Effect of temperature on the inhibitory effect induced by hydrogen peroxide on cell proliferation and osteogenic differentiation in preosteoblast MC3T3-E1 cells
Objective To investigate the effect of temperature on cell proliferation and osteogenic differentiation inhibition of preosteoblast induced by hydrogen peroxide(H2 O2).Methods The MC3T3-E1 cells in the logarithmic phase were randomly divided into 0,450,500,550,600,650 μmol·L-1 H2O2 intervention groups and incubated with 0,450,500,550,600,650 μmol·L-1 H2O2 for 2 h,respectively.Other MC3T3-E1 cells in the logarithmic phase were selected and randomly divided into the control group,model group,low-temperature group,and high-temperature group.Cells in the control group were cul-tured in an incubator with 5％CO2 for 24 h at 37 ℃;cells in the model group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 37 ℃;cells in the low-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 32 ℃;cells in the high-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5％CO2 for 24 h at 40 ℃.The cell proliferation in all groups was detected by cell counting kit-8.The expression levels of Runt-related transcription factor 2(RUNX2),osteopontin(OPN)and osteocalcin(OC)mRNA were detected by real-time fluorescence quantitave polymerase chain reaction;and the expression levels of RUNX2,OPN and OC protein were detected by Western blot.Results There was no statistically significant difference in cell proliferation among the 0,450 and 500 μmol·L-1 H2O2 intervention groups(P＞0.05);the cell proliferation rate in the 550,600 and 650 μmol·L-1 H2O2 intervention groups was significantly lower than that in the 0,450 and 500 μmol·L-1 H2O2 intervention groups,showing a significant decrease in cell proliferation with the increase of H2O2 concentrations(P＜0.05).In order to ensure that there were enough cells to perform the following experiments,550 μmol·L-1 H2 O2 was chosen.The cell proliferation rate in the model group and the low-temperature group was significantly lower than that in the control group and high-temperature group(P＜0.05);there was no significant difference in the cell proliferation rate between the control group and high-temperature group(P＞0.05).The relative expression of RUNX2 mRNA in the model group and high-temperature group were significantly higher than that in the control group and low-temperature group(P＜0.05);the relative expression of RUNX2 mRNA in the low-temperature group was significantly lower than that in the control group(P＜0.05);there was no significant difference in the relative expression of RUNX2 mRNA between the model group and high-temperature group(P＞0.05).The relative expression of OPN mRNA in the model group,low-temperature group and high-temperature group was significantly higher than that in the control group(P＜0.05);the relative expression of OPN mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P＜0.05);the relative expression of OPN mRNA in the low-tem-perature group was significantly higher than that in the high-temperature group(P＜0.05).The relative expression of OC mRNA in the model group,low-temperature group and high-temperature group was significantly than that in the control group(P＜0.05);the relative expression of OC mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P＜0.05);there was no significant difference in the relative expression of OC mRNA between the low-temperature group and high-temperature group(P＞0.05).The relative expressions of RUNX2,OPN and OC protein the model group,low-temperature group and high-temperature group were significantly lower than those in the control group(P＜0.05);the relative expressions of RUNX2 and OPN protein in the low-temperature group were significantly lower than those in the model group and high-temperature group(P＜0.05);the relative expression of OC protein was significantly lower than that in the high-temperature group(P＜0.05);and there was no siqnificantly difference in the relatiwe experesson of OC protein between the low-temperature group and model group(P＞0.05);the relative expressions of RUNX2,OPN and OC protein in the high-temperature group were significantly higher than those in the model group(P＜0.05).Conclusion The inhibitory effects of H2O2 on cell proliferation and osteogenic differentiation are observed in MC3T3-E1 cells;low-tempera-ture incubation can enhance the inhibition of H2O2 on cell proliferation and osteogenic differentiation in MC3T3-E1 cells,while high-temperature incubation can relieve its inhibitory effect on cell proliferation and osteogenic differentiation.RUNX2,OPN and OC protein might play an important role in cell proliferation and osteogenic differentiation mediated by temperature.
维普
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