Shikonin promotes osteogenesis and suppresses osteoclastogenesis in vitro

Abstract

Shikonin, as a traditional Chinese herbal medicine with a role of anti-cancer, anti-inflammatory, anti-bacterial and other effects. However, there are few studies on the effect of shikonin on osteoporosis. Therefore, the purpose of this study aims to investigate the role and mechanism of shikonin on differentiation of BMSCs and BMMs into osteoblasts and osteoclasts formation. In our study, we treated the cells with different concentrations of shikonin, and then illuminated its effect on oteogenesis and osteoclast differentiation by ALP/alizarin red staining, ALP activity, qRT-PCR, immunofluorescence, Western blot, and TRAP staining. The result showed that shikonin may promote BMSCs differentiate into osteoblasts through the Wnt/β-catenin signaling pathway. At the same time, it may also inhibit the formation of osteoclasts mediated by RANK/RANKL/OPG pathway in vitro. Our research explains excellently the mechanism of shikonin alleviating osteoporosis in vitro, which maybe contributing to the exploration of a new way to prevent osteoporosis.

Keywords: OPG; RANKL; Shikonin; Wnt; mesenchymal stem cells; osteoporosis; β-cantenin.

Figure 1

(A) The molecular structure of shikonin. High concentration of shikonin restrained the proliferation of BMSCs. Primary BMSCs were treated with shikonin before measuring cell viability. The CCK-8 assay (B) and live/dead staining (C) showed that the number of dead BMSCs decreased significantly after 48 hours of shikonin treatment at a concentration higher than 250 nM. Cytotoxicity (live/dead) assay images show the live (green) and dead cells (red) in a cellulose sample.

Figure 2

Effect of shikonin on osteogenic differentiation in BMSCs. A. BMSCs were cultured with OM (control) and shikonin. All the assays were performed using ALP staining assay after 7 days. B, C. BMSCs were cultured with OM (control), with or without shikonin for 14 days. All the assays were performed and quantified by Alizarin Red S staining. *P < 0.05 and #P < 0.01 vs the control group (n=3). D. ALP activity was tested on BMSCs cultured with OM and shikonin for 7 days. *P < 0.05 and #P < 0.01 vs the control group (n=3). E-G. BMSCs cultured with OM (control), shikonin (50, 150, 250 nM) were harvested on 3 day and 5 day. The mRNA expression levels of ALP, col-1 and OCN were assessed by qRT-PCR and quantified *P < 0.05 and #P < 0.01 vs the control group.

Figure 3

Shikonin stimulated the osteogenesis differentiation in BMSCs through Wnt/β-catenin signalling pathway. A. BMSCs cultured with OM (control), shikonin (50, 150, 250 nM) were harvested on days 3. The protein expression levels of wnt 1, β-catenin, GSK-3β and p-GSK-3β were assessed by WB and quantified *P < 0.05 and #P < 0.01 vs the control group. B. The protein expression levels of wnt 1 and β-catenin were quantified *P < 0.05 and #P < 0.01 vs the control group. C. BMSCs cultured with OM (control), with or without shikonin (250 nM) or ICG-001 were harvested on 48 h, the protein expression of ALP, col-1 and OCN were quantified. *P < 0.05 and #P < 0.01 vs the control group. D. BMSCs cultured with OM (control), with or without shikonin were harvested at 48 h. The mRNA expression levels of β-catenin and Runx2 were assessed by qRT-PCR and quantified. *P < 0.05 and #P < 0.01 vs the control group. E. Immunofluorescence detection of β-catenin translocation in cultured. BMSCs were treated with OM (control) or shikonin (50, 150, 250 nM). β-catenin expressed in both the cytoplasm and nucleus, as well as the fluorescent density and intensity was increased dose dependently for 5 days. The nuclei were stained with DAPI and were shown as blue fluorescence. Scale bar =100 µm. F. BMSCs cultured with OM (control), with or without shikonin or ICG-001 were harvested on 48 h, ALP staining and Alizarin red S staining were assessed. G. BMSCs treated with OM (control), shikonin were harvested on 48 h. The protein expression levels of RANKL and OPG were assessed by WB and quantified *P < 0.05 and #P < 0.01 vs the control group. H. BMSCs treated with shikonin (250 nM) were harvested on 12, 24 or 36 h. The protein expression levels of RANKL and OPG were assessed by WB and quantified *P < 0.05 and #P < 0.01 vs the control group.

Figure 4

Shikonin restrained the proliferation and differentiation of BMMs directly. A. Effects of shikonin on BMMs viability at 24, 48 or 72 h. B. Live/dead staining obtained for the activity of shikonin against BMMs at 72 h. *P < 0.05 and #P < 0.01 vs the control group. C-E. TRAP-positive BMMs treated with different concentrations of shikonin followed by the stimulation with M-CSF and RANKL for 5 days. Quantification of TRAP-positive multinuclear cells, area of osteoclasts. F. NFATc1, c-Fos, CTSK and TRAP expression in BMMs treated with the indicated shikonin concentrations for 2 days and quantified. *P < 0.05 and #P < 0.01 vs the control group. G. NFATc1, c-Fos and TRAP expression levels in BMMs treated with shikonin for 12 h, 24 h or 48 h. *P < 0.05 and #P < 0.01 vs the control group. H. Immunofluorescence detection of TRAP-positive BMMs in cultured for 5 days.

Figure 5

Shikonin inhibited the differentiation of BMMs indirectly. A. NFATc1 and c-Fos expression in BMMs treated with the indicated concentrations of supernatant for 48 h and quantified. *P < 0.05 and #P < 0.01 vs the control group. B-D. TRAP-positive BMMs treated with indicated concentrations of supernatant followed by the stimulation with M-CSF and RANKL for 5 days. Quantification of TRAP-positive multinuclear cells, area of osteoclasts.