Salvia MiltiorrhizaInduces VEGF Expression and Regulates Expression of VEGF Receptors in Osteoblastic Cells

Abstract

This study investigated in vitro whether Salvia Miltiorrhiza Bunge (SM) induces vascular endothelial growth factor (VEGF) expression and regulates expression of VEGF receptors 1 (VEGFR-1) and 2 (VEGFR-2) on osteoblasts. MC3T3-E1 cells were cultured with SM and VEGF at points 24, 48 and 72 h. A blank control was included. The mRNA expression of VEGF, VEGFR-1 and VEGFR-2 was examined using real-time polymerase chain reaction. VEGF protein expression was examined using enzyme linked immunosorbent assay. SM increased VEGF mRNA expression by 21% at 24 h (p < 0.05), 5% (p < 0.05) at 48 h and 74% (p < 0.001) at 72 h, while external VEGF intervention decreased the internal VEGF expression by 51% at 24 h (p < 0.001) and increased it by 126% at 72 h (p < 0.001). SM increased VEGF protein at 72 h (p < 0.05). VEGFR-1 mRNA expression, in response to SM, decreased by 8% (p < 0.05) at 72 h and decreased following VEGF at 24 and 72 h by 20% (p < 0.001) and 15%, respectively, (p < 0.001). VEGFR-2 mRNA expression increased following SM at 24 and 48 h by 25% (p < 0.05) and 73% (p < 0.05), respectively, and decreased at 72 h by 13% (p < 0.05). It was concluded that SM modulated expression of VEGF and its receptors in MC3T3-E1 cells.

Effects of VEGF on Bone Cells’ Metabolism and Activity

“…For bone formation, ALP, OPN and OCN were found increasing at different phases of intervention [8,20]. The process of osteogenesis consists of cell proliferation, cell differentiation, and mineralization, and ALP, OPN and OCN are related to the different stages of this process: ALP is an early marker, which is known to be responsible for osteoblastic differentiation; while OPN and OCN are regarded as late markers, as OPN is a major non-collagenous bone matrix protein secreted into the mineralizing extracellular matrix, and OCN is also closely related to calcification [17][18][19].…”

“…OCN is closely related to calcification [19]. [8,20] It was found that the mRNA expression of ALP increased by 73% after VEGF incubation for 24 hrs (p<0.001), but reduced by 14% and 41% at 48 hrs (p<0.001) and 72 hrs (p<0.05) respectively when compared to the control [8]. For the other two factors, OPN expression decreased by 11% (p<0.001) and OCN dropped by 41% (p<0.001) at 24 hrs; both the expression of OPN and OCN did not show any change at 48 hrs; but at 72hrs, OPN increased by 94% (p<0.001) and OCN grew by 149% (p<0.001) [8,20].…”

“…The process of osteogenesis consists of cell proliferation, cell differentiation, and mineralization, and ALP, OPN and OCN are related to the different stages of this process: ALP is an early marker, which is known to be responsible for osteoblastic differentiation; while OPN and OCN are regarded as late markers, as OPN is a major non-collagenous bone matrix protein secreted into the mineralizing extracellular matrix, and OCN is also closely related to calcification [17][18][19]. It was shown that the increase of ALP happened at the early stage (24 hrs) [8], while OPN and OCN had a significant increase at late stage (72 hrs) [8,20], consistent with the different time-serial function of ALP, OPN and OCN, indicating that VEGF play different regulation roles in different bone formation stages.…”