Epigallocatechin-3-gallate Promotes Osteoblastic Activity In Human Osteoblast-like Cells

PurposeTo explore Epigallocatechin-3-gallate(EGCG)on osteoblast proliferation and the role of bone to differentiation,and discuss the possible action mechanism.Methods1.Determined by MTT experiment,flow cytometry,Edu experiment,The Real time PCR and Western Blot method to detect different concentration Epigallocatechin-3-gallate of MC3T3 E1 osteoblast toxic effects and specific concentration Epigallocatechin-3-gallate effects on cell proliferation of MC3T3 E1 osteoblast.2.Determined by alkaline phosphate(ALP)staining test,Determination ALP,Alizarin red staining test,the Real time PCR and Western Blot method to testify the Epigallocatechin-3-gallate on the function of MC3T3 El osteoblast to differentiation of bone.3.Determined by Determination ALP,The Real time PCR and Western Blot method to discuss the possible action mechanism of Epigallocatechin-3-gallate on osteoblast proliferation differentiation.Result1.5μM-50μM Epigallocatechin-3-gallate of MC3T3 El osteoblast have no obvious toxic effects,and including 100μM,200μM two concentration group of Epigallocatechin-3-gallate have toxic effects to MC3T3 E1 osteoblast.Including 15μM,30 pM,the OD value of the concentration of two groups obviously higher than blank control group,prompt the Epigallocatechin-3-gallate in a certain concentration range may have promote proliferation of MC3T3 El osteoblast effect.Concentration of 30μM Epigallocatechin-3-gallate can promote MC3T3 E1 osteoblast proliferation and obviously promote the process of cell cycle S period.Edu experiments also show that including 30μM concentration of Epigallocatechin-3-gallate effect of MC3T3 El osteoblast cells in S phase proportion is about 51 plus or minus 8%,and the blank control group S phase proportion of about 38 plus or minus 5%,Epigallocatechin-3-gallate cell S phase proportion of treatment group was obviously higher than that of blank control group,two groups of cells in S phase proportion distribution has significant difference.30μM concentration of Epigallocatechin-3-gallate in the processing of MC3T3 E1 osteoblast cell cycle D1 protein expression level of mRNA and protein expression levels were significantly higher than the blank control group.2.The ALP staining showed that Epigallocatechin-3-gallate group of ALP positive cells is obviously more than the blank control group,statistically significant difference.ALP activity test,according to Epigallocatechin-3-gallate and ALP activity in cells of treatment group was obviously higher than that of blank control group,statistically significant difference.The result of Real time PCR and Western Blot test show that the mRNA expression level and the protein expression level of ALP in Epigallocatechin-3-gallate group processing cells is significantly higher than the blank control group.Alizarin red staining showed that 7 days or 14 days,the Epigallocatechin-3-gallate group processing cells of mineralized nodules were significantly higher than the blank control group,statistically significant difference.3.The result of Real time PCR and Western Blot test show that the mRNA expression level and the protein expression level of beta catenin in Epigallocatechin-3-gallate group processing cells obviously is higher than the control group.The ER antagonists ICI 182780 and Epigallocatechin-3-gallate jointly handle cell,beta catenin expression level of mRNA and protein in Epigallocatechin-3-gallate group and the control group have no difference.Also,There is no difference between the activity of ALP in The ER antagonists ICI 182780 and Epigallocatechin-3-gallate jointly handle cell and the control group.ConclusionA certain concentration of Epigallocatechin-3-gallate can promote osteoblast proliferation and bone to differentiation may be through the interaction with estrogen receptor activating Wnt signaling pathways of beta-catenin to complete.