Cajanolactone A Promoted Osteogenic Differentiation In Human Mesenchymal Stem Cells Via Activating Wnt/β-catenin/runx-2 Sigaling Pathway

Osteoporosis (OP) is bone metabolism disorder that is characterized by bone mass loss and bone tissue damage, which increases the risk of fragility fractures. OP, caused by reduced bone formation and/or enhanced bone resorption, seriously affects human health and the quality of normal life. Wnt/β-catenin/Runx-2 signaling transduction pathway regulates the process of osteoblast differentiation, cell growth and apoptosis, playing an important role in maintenance of bone metabolic balance.Objective:In previous study, cajanolactione A (CLA) was isolated from the hydrophobic extract of Cajanus cajan. CLA showed the ability to promote osteoblast differentiation and enhance bone density in mice with bone loss. To further unravel the anti-osteoporotic mechanism of CLA,, effect of CLA on Wnt/β-Catenin signaling pathway in the process of osteogenic differentiation is investigated in this study, by using human mesenchymal stem cells (hBMSCs). Our study might provide experimental basis for the development of CLA as a clinic anti-osteoporosis drug.Methods:(1) Effect of CLA on proliferation of hMSCs:in growth medium, well grown hMSCs (passage 4-6) were incubated with different concentrations of CLA for 38 or 72h. CCK-8 assay was used for detection of cell viability. (2) Effect of CLA on osteogenic differentiation:hBMSCs (passage 5) were induced for osteogenic differentiation in the presence or absence of various cocentrations of CLA. Osteoblast activity and the calcium nodules were monitored by alkaline phosphatase (ALP) staining and alizarin red S staining.(3) Effect of CLA on Wnt/beta-catenin/runx-2 signaling pathway:hMSCs (passage 5) were induced for osteogenic differentiation in the presence or absence of different concentrations of CLA. Cells without induction were used as negative control. Cells induced for 0.5 d,3d,6 d,9 d, or 12 d. were extracted for total RNA and mRNA levels of genes encoding Wnt/β-catenin/runx-2 siganal transducer, such as Runx-2 and Osterix (bone early transcription factors), Wnt-3a, LRP-5/6 (low density lipoprotein receptor related protein 5/6), Fz (frizzled), β-catenin were quantified by employment of RT-PCR trchnology. While cells induced for 15 d were extracted for total protein and the protein levels of Runx-2, β-catenin, collagen Ⅰ were deteced by Western Blotting.Results:(1) Results from proliferation assay:CLA at 31 μ mol/L partially inhibited the growth of hBMSC, but at 10-0.6 μmol/L it did not affect the proliferation of hBMCSs, after incubation with the cells for 38 h or 72 h. (2) Results from Alkaline phosphatase and alizarin red S staining:hBMSCs induced for osteogenic differentiation in the presence of CLA (4,2 or 1 μ mol/L) showed increased staining of Alkaline phosphatase and alizarin red S, implying promoted osteoblast differentiation by CLA, but dose-depedent effect was not observed between cells treated with 4 μmol/L and 2 μmol/L of CLA. (3) Results from RT-PCR analysis:compared with the negative control, cells induced for osteogenic differentiation expressed obviously upregulated mRNA levels of Wnt-3a, LRP-5, Frizzled, β-catenin, Runx-2 and Osterix gene in the whole period of differentiation induction, and the presence of CLA (2,1,0.5 μmol/L) further stimulated the expression of these genes. Gene expression reached the peaks at the 3rd day of the induction, in a dose-depedent manner in respond to CLA, then decreased to relatively lower and stable levels. (4) Results from Western blotting:CLA treatment significantly stimulated the expression of Runx-2, Collagen Ⅰ and β-catenin at the protein levels.Conclution:CLA could promote the osteogenic differentiation in hBMCSs, and this might be achieved by activation of classical Wnt/β-catenin signaling pathway with transcription factor of Runx-2,Osterix.