Osthole stimulates bone formation, drives vascularization and retards adipogenesis to alleviate alcohol-induced osteonecrosis of the femoral head

Abstract

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol-induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β-catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well-constructed rat model of ethanol-induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro-CT scanning, haematoxylin-eosin staining, TdT-mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol-induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.

Keywords: ethanol; osteonecrosis of the femoral head; osthole; vascularization.

Figure 1

Osthole alleviated the ethanol‐induced inhibition on proliferation and osteogenesis of BMSCs. A, The proliferation of BMSCs incubated in medium supplemented with ethanol and osthole was determined by CCK 8 assay. Results were means ± SEM of four independent experiments in duplicate. B, The ALP activity of BMSCs cultured in osteogenic medium supplemented with ethanol and osthole was tested at day 7 and 14. Results were means ± SEM of four independent experiments in duplicate. C‐E, The mRNA level of COL I, OCN and OPN was decreased in BMSCs after 48 h incubation with ethanol but was substantially up‐regulated when treated with osthole. GAPDH was set as a normalization control. Results were means ± SEM of four independent experiments in triplicate. F, Runx2 was decreased by ethanol but was strengthened by osthole in BMSCs. Results were means ± SEM of four independent experiments. G, Immunofluorescence staining of COL I and OCN showed osthole reversed the anti‐osteogenic effect of ethanol in BMSCs. BMSCs were cultured for 48 h in Osteogenic medium supplemented with ethanol and osthole. Cytoskeletons were stained with phalloidine (red), and the nucleus was stained with DAPI (blue)

Figure 2

Osthole abolished the ethanol‐induced inhibition on mineralization via Wnt/β‐catenin‐dependent pathway. A, The Alizarin red and ALP staining of BMSCs incubated for 21 days in osteogenic medium with supplemented ethanol and osthole, showed the inhibition role of ethanol and the protection of osthole on extracellular mineralization. B,C, β‐catenin was decreased by ethanol and increased by osthole in BMSCs. Results were means ± SEM of four independent experiments. D,E, Selective Wnt/β‐catenin antagonist JW74 abolished the protective effects of osthole. Results were means ± SEM of four independent experiments. F, JW74 abolished the reverse effects of osthole on ethanol‐induced decreased mineralized nodules deposition in BMSCs, revealed by ARS and ALP staining

Figure 3

Osthole retarded the ethanol‐promoted adipogenic differentiation of BMSCs. A, The oil red O staining of BMSCs, which were incubated for 21 days with respective conditions, showed the effect of ethanol and osthole on adipogenesis. B,C, Leptin and PPARγ mRNA were substantially up‐regulated in BMSCs after 7 days incubation with ethanol; however, they were abolished when supplemented with osthole. Results were means ± SEM of four independent experiments in triplicate. D,E, The protein of PPARγ was increased by ethanol but was counteracted by additional osthole in BMSCs. Results were means ± SEM of four independent experiments

Figure 4

Osthole alleviated the ethanol‐induced inhibition on proliferation and vasculogenesis of HUVECs. A, Proliferation of HUVECs incubated in medium supplemented with ethanol and osthole as indicated by CCK 8 assay. Results were means ± SEM of four independent experiments in duplicate. B, The tube formation image of HUVECs. C,D, Quantitation of tubes and branch points. Results were means ± SEM of four independent experiments in duplicate. E,F, Both mRNA and protein levels of VEGF in HUVECs were decreased after 48 h incubation with ethanol but were substantially up‐regulated when supplemented with osthole. GAPDH was set as a normalization control. Results were means ± SEM of four independent experiments

Figure 5

Micro‐CT scanning and analysis. A, Micro‐CT scanning images of the femoral head divided by group and section. Subchondral trabecular bone was dramatically impaired in the ethanol group; however, the trabecular structure was largely restored in the ethanol + osthole group. B‐E, BMD, Tb.N, BV/TV and Tb.Th were calculated based on reconstructed CT images. Results were means ± SEM of five specimen. BMD, bone mineral density; BV/TV, bone volume/tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness

Figure 6

Osthole ameliorated ethanol‐induced ONFH in the rat model. A, H & E staining indicated obvious osteonecrosis in the ethanol group. Empty lacunae in the subchondral trabeculae (black arrows) were observed in the ethanol group. B, TUNEL showed increased apoptosis in the ethanol group, which was alleviated by osthole treatment. The TUNEL positive cells were indicated in the trabeculae of the femoral head (red arrows). C, Immunohistochemical staining of COL I and OCN. Fewer cells in the trabeculae (black arrows) were positive for COL I and OCN in the ethanol groups; however, the staining was substantially improved with osthole treatment

Figure 7

The protective effects of osthole against ethanol‐induced ONFH in the rat model. The fluorochrome labelling indicated significantly reduced new bone formation in the ethanol‐treated rat. Bone formation capacity was substantially restored with osthole treatment