Procyanidin C1 effectively inhibits hydrogen peroxide stimulation-induced senescence of osteoblasts
Objective To investigate the protective effect and mechanism of procyanidin C1(PCC1)on osteoblast senescence.Methods Osteoblast precursor cell line MC3T3 was purchased from Wuhan Pricella Life Technology Co.Osteoblast viability was detected by cell counting kit-8(CCK-8)assay after applying different concentrations of hydrogen peroxide to stimulate the osteogenic precursor cell line MC3T3,and the concentration with an activity inhibition rate of about 50％was chosen for inducing senes-cence in MC3T3 cells.Based on different treatment measures,the cells were divided into control,senes-cence and PCC1 groups.Complete medium was added to the normal group,complete medium containing 1 μmol/L H2O2 to the senescent group,and complete medium containing 1 μmol/L H2O2 and 5 μmol/L PCC1 to the PCC1 group.The CCK-8 assay was used to detect osteoblast viability.Real-time quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect the expression of senes-cence-related genes p53,pl6INK4a,p21,matrix metalloproteinase(MMP)-3 and MMP-13 in MC3T3 cells.Western blotting was used to detect the expression levels of senescence-related proteins in each group.β-galactosidase staining was used to detect the expression of β-galactosidase-positive cells in osteo-blasts.Results PCC1 could significantly inhibit the reduction of cell viability induced by hydrogen perox-ide stimulation,and the senescent group showed a significantly lower reduction in cell activity than the PCC1 group[(44.54±2.38)％vs.(30.45±2.84)％,t=7.60,P＜0.05].The RT-qPCR results showed that the expression of senescence-related genes(p53,p16INK4a,p21,MMP-3,MMP-13)was significantly higher in the senescence group than in the control group[p53:(2.00±0.06 vs.1.00± 0.02,t=28.13,P＜0.05);p16INK4a:(1.65±0.07 vs.1.00±0.01,t=16.01,P＜0.05);p21:(2.58±0.14 vs.1.00±0.01,t=19.05,P＜0.05);MMP-3(2.52±0.13 vs.1.00±0.02,t=19.16,P＜0.05);MMP-13:(1.83±0.06 vs.1.00±0.02,t=21.75,P＜0.05).The expression of the related genes was significantly suppressed in the PCC1 group[p53:(1.35±0.10 vs.1.00±0.02,t=9.47,P＜0.05);p16INK4a:(1.18±0.05 vs.1.00±0.01,t=9.62,P＜0.05);p21:(1.56± 0.11 vs.1.00±0.01,t=9.83,P＜0.05);MMP-3(1.66±0.07 vs.1.00±0.02,t=9.74,P＜0.05);MMP-13(1.35±0.07 vs.1.00±0.02,t=8.85,P＜0.05)].The results of Western blotting showed that the expression of relevant proteins was elevated in the senescent group[p53:(1.35±0.02 vs.1.65±0.07,t=9.22,P＜0.05);p16INK4a:(0.80±0.02 vs.0.39±0.05,t=12.55,P＜0.05);p21:(1.48±0.08 vs.0.85±0.03,t=13.28,P＜0.05);MMP-3:(0.41±0.01 vs.0.57±0.02,t=10.19,P＜0.05).The expression of senescence-associated proteins in the PCC1 group was significantly decreased in comparison to that of the senescence group[p53:(1.38±0.05 vs.1.65±0.07,t=7.40,P＜0.05);p16INK4a:(0.53±0.04 vs.0.80±0.02,t=11.64,P＜0.05);p21:(1.14±0.12 vs.1.48±0.08,t=3.98,P＜0.05);MMP-3:(0.43±0.02 vs.0.57±0.02,t=7.87,P＜0.05)].β-galactosidase staining showed a significant decrease in β-galactosidase-positive cells in the PCC1 group compared to the senescent group.Conclusion PCC1 slows down osteoblast senescence and improves oste-oblast activity.
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