Figure 2.

Kaempferol-induced osteogenic differentiation is mediated by ER signaling cultured osteoblasts. A: Application of 17β-estradiol (100 nM) or kaempferol (30 - 300 μM) in cultured osteoblasts for 3 days increased ALP activity in a dose-dependent manner. The stimulatory effect was abolished upon pre-treatment with ICI 182,780 (100 nM) for 1 hour. The ALP activities detected after the pre-treatment of ICI 182, 780 were compared with the ALP activities detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol (P = 0.0412), kaempferol at 30 μM (P = 0.0485), 100 μM (P = 0.0081) and 300 μM (P = 0.0086). B: Cultured osteoblats were treated with 17β-estradiol (100 nM) or kaempferol (10 μM) for 2 days, with or without pre-treatment with ICI 182,780 (100 nM) for 1 hour. Total RNAs were extracted from the cultures to perform quantitative PCR for osteogenesis-associated genes, including type I collagen (COL1A1), osteonectin, osteocalcin, osterix and Runx2 mRNAs. The mRNA amounts of osteogenesis-associated genes detected after the pre-treatment of ICI 182, 780 were compared with the mRNA amounts detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol (P = 0.0012 for COL1A1; P = 0.0070 for osteonectin; P = 0.0033 for osteocalcin; P = 0.0441 for osterix and P = 0.0023 for Runx2) and kaempferol (P = 0.0065 for COL1A1; P = 0.0063 for osteonectin; P = 0.0072 for osteocalcin; P = 0.0068 for osterix and P = 0.0064 for Runx2). C: Cultured osteoblasts underwent mineralization upon the addition of 17β-estradiol (100 nM) or kaempferol (10 μM) in the presence of β-glycerophosphate (5 mM). After 21 days of treatment, nodules were found, as shown by Alizarin Red staining. The mineralization process was hindered by pre-treatment with ICI 182,780 (100 nM). D: From the cultures of (C), Alizarin Red staining was quantified using a solution of 20% methanol and 10% acetic acid in water, and the reading was done on a spectrophotometer at 450 nm. The normalized alizarin red amounts detected after the pre-treatment of ICI 182, 780 were compared with the amount detected without the pre-treatment. The statistically significant results include the blocking effects of 17β-estradiol (P = 0.0093) and kaempferol (P = 0.0085). Values in all panels are expressed as the fold increase from the basal reading (control culture; 0.02% DMSO); mean ± SD, n = 5, each with triplicate samples.