Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells

Abstract

Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1-1 μM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPAR γ ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

Figure 1

Biochanin A (BA) inhibited the adipogenic differentiation of ADSCs. ADSCs were treated for 12 days with a basic medium (negative control) or an adipogenic medium (MDI) in the presence of 0.1–1 μM biochanin A. (a) After 12 days of incubation, the cells were fixed and adipogenic differentiation was determined by the Oil red O staining of lipid droplets. Morphological changes in cells were observed using a light microscope (upper and lower panel, original magnification: 200x). Lipid droplets were quantified using Oil red O dissolved in isopropanol and by determining absorbance at 490 nm. (b) After 12 days of incubation, the cells were harvested, fixed, and stained with Nile red solution. Percentage of Nile-red-stained cells in the total population of each sample was quantified with FACScan flow cytometry. All results are expressed as the mean ± SD of three independent experiments. *P < 0.05 compared with the control.

Figure 2

Biochanin A inhibited the expression of adipogenesis-related factors in ADSCs. ADSCs were treated for 12 days with a basic medium (negative control) or an adipogenic medium (MDI) in the presence of 0.1–1 μM biochanin A. After 12 days of incubation, the expression of PPARγ, LPL, leptin, and OPN mRNA was measured by RT-PCR. GAPDH expression was used for normalization. All results are expressed as the mean ± SD of three independent experiments. *P < 0.05 compared with the control.

Figure 3

Biochanin A inhibited the expression of adipogenesis-related cytokines in ADSCs. ADSCs were treated for 12 days with a basic medium (negative control) or an adipogenic medium (MDI) in the presence of 0.1–1 μM biochanin A. After 12 days of incubation, expression of TNFα and IL-6 mRNA was measured by RT-PCR. GAPDH expression was used for normalization. All results are expressed as the mean ± SD of three independent experiments. *P < 0.05 compared with the control.

Figure 4

Biochanin A enhanced the osteogenic differentiation in ADSCs. ADSCs were treated for 12 days with a basic medium (negative control) or an osteogenic medium (OM) in the presence of 0.1–1 μM biochanin A. (a) Osteogenic differentiation was determined by alizarin red S staining of calcium deposits (mineralization) within the cell monolayer. (b) ALP activity was determined using the ALP assay kit according to the manufacturer's protocol. All results are expressed as the mean ± SD of three independent experiments. *P < 0.05 compared with the control.

Figure 5

Biochanin A enhanced the expression levels of genes and proteins that regulate osteogenic differentiation in ADSCs. ADSCs were treated for 12 days with a basic medium (negative control) or an osteogenic medium (OM) in the presence of 0.1–1 μM biochanin A. (a) After incubation, expression of ALP, OCN, and GAPDH mRNA was measured by RT-PCR. Expression of ALP and OCN genes was normalized to that of GAPDH. (b) After incubation, total proteins were isolated and analyzed for the expression of OPG, Runx2, Sirt1, and RhoA proteins by western blot analysis. β-actin was used as the internal control. All results are expressed as the mean ± SD of three independent experiments. *P < 0.05 compared with the control.