Effects Of Pinoresinol Di-O-β-D-glucopyranoside On The Expressions Of Receptor Activator Of Nuclear Factor-κB Ligand And Osteoprotegerin In Murine Osteoblasts MC3T3-E1in Vitro

Objective Osteoporosis (OP) is an irreversible systemic skeletal disorder that affects bone density and quality, leading to bone fragility and increasing the risk of fractures. Bone mass and strength decrease by age. OP is becoming a major, worldwide health problem, but the pathology is complex and poorly understood. In recent years, many researchers from abroad and China have made much effect to study on the Eucommia cortex. According to the results, it was known that Eucommia ulmoides could be benefit for the bone remolding by up-regulating OPG/RANKL ratio of osteoblasts. As we know, lignans and iridoid compounds are the characteristic constituents of Eucommia ulmoides. There are two lignans which have Pinoresinol di-O-β-D-glucopyranoside (PDG) and syringaresinol di-O-β-D-glucopyranoside, and PDG is the major component in Eucommia ulmoides. Based on the Chinese Pharmacopoeia (2010), the content of PDG in Eucommia ulmoides is no less than0.10%. In this paper, the effects of PDG on MC3T3-E1cells were determined in vitro to explore the mechanism.MethodsPart1The cells were suspended in the growth medium and plated into a96-well culture dish. MC3T3-E1cells were cultured with different concentration of PDG(0-7mg/ml、2×10-8g/ml、4×10-9mg/ml). Cell proliferation was assessed by OD that analysed through MTT colorimetric assay. Given three different concentration of PDG in MC3T3-E1cells, the clear supernatant was used to measure the ALP activity to test the differentiation of MC3T3-E1cells. And then the Alizarin red calcium deposition assay was used to see the mineralization of MC3T3-E1cells.Part2The expressions of OPG, RANKL mRNA in MC3T3-E1cells were obtained with semi-quantative RT-PCR, after cultured with three different concentration of PDG(0-7mg/ml、2×10-8mg/ml、4×10-9mg/ml). ResultsPart11. Growth of MC3T3-E1cells was good with three different concentration of PDG(0"7mg/ml、2×10-8mg/ml、4×10-9mg/ml).2. Growth of MC3T3-E1cells was promoted by stimulation with PDG (0-7mg/ml,2×10-8mg/ml,4×10-9mg/ml) significantly compared with control group (P<0.01), and there was a statistically significant difference (P<0.01) among the PDG groups.3. PDG also demonstrated to promote ALP activity. The cultured cells in the presence of PDG (4×10-9,2×10-8and10-7mg/ml) caused a significant increase in the ALP activity of osteoblastic cells (P<0.01).4. The MC3T3-E1cells were treated with various concentrations of PDG and the mineralization of osteoblasts was measured by Alizarin Red S staining. The increase of mineralization was significant at PDG concentrations of4×10-9in MC3T3-E1cell culture.Part21. Growth of MC3T3-E1cells was good with three different concentration of PDG(0-7mg/ml,2×10-8mg/ml,4×10-9mg/ml).2. Semi-quantative RT-PCR examination revealed that OPG mRNA expression was increased in the PDG groups after72hour subculture, and there was a statistically significant difference (P<0.05) among the PDG groups.3. The RANKL mRNA expression was decreased in the PDG groups after72hour subculture (P<0.05).Conclusions The experiment showed that PDG promoted the proliferation of MC3T3-E1, and with increasing PDG concentrations, OD values raised in concentration-dependent. ALP, an important marker of osteoblasts was examined to investigate the effects of PDG on the differentiation of osteoblast cells. The results showed that PDG increased differentiation of osteoblastic MC3T3-E1cells. The results supported our hypothesis that PDG increases the osteogenic effect in osteoblastic MC3T3-E1cells and these stimulated osteogenic effects are mediated by increasing osteoblast proliferation and differentiation. What’s more, our data showed PDG increased the expression of OPG but inhibited RANKL. Because of the opposing nature of OPG on RANKL action, this was expected to reduce bone turnover and prevent bone loss.In summary, PDG increased the proliferation of osteoblasts and stimulated ALP activity and cellular calcium content, and these increases trigger osteoblastic differentiation. Moreover, our data indicate that PDG increased the ratio of OPG/RANKL and might result in a decrease of loss of bone. Thus, PDG may be a good candidate for the protection of osteoporosis. These results may contribute to the development of a therapeutic approach of PDG against OP.