Abstract
Many recent studies have suggested that bergapten (BP), a class of native compound with numerous biological activities such as anti-resorptive properties, may exert protective effects against postmenopausal bone loss. However, it remains unknown whether BP regulates or improves the osteogenic function of bone marrow stromal cells (BMSCs) in the treatment and prevention of osteoporosis. In our study, BMSCs were cultured in osteogenic induction medium with the addition of BP for 2 weeks and an ovariectomized mouse model of osteoporosis was used to investigate the anti-resorptive effect of BP by gavage administration for 3 months. The concentrations of BP used were 0.1, 1, and 10 μmol/L in vitro and the gavage dose was 20 mg/kg/d. The result of our study indicated that BP promotes the expression of alkaline phosphatase (ALP) by BMSCs in vitro in a dose-dependent manner, as revealed by ALP staining. Runt-related transcription factor 2 and osteocalcin were up-regulated both in vitro and vivo, while osterix and collagen Iα1, assessed by immunofluorescence and immunohistochemistry, were correspondingly raised in the presence of BP in BMSCs in vitro. In addition, a protective effect of BP against ovariectomy-induced bone loss was found by distal femur micro-CT scanning, with improvements of bone metabolism parameters such as bone mineral density, trabecular number, and trabecular separation. Furthermore, WNT/β-catenin signaling was activated in the presence of BP in BMSCs in osteogenic culture. Finally, BP promoted differentiation of BMSCs into osteoblasts by up-regulation of the WNT/β-catenin pathway.
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Acknowledgments
We thank Zhang Xiao-dong (Department of Medical Image, The Third Affiliated Hospital of Southern Medical University, Guangdong, PR China) for excellent technical support.
Author contribution
Design of the study: X J-j, Z W-j, Z X-t and Z W-l. Acquisition of data: X J-j, Z W-j, Z X-t, Z W-l, W X-x, Y S-h and J F. Interpretation of data: W X-x, Y S-h, J F, Z Y-x and C F-n . Manuscript preparation: X J-j, Z W-j, Z X-t, Z W-l and L S-l.
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This work was supported by Natural Science Foundation of China.
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Ethical approval was given by the Medical Ethics Committee of Southern Medical University.
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Ji-jie Xiao, Wen-ji Zhao, Xin-tao Zhang, and Wen-long Zhao have contributed equally to this work.
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11010_2015_2517_MOESM1_ESM.jpg
Supplementary Fig. 1. BP promotes the expression of RUNX2, OCN, β-catenin and GSK-3β. BMSCs were cultured in OIM alone without DMSO and BP or supplemented with BP (0.1 μM) for 2 weeks. BMSC lysates (20 μg protein) were prepared and subjected to western blot analysis using antibodies specific for OCN (a, b), RUNX2 (a, c), β-catenin (a, d) and GSK-3β (a, e) as described in the methods. The expression of OCN (b), RUNX2 (c), β-catenin (d) and GSK-3β (e) increased compared to the OIM group. Data are expressed as fold change versus OIM group. The results show that BP promotes the expression of RUNX2, OCN, β-catenin and GSK-3β compared to the OIM group. * P < 0.05 compared to OIM alone. Columns represent the mean ± SEM from nine wells per group (b–e). Below is the link to the electronic supplementary material. Supplementary material 1 (JPEG 192 kb)
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Supplementary Fig. 2. BP has no effect on the protein expression of RUNX2, OCN, β-catenin and GSK-3β in the absence of differentiation medium. BP (1–10 μM) did not alter the protein expression of OCN (a, b), RUNX2 (a, c), β-catenin (a, d) or GSK-3β (a, e) when cells were cultured for 14 days in the absence of differentiation medium. Columns represent the mean ± SEM from nine wells per group (b–e). Supplementary material 2 (JPEG 202 kb)
11010_2015_2517_MOESM3_ESM.jpg
Supplementary Fig. 3. BD did not have significant effect on endogenous stem cells as results of IHC. Through the characteristic markers of marrow stromal cells, such as CD105 (a-a’’ and c) and CD106 (b-b’’ and d), endogenous stem cells did not change significantly after BD treatment. Compared to OVX+BP group (a’’ and b’’), the expression intensity of CD105 and CD106 in OVX group (a’ and b’) and Sham group (a and b) got even distribution under microscope in IHC staining and had no significant difference between each other. n = 10. All values are shown as mean ± SEM of data from three independent experiments (c–d). Supplementary material 3 (JPEG 606 kb)
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Xiao, Jj., Zhao, Wj., Zhang, Xt. et al. Bergapten promotes bone marrow stromal cell differentiation into osteoblasts in vitro and in vivo. Mol Cell Biochem 409, 113–122 (2015). https://doi.org/10.1007/s11010-015-2517-9
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DOI: https://doi.org/10.1007/s11010-015-2517-9