Ginsenoside Compound K Enhances Fracture Healing via Promoting Osteogenesis and Angiogenesis

Abstract

Fractures have an extraordinarily negative impact on an individual's quality of life and functional status, particularly delayed or non-union fractures. Osteogenesis and angiogenesis are closely related to bone growth and regeneration, and bone modeling and remodeling. Recently Chinese medicine has been extensively studied to promote osteogenic differentiation in MSCs. Studies have found that Ginseng can be used as an alternative for tissue regeneration and engineering. Ginseng is a commonly used herbal medicine in clinical practice, and one of its components, Ginsenoside Compound K (CK), has received much attention. Evidence indicates that CK has health-promoting effects in inflammation, atherosclerosis, diabetics, aging, etc. But relatively little is known about its effect on bone regeneration and the underlying cellular and molecular mechanisms. In this study, CK was found to promote osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) by RT-PCR and Alizarin Red S staining in vitro. Mechanistically, we found CK could promote osteogenesis through activating Wnt/β-catenin signaling pathway by immunofluorescence staining and luciferase reporter assay. And we also showed that the tube formation capacity of human umbilical vein endothelial cells (HUVECs) was increased by CK. Furthermore, using the rat open femoral fracture model, we found that CK could improve fracture repair as demonstrated by Micro-CT, biomechanical and histology staining analysis. The formation of H type vessel in the fracture callus was also increased by CK. These findings provide a scientific basis for treating fractures with CK, which may expand its application in clinical practice.

Keywords: CK; Wnt/β-catenin; angiogenesis; fracture healing; osteogenesis.

FIGURE 1

CK enhanced cell viability, osteogenesis and angiogenesis in vitro. (A) The chemical structure depiction of CK. (B) Optical density (OD) values indicating the effects of CK treatment for 48 h and 72h on cell viability of BMSCs, *p < 0.05, n = 3, compared with 0 μM CK group. (C–F) Real-time PCR results of osteogenesis-related genes at 3 days treated with different concentrations of CK in OIM, *p < 0.05, n = 3, compared with OIM group. (G) Representative staining images and (H) Quantification of Alizarin Red S of BMSCs incubated with different concentrations of CK in OIM for 14 days, *p < 0.05, n = 3, compared with OIM group. (I) Optical density (OD) values indicating the effects of CK treatment for 48 h and 72h on cell viability of HUVECs, *p < 0.05, n = 3, compared with 0 μM CK group. (J) Representative images of tube formation assay and (K-L) Quantitative analysis of tube length and branch points in Matrigel MSCs and HUVEC co-culture system, Scale bars < 100 μm, *p < 0.05, compared with the Control group, n = 3.

FIGURE 2

CK increased β-catenin and Runx2 expression in BMSCs. (A) Representative images and (B) Quantification of immunofluorescence staining of β-catenin and Runx2 treated with 10 μM CK for 24 h. Scale bars = 50 μm, *p < 0.05, compared with the OIM group, n = 3 (C) The TOPflash luciferase activity was measured in BMSCs after treatment of CK (10 μM). *p < 0.05, compared with the Control group, n = 3.

FIGURE 3

CK accelerated the progression of fracture healing. (A) Schematic illustration of time points of animal modeling and sample collection. (B) Representative 3-dimensional micro-CT images of femurs in each group. (C–E) Quantitative analysis of parameters, including CV, BMD, and BV/TV, *p < 0.05, compared with the PBS group, n = 3 (F–G) Biomechanical properties of the fractured bones by 3-point bending test, *p < 0.05, compared with the PBS group, n = 6.

FIGURE 4

H&E, Safranin O-Fast Green, and immunohistochemical examination of fracture callus. (A) Representative images of H&E staining of fracture calluses in PBS and CK groups. Insets indicate the regions shown in the enlarged images (lower). Scale bar: 500 μm. (B) Representative images of Safranin O-Fast Green staining of fracture calluses in PBS and CK groups. Insets indicate the regions shown in the enlarged images (lower). (C–F) Representative images of immunohistochemical analysis of OPG, ALP, RANKL and OPN of fracture calluses in PBS and CK groups. Scale bar = 50 μm.

FIGURE 5

CK regulated H-type vessel formation in fracture callus. (A) Representative immunofluorescence double staining and (B) Quantification of CD31 (green), EMCN (red) of fracture calluses in PBS and CK groups, Scale bars = 50 μm, *p < 0.05, compared with the PBS group, n = 3.

FIGURE 6

CK up-regulated β-catenin and Runx2 in fracture callus. (A) Immunofluorescence staining images of β-catenin and Runx2 in PBS and CK groups and (B) Quantification of the immunofluorescence density of β-catenin and Runx2 of fracture calluses in PBS and CK groups, Scale bars = 50 μm, *p < 0.05, compared with the PBS group, n = 3.