Salvia miltiorrhiza promotes osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast by the extracellular signal-regulated kinase pathway

Abstract

Objective To study whether or not Salvia miltiorrhiza (SM) can promote osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast, and examine the relationship between the mechanism and extracellular signal-regulated kinase (ERK) signal pathway. Methods Different groups with different Salvia miltiorrhiza concentration (75, 150, 300 mg/L), the control group without Salvia miltiorrhiza, the proliferation of MC3T3-E1 cell was analyzed by cell counting kit-8 (CCK-8) assy, After 21days culture, the mineralization of MC3T3-E1 was examined by alizarin red staining, and the calcium nodes were counted. The mRNA expressions of bone gla protein (BGP) and runt related transcription factor 2 (Runx2) were examined by real-time fluorescence quantitative PCR (FQ-PCR). MC3T3-E1 cells were treated with SM in high concentration (300 mg/L), the protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) were detected by Western bloting. After added inhibitor of ERK1/2 pathway, the mRNA expressions of BGP and Runx2 were examined by qPCR again. Results Compared to control group, MC3T3-E1 preosteoblasts treated with SM had higher level of proliferation, and SM in 300 mg/L showed extremely significant difference (P=0.006). MC3T3-E1 cells treated with SM in different concentration had more calcium nodes than the control group (P=0.000), the quantity was (11.40±2.30), (21.00±2.24), (35.60±1.52), (6.40±1.14) (control group) respectively. In general, the groups treated with SM had higher level of the mRNA expressions of osteoblasts-related gene BGP and Runx2. For BGP mRNA, the raletive expression were (1.02±0.04), (1.11±0.07), (1.26±0.05)respectively, the two groups (treated with SM in 150 mg/L and 300 mg/L) haded significant statistical differences (P=0.019, P=0.000). For Runx2 mRNA, the raletive expression were (1.38±0.25), (2.28±0.13, (2.48±0.06)respectively, the group treated with SM in 75 mg/L showed significant differences (P=0.012), while the two groups treated with SM in 150 mg/L and 300 mg/L respectively, showed very significant statistical differences (P=0.000). The expression level of protein p-ERK1/2 was also remarkably elevated in SM group compared to the control group (P=0.014), and the quantity were (0.14±0.01), (0.10±0.01)respectively. The two groups were compared (300 mg/L SM, 300 mg/L SM+ PD98059), after added inhibitor of ERK pathway (PD98059), the mRNA expressions of BGP (1.87±0.05, 1.39±0.07)and Runx2 (1.87±0.05, 1.39±0.07) obviously decreased. Conclusion Salvia miltiorrhiza can promote osteogenic differentiation, proliferation and mineralization of MC3T3-E1 preosteoblast by the ERK signal pathway. Key words: Salvia miltiorrhiza; MC3T3-E1 preosteoblast; Extracellular signal-regulated kinase; Signal pathway