The effects of pioglitazone on proliferation, apoptosis and expressions of functional protein in normal osteoblastic cells MC3T3-E1

Abstract

Objective To investigate the effects of pioglitazone(PIO) on proliferation,apoptosis and expressions of functional proteins in mouse preosteoblastic cells MC3T3-E1 exposed to normal glucose concentration.Methods MC3T3-E1 cells were cultured and divided into control group,PIO administration group with different PIO concentrations(5,10,20 and 40 μmol /L,respectively) and were exposed for 24 or 48 hours.Cell proliferation was tested with CCK-8.Cell apoptosis was detected using quantitative flow cytometry.The secretions of osteocalcin(OCN) and alkaline phosphatase(ALP) were measured using RIA or ELISA,respectively.The mRNA expressions of peroxisome proliferator-activated receptor gamma(PPARγ),osteogenic factor runt related gene 2(Runx2),and bone morphogenetic protein-2(BMP-2) was detected using semi-quantitative RT-PCR.Results(1) Compared to the control group,cell proliferation was promoted in PIO 5 μmol /L group(P 0.05).In cultures administered with PIO concentration higher than 5-10 μmol /L,cell proliferation was decreased significantly compared to that in control group(P 0.05).Extending the interfering time from 24 h to 48 h,cell proliferation in each group increased in a certain extent;however the increase was not statistically significant in 20 and 40 μmol /L PIO groups.(2) In cultures of control and PIO groups exposed for 24 h,the apoptotic rates were 1.97%,0.43%,13.0%,48.30%,and 81.00%,respectively.(3) In cultures with PIO concentration from 0 to 40 μmol /L,PPARγ mRNA expression levels of the cell showed a dose-dependent increase(P 0.05).Runx2 mRNA expression increased in PIO 5 μmol /L group,but it decreased in groups with PIO doses higher than 10 μmol /L.(4) The secretions of ALP,OCN,and the expression of BMP-2 were the highest in PIO 5 μmol /L group.Those were decreased dose-dependently when PIO concentrations were more than 10 μmol /L(P 0.05).Expressions of ALP and BMP-2 increased in each group when the exposing time extended from 24 to 48 h(P 0.05),but the expression of OCN did not change(P 0.05).Conclusion PIO had a biphasic effect on mouse preosteoblastic cell MC3T3-E1 exposed to normal glucose.Low concentrations of PIO promoted cell proliferation,while high concentrations of PIO stimulated cell apoptosis.Appropriate activation of PPARγ could stimulate cells to increase functional protein synthesis by increasing Runx2.Over activation of PPARγ showed cellular toxicity.