| Objective: Osteoporosis is a common disease affecting the elderly health.As an ageing population,the prevalence of osteoporosis is expected to annually increase.How to effectively prevent and cure osteoporosis has become a research hotspot.Osteoporosis is closely related to the disorder between bone formation and bone resorption balance.Until now,osteoporosis treatments have included mostly anti-resorption drugs and few anabolic agents.However,anti-resorption drugs reduce bone loss but do not restore lost bone mass and damaged skeletal microstructure.In addition,anti-resorption drugs confer multiple complications for long-term osteoporosis therapy.Therefore,it is becoming particularly important to find novel safe and effective bone reformation drugs and to provide new options for the treatment of osteoporosis.The active substances extracted from traditional Chinese medicines and natural plants are more economical,safe and effective and they have little side effects for long-term use.The active substances have been shown to be effective source for the new drug development.Arbutin is a natural compound extracted from various plants,including bearberry leaves,that exerts multiple effects including skin whitening,anti-inflammatory and oxidative stress-protective properties.However,the effects of arbutin on osteoblasts remain unknown.The aim of the present study was to investigate the function and the mechanisms of arbutin on the proliferation and differentiation of MC3T3-E1 mouse osteoblast precursor cells in vitro.Methods: 1.The present study use MC3T3-E1 cell which are mouse calvarial pre-osteoblasts.The effect of arbutin on MC3T3-E1 cells apoptosis was assessed using Annexin V-FITC/PI flow apoptosis analysis.2.The proliferation of MC3T3-E1 cells treated with arbutin was assessed using a Cell Counting Kit-8 assay and a 5-ethynyl-2'-deoxyuridine labeling assay.3.The effect of arbutin on the cell cycle of MC3T3-E1 cell was examined using flow cytometry analysis.4.The effect of arbutin on activity of alkaline phosphatase(ALP)was observed by ALP staining.5.The effect of arbutin on the gene expression levels of collagen type I ?1 chain(COL1A1),osteocalcin(BGLAP)and SP7,Runt-related transcription factor 2(RUNX2)and ?-catenin in MC3T3-E1 cells was detected by reverse transcription-quantitative polymerase chain reaction(RT-q PCR).6.The effect of arbutin on the protein expression levels of RUNX2 and ?-catenin in MC3T3-E1 cells was detected by western blotting.To further investigate the molecular mechanism underlying arbutin function in promoting osteogenesis,the Wnt signaling pathway inhibitor DKK-1 was used to verify the effect of arbutin on Wnt/?-catenin signaling pathway in MC3T3-E1 cells.Results: 1.The results of Annexin V-FITC/PI apoptosis assay showed that there was no obvious toxic effect on MC3T3-E1 cells when the concentration of arbutin was below 100 ?M.2.The results of CKK-8 and Ed U labeling assay showed that Arbutin significantly promoted MC3T3-E1 cell proliferation.3.Cell cycle assay showed that arbutin increased in the percentage of MC3T3-E1 cells in S-phase and decreased cells in G1 phase.4.The alkaline phosphatase staining showed that arbutin increased alkaline phosphatase activity in MC3T3-E1 cells.5.RT-q PCR showed that arbutin up-regulated the m RNA expression levels of COL1A1,BGLAP,SP7,RUNX2 and ?-catenin in MC3T3-E1 cells.6.Western blotting showed that arbutin treatment up-regulated the protein expression levels of RUNX2 and ?-catenin,and Wnt pathway inhibitor DKK-1 inhibited the increase of RUNX2 protein caused by arbutin.Conclusion: 1.Arbutin was able to promote the proliferation of MC3T3-E1 cells and did not affect the apoptosis of osteoblasts.2.Arbutin increased the activity of alkaline phosphatase and the expression levels of bone specific matrix proteins and transcription factors during osteoblast differentiation.3.Arbutin promotes osteoblast proliferation and differentiation via the Wnt/?-catenin pathway. |
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