RESUMO
Objective WNT signaling pathway plays an important role in the formation, differentiation and maturation of bone cells, it is a classical intracellular signaling pathway involved in bone metabolism. DKK1 and Sost play a negative regulatory role in regulating bone mass and osteoblast differentiation, and are negative regulators of WNT signaling pathway. Estrogen-related receptor alpha (ERRα) regulates the functional activity of osteoblasts. The aim of study was to investigate the effect of ERRα on the transfection of MG63 cells and related proteins by the WNT signaling pathway inhibitor Dickkopf (DKK)1 and sclerostin (SOST) adenovirus vectors.Methods The cultured MG63 cells were divided into blank control group, silencing DKK1 group, silencing SOST group, silencing (DKK1+SOST) group, ERRα intervention empty adenovirus group, ERRα intervention silencing DKK1 group, ERRα intervention silencing SOST group, ERRα intervention silencing (DKK1+SOST) group. MG63 cells were transfected with packaged silencing DKK1 and SOST adenovirus vectors according to different groups. The activity of MG63 cells was detected by MTT assay, the activity of ALP was detected by alkaline phosphatase kit, and the concentration of calcium ion was analyzed by flow cytometry. Western blot was used to detect the expressions of low density lipoprotein associated protein 5 (LRP5), bone morphogenetic protein 2 (BMP2), osteopontin (OPN), osteoprotegerin(OPG).Results (1) Compared with blank control group, silencing DKK1, SOST, DKK1+SOST group and ERRα overexpression in the empty adenovirus group could increase cell activity, ALP activity, and decrease calcium ion concentration and increase the expressions of LRP5, BMP2, OPN, and OPG. Differences between groups were statistically significant(P0.05).Conclusion ERRα Overexpression can increase the activity of MG63 cells, ALP activity, LRP5, BMP2, OPN, and OPG proteins, and decrease the calcium ion concentration in silencing DKK1 and SOST adenovirus-transfected cells.
RESUMO
BACKGROUND: Chinese herbs have been shown to affect osteoblasts through Wnt/β-catenin signaling pathway. Sclerostin (Sost) is an inhibitor of Wnt signaling pathway and an inhibitor of osteogenesis, and it is also an antagonistic factor that regulates the differentiation and proliferation of osteoblasts. Bushen Jianpi Huoxue decoction (BJHD) is the empirical formula in the prevention and treatment of osteoporosis at the Affiliated Orthopedics Hospital, Guangzhou University of Chinese Medicine. BJHD can promote osteogenesis and effectively treat osteoporosis. OBJECTIVE: To observe the effect of BJHD on the cell proliferation, alkaline phosphatase activity and expression levels of related proteins in Sost-overexpressed adenovirus transfected osteoblasts. METHODS: The osteoblasts were divided into two groups: blank control group (empty adenovirus transfection) and Sost overexpressed group (Sost overexpression and adenovirus transfection). Two groups were cultured with blank serum and medicine serum. The cell proliferation was tested by cell counting kit-8 assay, and alkaline phosphatase activity was tested by alkaline phosphatase kit. Expression levels of bone regulation-related proteins were tested by western blot assay. RESULTS AND CONCLUSION: Compared with the blank serum, medicine serum could increase the cell viability at 24, 48 and 72 hours in both groups (P ＜ 0.01). Compared with the blank serum, medicine serum increased the alkaline phosphatase activity (P ＜ 0.01). Compared with the blank serum, medicine serum in the blank control group decreased the expression of low-density lipoprotein receptor-related protein 5 and osteoprotegerin (P ＜ 0.01), but showed no effect on the expression of osteopontin and tumor necrosis factor α. Medicine serum in the Sost-overexpressed group decreased the expression of low-density lipoprotein receptor-related protein 5, osteoprotegerin and osteopontin, increased the expression of tumor necrosis factor α (P ＜ 0.01). These results indicate that BJHD can increase the proliferation of recombinant adenovirus transfected Sost-overexpressed osteoblasts and alkaline phosphatase activity, and regulate the expression of low-density lipoprotein receptor-related protein 5, osteoprotegerin, osteopontin, and tumor necrosis factor α.