Beta-ecdysterone promotes in vitro proliferation and osteogenic differentiation of MC3T3-E1 cells

doi:10.3969/j.issn.2095-4344.2781

Abstract

Abstract:

BACKGROUND: β-ecdysterone as a “phytoestrogen” has the ability to stimulate protein synthesis, promote carbohydrate and lipid metabolism, relieve hyperglycemia and hyperlipidemia, protect endothelial cells from apoptosis and induce their proliferation. Some scholars have reported that it also plays an important role in the treatment of osteoporosis, fractures and other bone inflammatory diseases.

OBJECTIVE: To observe the effect of β-ecdystrone on the proliferation of mouse pre-osteoblasts (MC3T3-E1 cells) in vitro, and to explore whether β-ecdysterone can induce osteogenic differentiation of MC3T3-E1 at a safe dose.

METHODS: The fourth generation MC3T3-E1 cells were cultured in the osteogenic induction medium for 7, 10, 14, 21, and 28 days. The osteogenic differentiation proteins (alkaline phosphatase, type I collagen, osteopontin, and calcified nodules) were detected at different time points, to identify whether the cells have the ability of osteogenic differentiation. MC3T3-E1 cells were then seeded into the induction medium containing different final concentrations of β-ecdysterone (0.01, 0.1, 1, 10, 100 μmol/L). The proliferation activity of the cells was detected by cell counting kit-8 method at days 1, 2, 3, 4, 5, 6, and 7 after induction. The control group (general induction medium group) and the experimental group (general induction medium + β-ecdysterone) were cultured under the same conditions, and the expression levels of osteogenic marker proteins in each group of cells at different time periods were determined.

RESULTS AND CONCLUSION: In the MC3T3-E1 cells stimulated by the osteogenic induction medium, alkaline phosphatase staining and type I collagen florescence staining showed higher expression at day 10 of induction, and this was also confirmed by detection of alkaline phosphatase activity (P < 0.05). At day 14 of induction, osteopontin immunocytochemical staining also indicated significant expression. Alizarin red staining results demonstrated that the number of calcified nodules increased significantly after osteogenic induction, and there were more calcified nodules with larger diameter and darker color at day 28 than at day 21. After treatment with β-ecdysterone, the proliferative activity of MC3T3-E1 cells reached the peak at day 5 after induction. The optimal concentrations of β-ecdysterone were 0.01 and 0.1 μmol/L. There was no significant difference between the two concentrations (P > 0.05). The expression of alkaline phosphatase and type I collagen was higher in the experimental group than in the control group at day 10 of induction. The expression of osteopontin and osteocalcin in the cells was higher at day 14 of induction, and there was no significant difference in the calcified nodule staining between the two groups at day 28 of induction. These findings indicate that β-ecdysterone can promote the proliferation of MC3T3-E1 cells in vitro and induce MC3T3-E1 cells to differentiate into osteoblasts at a safe dose.

Key words: β-ecdysterone, MC3T3-E1 cells, osteogenic differentiation, alkaline phosphatase, type I collagen, osteopontin, osteocalcin

CLC Number:

Yan Caiping, Chen Lu, Deng Changgong, Chen Qian, Jiang Ke, Yi Yuanyuan, Li Yuling. Beta-ecdysterone promotes in vitro proliferation and osteogenic differentiation of MC3T3-E1 cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(29): 4605-4612.

Figures/Tables 15

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