Puerarin promoted osteogenesis in osteoblast-like MC3T3-E1 cells. (A) Puerarin promoted MC3T3-E1 osteoblastic cells proliferation. The cells were incubated with puerarin at the concentration of 0.1, 1, 10 and 20 μM for 48 hours before CCK-8 assay. DMSO was served as control. (B) Effect of puerarin on ALP activity of MC3T3-E1 cells. MC3T3-E1 cells were cultured with vehicle or various concentrations of puerarin for 6 days. The data are expressed as percentage of positive control (ctl+) that was induced by osteoblastic differentiation. (C) Effect of puerarin on the mineralization of extracellular matrix by MC3T3-E1 cells. AR-S staining was performed for the demonstration of mineralized nodule formation at days 8. (D) AS-R was then eluted from the matrix and measured by spectrophotometry at 562 nm. (E) Secreted Osteocalcin (OC) levels in the media were also measured after 6-day induction. (F-G) The protein expression of RUNX2, OPN, OPG was detected by western blot assay and quantification of immunoblots. * P < 0.05, ** P < 0.01 compared with the cells without treatment by puerarin (n = 3).