Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis

Abstract

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.

Keywords: angiogenesis; astragaloside IV; bone marrow mesenchymal stem cell; distraction osteogenesis; preosteoclast.

Figure 1

Effect of AS-IV on osteoclast differentiation of RAW264.7 cells induced by RANKL. (A) Representative images of TRAP staining showing osteoclast and preosteoclast formation from RAW264.7 cells treated with vehicle, RANKL, and RANKL + various concentrations of AS-IV. Scale bar: 200 µm. (B) Quantitative analysis of the area of osteoclasts and the number of preosteoclasts. (C) qRT-PCR analysis of CTSK, Atp6v0d2, and PDGF-BB expression levels in RAW264.7 cells treated with vehicle, RANKL, and RANKL + AS-IV (40 µM). (D) Detection of PGDF-BB concentration in conditioned media from RAW264.7 cells treated with vehicle, RANKL and RANKL + AS-IV (40 µM) by ELISA. (E) Western blot of AKT, p-AKT, GSK-3β, p-GSK-3β, β-catenin, p-β-catenin, and NFATc1 in RAW264.7 cells treated with vehicle, RANKL, and RANKL + various concentrations of AS-IV. (F) Quantitative analysis of the phosphorylated levels of AKT, GSK-3β, and β-catenin and the protein level of NFATc1 relative to GAPDH. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. *P < 0.05; ^**P < 0.01.

Figure 2

AS-IV augments the pro-angiogenic effects of preosteoclasts on endothelial cells. (A, B) Representative images (A) and quantification of tube formation (B) in Ea.hy926 cells stimulated with CM from each group and PDGF-BB-neutralizing antibody of IgG isotype control antibody. Scale bar: 200 µm. (C-F) Endothelial cell motility in different treatment groups was evaluated using the Transwell migration assay (C, D) and the scratch wound assay (E, F). Scale bar: 200 µm. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. *P < 0.05; ^**P < 0.01.

Figure 3

AS-IV promotes osteogenic differentiation, cell viability, and the expression of angiogenic genes of BMSCs. (A-D) Osteogenesis of BMSCs treated with OIM and different concentrations of AS-IV were determined with ALP staining (A), ALP activity assays (B) and alizarin red staining (C). Calcium deposition was assessed by measuring the optical density (D). Scale bar: 200 µm. (E) CCK-8 analysis of BMSC proliferation in different treatment groups. (F, G) Expression of osteogenic-specific genes (F) and angiogenic-specific genes (G) of BMSCs treated with OIM+AS-IV (40 µM) were assessed with qRT-PCR. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. *P < 0.05; ^**P < 0.01; ^#P < 0.05 vs 0 µmol/L group; ^##P < 0.01 vs 0 µmol/L group; ^ΔP < 0.05 vs 40 µmol/L group; ^ΔΔP < 0.01 vs 40 µmol/L group.

Figure 4

AS-IV activates the AKT/GSK-3β/β-catenin pathway in BMSCs. (A) Western blot of AKT, p-AKT, GSK-3β, p-GSK-3β, β-catenin, and p-β-catenin in BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206. (B) Quantitative analysis of the phosphorylated levels of AKT, GSK-3β, and β-catenin. (C) Representative immunocytochemistry images showing the expression and nuclear translocation of β-catenin in BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206. Scale bar: 100 µm. (D) Quantitative analysis of the expression and nuclear translocation of β-catenin. ^**P < 0.01. MFI, mean fluorescence intensity. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. ^NSP > 0.05; ^**P < 0.01.

Figure 5

MK2206 inhibits the effects of AS-IV on osteogenic differentiation, cell viability, and angiogenic gene expression of BMSCs. (A-D) Osteogenesis of BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206 was determined with ALP staining (A), ALP activity assays (B) and alizarin red staining (C). Calcium deposition was assessed by measuring the optical density (D). Scale bar: 200 µm. (E) CCK-8 analysis of BMSC proliferation in different treatment groups. (F, G) Expression of osteogenic-specific genes (F) and angiogenic-specific genes (G) of BMSCs in different treatment groups were assessed using qRT-PCR. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. ^**P < 0.01; ^#P < 0.05 vs control group; ^##P < 0.01 vs control group; ^ΔΔP < 0.01 vs AS-IV group.

Figure 6

AS-IV administration accelerates bone consolidation during distraction osteogenesis in rats. (A) Mechanical properties, including E-modulus, ultimate load, and energy to failure of distraction regenerates in control and AS-IV groups. The values were normalized to the corresponding contralateral normal tibias. (B) Representative X-rays of distraction regenerates at various time points. (C, D) Representative 3D and longitudinal images (C) and quantitative analysis (D) of micro-CT data, including bone mineral density and bone volume/tissue volume, of the tibial distraction zone after 2 and 4 weeks of consolidation and treatment. The data were confirmed by Student's t-test between control group and AS-IV group and are presented as the means ± SD. ^*P < 0.05; ^**P < 0.01.

Figure 7

AS-IV improves vascularized bone regeneration in the tibial distraction zone. (A) Representative images of H&E, Masson, and Safranin O-Fast Green staining and immunohistochemical analysis of OCN. Scale bar: 200 µm. Black arrows, cartilaginous tissue. Dotted arrows, fibrous-like tissue. White arrows, newly formed trabecular bone. (B) Micro-CT observation of the newly formed blood vessels perfused with Microfil in the distraction regions. (C) Quantitative analysis of the vessel volume fractions within the distraction gaps from the two groups. (D) Immunofluorescence staining images of CD31 and EMCN for the distraction area sections from the two groups. Scale bar: 100 µm. (E) Quantitative analysis of CD31^hiEMCN^hi cells per mm² from the staining results. (F) Immunohistochemical analysis of β-catenin in control and ADM2 groups. Scale bar: 500 µm. (G) Quantitative analysis of the immunohistochemical staining of β-catenin. The data were confirmed by Student's t-test between control group and AS-IV group and are presented as the means ± SD. ^**P < 0.01.

Figure 8

Working model of AS-IV promoting osteogenesis along with preosteoclast-induced angiogenesis during DO by activating AKT/GSK-3β/β-catenin pathway. Under the administration of AS-IV, AKT/GSK-3β/β-catenin signaling is activated in BMSCs and macrophages. In BMSCs, the osteogenic potential is enhanced, and the expression of angiogenic genes is up-regulated. Additionally, in RANKL-induced macrophages, the activated AKT/GSK-3β/β-catenin signaling inhibits the maturation of multinucleated osteoclasts, increasing the production of PDGF-BB from preosteoclasts, thus enhancing the angiogenesis of ECs. In conclusion, AS-IV could directly promote osteogenesis and indirectly improve angiogenesis by activating AKT/GSK-3β/β-catenin signaling in BMSCs and macrophages simultaneously, thus enhancing the coupling of osteogenesis and angiogenesis and accelerating bone regeneration during DO. AS-IV: astragaloside IV. AKT: protein kinase B. GSK-3β: glycogen synthase kinase-3β. BMSCs: bone marrow derived mesenchymal stem cells. DO: distraction osteogenesis. RANKL: receptor activator for nuclear factor κB ligand. PDGF-BB: platelet-derived growth factor-BB. ECs: endothelial cells.