qPCR examination, western blotting, and luciferase activity assays. Using the femur fracture mice model, the in vivo effect of Aucubin on bone formation was monitored by X-ray, micro-CT, histomorphometry, and immunohistochemistry staining.

Results: In the present study, Aucubin was found to significantly promote osteogenic differentiation in vitro and stimulated bone formation in vivo. Regarding to the underlying mechanism, H19 was found to be obviously upregulated by Aucubin in MSCs and thus induced the activation of Wnt/β-catenin signaling. Moreover, H19 knockdown partially reversed the Aucubin-induced osteogenic differentiation and successfully suppressed the activation of Wnt/β-catenin signaling. We therefore suggested that Aucubin induced the activation of Wnt/β-catenin signaling through promoting H19 expression.

Conclusion: Our results demonstrated that Aucubin promoted osteogenesis in vitro and facilitated fracture healing in vivo through the H19-Wnt/β-catenin regulatory axis.

Figure 1

Aucubin promoted osteogenic differentiation of BM-MSCs. (a) Qualitative and quantitative examination of LP activity on day 7. (b) Qualitative and quantitative assays of ARS staining on day 21. Scale bar: 200 μm. (c) The expression of osteogenic marker genes was measured on day 7. All experiments were repeated three times.  ^∗P  < 0.05;  ^∗∗P  < 0.01;  ^∗∗∗P  < 0.001.

Figure 2

H19 was significantly upregulated by Aucubin in BM-MSCs. (a) Several lncRNAs related to osteogenesis were screened for examining their expression profiling in the MSC treated with Aucubin. (b) The H19 expression was further detected in the Aucubin-treated MSCs (0, 2.5, 5, 10, 20 μM). (c) The H19 expression was examined during osteogenesis. n = 3;  ^∗P  < 0.05;  ^∗∗P  < 0.01;  ^∗∗∗P  < 0.001.

Figure 3

Aucubin stimulated the activation of Wnt/β-catenin signaling in osteogenic differentiation. (a) Luciferase activity was examined in the MSCs transfected with TOPFlash firefly luciferase reporter and TK-Renilla reporter with Aucubin treatment. (b) The expression levels of total, nuclear, and cytoplasmic β-catenin were detected by western blotting with Aucubin (0, 2.5, 5, 10, 20 μM) treatment. (c) β-catenin expression was also detected by immunofluorescence staining. (d–g) The expression of several downstream genes of Wnt/β-catenin signaling was detected by qRT-PCR at day 7. All experiments were repeated three times.  ^∗P  < 0.05;  ^∗∗P  < 0.01;  ^∗∗∗P  < 0.001.

Figure 4

Aucubin facilitated bone fracture healing in vivo. The femoral fracture was created in C57BL/6J mice and subsequently treated with Aucubin for 4 or 6 weeks of low-dose (0.5 mg/kg), high-dose (1.0 mg/kg), or stroke-physiological saline solution. (a) Representative X-ray images were taken during the fracture healing processes at weeks 4 and 6. (b, c) Micro-CT examination of the femur fractured zone in Aucubin-treated groups. (d) The statistical diagram of TV and BV/TV at weeks 4 and 6. n = 3.  ^∗∗P  < 0.01 and  ^∗∗∗P  < 0.001.

Figure 5

Aucubin faciliatted fracture healing by histological examination. (a) H&E staining assays for the fractured femur section at weeks 4 and 6. Immunohistofluorescence examination of OCN (b), OSX (c), and β-catenin (d) in the fractured femur section at weeks 4 and 6. Scale bars: 200 μm (upper panel) and 50 μm (lower panel, inset).

Figure 6

Aucubin promoted osteoblast differentiation via the H19-Wnt/β-catenin regulatory axis. (a) The ALP activity and ARS staining were examined in the Aucubin-treated shH19 MSCs. (b) The expression levels of osteogenic marker genes in the Aucubin-treated shH19 MSCs was examined. (c, d) The protein levels of total, nuclear, and cytoplasmic β-catenin were examined in the shH19 MSCs with Aucubin treatment. (d) The downstream genes of Wnt signaling were also detected in this Aucubin-treated shH19 MSCs. n = 3;  ^∗P  < 0.05;  ^∗∗P  < 0.01;  ^∗∗∗P  < 0.001.

Figure 7

A schematic diagram of the mechanism illustrated in this study. Aucubin promoted osteogenic differentiation and facilitated bone formation through the lncRNA-H19 driven Wnt/β-catenin signaling regulatory axis, in which H19 was obviously upregulated by Aucubin, and stimulated the activation of Wnt/β-catenin signaling, eventually leading to facilitating osteogenic differentiation.