Schisandrin B regulates MC3T3-E1 subclone 14 cells proliferation and differentiation through BMP2-SMADs-RUNX2-SP7 signaling axis

Schisandrin B (SchB) is the highest content of biphenyl cyclooctene lignans in Schisandra chinensis. It has been reported to have a variety of pharmacological effects, including anti-inflammatory, anti-oxidant, anti-cancer, heart protection, liver protection. In this study, we found that SchB can promote the proliferation of MC3T3-E1 subclone 14 cells. Meanwhile, we found that SchB can regulate the BMP2-SMADs signaling pathway by increasing gene and protein expression of those relative biomolecules. Furthermore, SchB can raise the RUNX2 and SP7 expression in both mRNA and protein levels. Since the role of BMP2-SMADs-RUNX2-SP7 signaling axis in osteoblast proliferation and differentiation has been well documented. The present experimental findings indicate that SchB could promote the proliferation and differentiation of osteoblasts through BMP2-SMADs-RUNX2-SP7 signaling axis.

SchB promotes BMP2, SMADs and Runx2 protein expression. Based on the findings at the mRNA level, we further examined the effects of SchB on these molecules at the protein level. Given that 2.5 μM SchB has no significant effect on BMP2-SMADs at the mRNA level, we chose 5-40 μM for protein level detection. The data showed that SchB could promote the protein expression of BMP2 in the concentration range of 5-40 μM. Meanwhile, SchB can significantly up-regulate the expression of SMAD4 protein only at the concentration of 20  SchB promotes SP7 mRNA and protein expression. Sp7 is an essential transcription factor for osteoblast differentiation, which induced by Runx2 12 . Bglap also known as osteocalcin, is an osteoblast marker. Sp7 directly regulates the expression of Bglap through Sp7-binding sites on the promoter region of the gene 15 . In the www.nature.com/scientificreports/ present study, we observed that SchB could upregulate the mRNA and protein expression of Sp7 while show no significant effect on Bglap (Fig. 4).

Discussion
In this study, we found that SchB can promote the proliferation of MC3T3-E1 subclone 14 cells and up-regulate the gene and protein expression of biomolecules in BMP2-SMADS signaling pathway. Firstly, SchB can promote the proliferation of MC3T3-E1 subclone 14 cells at the concentration of 1.25-40 μM. However, we note that extending the treating time of SchB does not enhance its effectiveness. This data indicated that SchB can achieve its strongest effect within 24 h. At the level of mRNA, SchB can promote the expression of BMP2, SMADs, Runx2, Sp7 genes a dose-dependent manner but have no impact on Bglap. The expression of BMP2, SMADs, Runx2, SP7 protein was up-regulated to varying degrees, but only SMAD4 and SP7 protein showed a dose-dependent relationship, while the expression of SMAD1 protein had no significant change. These results indicate that SchB may have a high selectivity for SMAD4 and SP7.
In addition, we noticed that 10-20 μM SchB markedly up-regulated the expression of Runx2, while 40 μM SchB had no significant effect on the protein expression of RUNX2 (Fig. 3K, L). Then we found 40 μM SchB also have no impact on mRNA expression of Sp7 (Fig. 4A). Given that SP7 induced by Runx2, this finding is reasonable. However, these series of results suggest that 40 μM SchB may be too high for MC3T3-E1 subclone 14 cells.
Taken together, our findings reveal the effect and mechanism of SchB on MC3T3-E1 subclone 14 cells (Fig. 5), and further confirm its potential value in the treatment of bone-related diseases, especially osteoarthritis and rheumatoid arthritis, which are closely related to inflammation and osteoblasts. Based on the findings of this study, we will further examine the potential role of SchB in the treatment of osteoarthritis and rheumatoid arthritis in vitro and in vivo.  Endogenous gene expression. MC3T3-E1 subclone 14 cells were seeded into 60 mm dishes and grown for 24 h in medium containing 10% FBS. Cells were then treated with DMSO or SchB with the indicated concentrations for 24 h. RNA was extracted and purified using the TrIzol Reagent. cDNA was prepared from 1 μg  Western blot. MC3T3-E1 subclone 14 cells were seeded into 60 mm dishes and grown for 24 h in medium containing 10% FBS. Then cells received fresh medium containing the indicated treatments. Whole cell extracts were prepared after 24 h of treatment using RIPA buffer supplemented with 1 mM PMSF. 40 μg of protein per lane was analyzed on 10% SDS-PAGE gels and transferred to PVDF transfer membranes. BMP2, SMADs, Runx2, SP7 protein was detected using antibodies listed above. Images were captured using the Chemidoc CD Touch (Bio-Rad, USA), and images analyzing and processing using the Image Lab 6.0 (Bio-Rad, Chinese edition).

Statistical analysis.
All results were presented as mean ± standard deviation (SD). Statistical significance was determined with One-Way ANOVA. p < 0.05 was considered statistically significant.