The Effects Of Pioglitazone On Proliferation,apoptosis And Expressions Of Functional Protein In Normal Osteoblastic Cell MC3T3-E1

Objective:To investigate the effects of pioglitazone(PIO) on proliferation,apoptosis and expression of functional protein in mouse preosteoblastic cells MC3T3-E1 exposed to normal glucose and and approach the possible mechanisms, further provide some experiement reference frame for clinical medication.Methods:The MC3T3-E1 cells were incubated with different concentration(0, 5, 10, 20 and 40μmol/L separately) of PIO for different times(24 and 48 hours separately). CCK-8 was taken to test the cell proliferation; Annexin V-FITC/PI double staining flow cytometry was used to detect the cells apoptosis quantitatively; the secretions of osteocalcin(OCN) and alkaline phosphase(ALP) were measured by radioimmunoassay and enzyme-linked immunosorbent assay separately; the expressions of mRNA of peroxisome proliferator-activated receptor gamma(PPARγ),runt related gene 2(Runx2) and bone morphogenetic protein-2(BMP-2) was detected by semi-quantity RT-PCR.Results:When the MC3T3-E1 cells were treated with PIO:1 Compared with the control group, The cell proliferation was promoted in PIO 5μmol/L group (P<0.05); while the concentration of PIO was higher than 5μmol/L, it was decreased significantly(P<0.05); As extending of the interfering time(24-48h), cell proliferation in each group increased in various degrees, however the trend was not statistical significant in 20 and 40μmol/L PIO groups;2 In the experimental group, the apoptotic rate showed a rising trend after the first decline, apoptotic rates were as follow: 1.97%,0.43%,13.0%,48.30%,81.00%;3 Increasing of the PIO concentration from 0 to 40μmol/L, PPARγexpression level of cells showed an increasing trend; Runx2 expression increased in 5μmol/L group, while decreased in higher dose of PIO(≥10μmol/L), each group P<0.05;4 In each group, the secretions of ALP,OCN and expressions of BMP-2 were higher than the control group at 5μmol/L, lower and gradual descending when concentration above 5μmol/L(P<0.05); Expressions of ALP and BMP-2 were increasing with the time from 24 to 48 h (P<0.05), but not statistically significant of OCN.Conclusions:1 pioglitazone has a biphasic effect on mouse preosteoblastic cells MC3T3-E1 exposed to normal glucose: in a certain concentration(5μmol/L), it can inhibit cell apoptosis,promote cell proliferation and expressions of functional protein; while accelerate cell death,suppress cell proliferation and function obviously at higher concentrations(≥10μmol/L);2 under the normal glucose, pioglitazone can not only activate PPARγand reduce bone- related signaling pathways, leading to decreased activity of osteoblasts; but also activate other pathways to antagonize the role of PPARγ-induced bone injury;3 normal osteoblasts may have the self-regulatory mechanism, low-dose pioglitazone is able to start a protectional mechanism on osteoblasts.