In vitro study of effects and mechanisms of White Kwao Krua Pueraria mirifica extracted phytoestrogens and synthetic phytoestrogens on bone formation and resorption

Abstract

This study aimed to investigate the effects and mechanisms of actions of Pueraria mirifica (PM) extract, a Thai herb which contained various types of phytoestrogens, including genistein (GEN) and puerarin (PU), on bone formation and resorption process in two types of rat osteoblast cells, osteosarcoma UMR106 and primary cells. To develop the PM as an alternative drug for osteoporosis treatment in humans, the culture system of baboon primary osteoblast cells was first established. UMR106 rat osteosarcoma cells, rat and baboon primary osteoblast cells were incubated with PM extract, GEN and PU in different doses and time-courses. Cell proliferation, mRNA expression of genes associated with bone formation and resorption, and mineralization processes of osteoblast cells were determined by BrdU assay, qRT-PCR and Alizarin Red S staining, respectively. In the UMR106 cells, PM extract, GEN and PU decreased cell proliferation, increased mRNA expression of the gene associated with bone formation, i.e., alkaline phosphatase (ALP), and the gene associated with bone resorption, i.e., osteoprotegerin (OPG), while the mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) were varied, however, the RANKL/OPG ratios, an indicator of bone resorption process, were decreased. The in vitro calcium deposition was also increased. The effects of the PM extract and phytoestrogens were passed through the estrogen receptors (ERs) by observing the abolishment of ER antagonist (ICI182780) on expression of ALP mRNA. Further study in rat and baboon primary osteoblast cells indicated that the responses of these two cell types to the PM extract, GEN and PU were nearly in the same line. All three treatments stimulated cell proliferation and expression of genes associated with bone formation (ALP in rat, and ALP and type I collagen in baboon osteoblast cells). However, the expression of RANKL mRNA levels and the RANKL/OPG ratios in baboon osteoblasts were decreased, whilst no alterations of those genes were observed in primary rat osteoblasts. In conclusion, the PM extract induced bone formation by enhancing expression of genes associated with osteoblast differentiation and suppressing expression of genes associated with osteoclast differentiation, in an ER-dependent manner. This study corroborates the high potential of the PM herb to be developed as anti-osteoporotic drug for human use. In regard to the different responses of these three cell types to the PM extract, it suggests that research on effects and mechanisms of actions of PM extract in vitro, at least more than one cell types of osteoblasts should be used.