原花青素C1可有效抑制过氧化氢刺激所致的成骨细胞衰老

Procyanidin C1 effectively inhibits hydrogen peroxide stimulation-induced senescence of osteoblasts

目的探讨原花青素C1(PCC1)对成骨细胞衰老的保护作用。方法应用不同浓度过氧化氢刺激成骨前体细胞系MC3T3后通过细胞计数试剂盒(CCK-8)检测成骨细胞活性,选取活性抑制率约为50%的浓度用于诱导MC3T3细胞衰老。依据不同处理措施,将细胞分为对照组、衰老组和PCC1组。正常组加入完全培养基,衰老组加入含1μmol/L H_(2)O_(2)的完全培养基,PCC1组加入含1μmol/L H_(2)O_(2)和5μmol/L PCC1的完全培养基。CCK-8用于检测成骨细胞活性。实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质免疫印迹法(Western blot)用于检测经不同处理后MC3T3细胞中衰老相关基因p53、p16INK4a、p21、基质金属蛋白酶(MMP)-3、MMP-13的表达。采用β-半乳糖苷酶染色检测成骨细胞β-半乳糖苷酶阳性表达。两组之间采用t检验进行统计分析。结果PCC1可以显著抑制过氧化氢刺激而所致的细胞活性降低,衰老组细胞活性降低显著程度低于PCC1组[(44.54±2.38)%比(30.45±2.84)%,t=7.60,P<0.05];RT-qPCR结果显示,衰老组中p53、p16INK4a、p21、MMP-3、MMP-13等衰老相关基因表达显著高于对照组[p53(2.00±0.06比1.00±0.02,t=28.13,P<0.05),p16INK4a(1.65±0.07比1.00±0.01,t=16.01,P<0.05),p21(2.58±0.14比1.00±0.01,t=19.05,P<0.05),MMP-3(2.52±0.13比1.00±0.02,t=19.16,P<0.05),MMP-13(1.83±0.06比1.00±0.02,t=21.75,P<0.05)];衰老组中相关基因表达显著高于PCC1组[p53(1.35±0.10比1.00±0.02,t=9.47,P<0.05),p16INK4a(1.18±0.05比1.00±0.01,t=9.62,P<0.05),p21(1.56±0.11比1.00±0.01,t=9.83,P<0.05),MMP-3(1.66±0.07比1.00±0.02,t=9.74,P<0.05),MMP-13(1.35±0.07比1.00±0.02,t=8.85,P<0.05)]。Western blot结果显示,衰老组衰老相关蛋白表达显著高于PCC1组[p53(1.35±0.02比1.65±0.07,t=9.22,P<0.05),p16INK4a(0.80±0.02比0.39±0.05,t=12.55,P<0.05),p21(1.48±0.08比0.85±0.03,t=13.28,P<0.05),MMP-3(0.41±0.01比0.57±0.02,t=10.19,P<0.05)];PCC1组衰老相关蛋白表达量显著低于衰老组[p53(1.38±0.05比1.65±0.07,t=7.40,P<0.05),p16INK4a(0.53±0.04比0.80±0.02,t=11.64,P<0.05),p21(1.14±0.12比1.48±0.08,t=3.98,P<0.05),MMP-3(0.43±0.02比0.57±0.02,t=7.87,P<0.05)]。β-半乳糖苷酶染色显示,PCC1组相较于衰老组β-半乳糖苷酶阳性细胞显著减少。结论原花青素C1可以减缓成骨细胞衰老,提高成骨细胞活性。

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 senescence in MC3T3 cells.Based on different treatment measures,the cells were divided into control,senescence and PCC1 groups.Complete medium was added to the normal group,complete medium containing 1μmol/L H_(2)O_(2) to the senescent group,and complete medium containing 1μmol/L H_(2)O_(2) and 5μmol/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 senescence-related genes p53,p16INK4a,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 ofβ-galactosidase-positive cells in osteoblasts.Results PCC1 could significantly inhibit the reduction of cell viability induced by hydrogen peroxide 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 inβ-galactosidase-positive cells in the PCC1 group compared to the senescent group.Conclusion PCC1 slows down osteoblast senescence and improves osteoblast activity.