原花青素B2促进老龄小鼠骨折损伤愈合机制研究

Effect of procyanidin B2 on fracture healing in aged mice and its mechanism

目的探讨原花青素B2对老龄小鼠骨折损伤后愈合过程的影响及其作用机制。方法将老龄C57BL/6J小鼠分为假手术组(Sham组, 未行骨折处理)、模型组(Model组, 构建骨折模型)、低浓度原花青素B2组(PCB2-L组, 小鼠构建骨折模型术后灌胃1 mg/ml的原花青素B2)和高浓度原花青素B2组(PCB2-H组, 小鼠构建骨折模型术后开始灌胃10 mg/ml的原花青素B2)。采用酶联免疫吸附法检测各组小鼠血清骨钙素(BGP)和骨碱性磷酸酶(BALP)浓度;计算机断层成像检测各组小鼠骨痂中骨小梁数量、最大长度和最小长度变化;生物力学检测各组小鼠骨痂的生物力学强度变化;采用实时荧光定量聚合酶链反应(RT-qPCR)检测各组小鼠可聚蛋白多糖(Aggrecan)、X型胶原α1链(Col10a)、骨形态发生蛋白-2(BMP-2)、骨钙蛋白(OCN)和骨桥蛋白(OPN)基因表达。应用SPSS 18.1统计软件分析, 两组间比较行t检验, 多组间比较采用单因素方差分析, 组间两两比较采用SNK检验。结果 Model组老龄小鼠术后7、14、21 d血清中BGP和BALP浓度均显著低于Sham组小鼠, 差异有统计学意义(t=16.235、7.482、12.276、20.388、15.221、8.912, 均P<0.05)。PCB2-H组老龄小鼠骨折损伤后7、14、21 d BGP和BALP浓度均显著高于PCB2-L组小鼠, 差异有统计学意义(t=16.235、7.482、12.276、20.388、15.221、8.912, 均P<0.05)。PCB2-H组老龄小鼠骨折损伤后骨小梁数量和力学强度高于PCB2-L组小鼠[(6.59±1.51)个比(5.23±1.18)个、3.56±0.29比2.83±0.27], 骨痂最大长度[(2.11±0.19) mm比(2.74±0.26) mm]和最小长度[(1.86±0.12) mm比(1.99±0.15) mm]均显著低于PCB2-L组小鼠, 差异有统计学意义(t=16.238、14.009、10.562、13.682, 均P<0.05)。PCB2-H组老龄小鼠骨折损伤后Aggrecan和Col10a基因mRNA表达低于PCB2-L组小鼠(0.58±0.07比0.83±0.18、0.43±0.05比0.74±0.16), BMP-2、OCN和OPN基因mRNA表达高于PCB2-L组小鼠(2.78±0.18比1.93±0.11、3.24±0.21比1.88±0.12、2.86±0.17比1.99±0.15), 差异有统计学意义(t=6.811、10.932、9.209、13.477、15.761, P<0.05)。结论原花青素B2可促进老龄小鼠骨折损伤后愈合, 其机制可能与调控Aggrecan、Col10a、BMP-2、OCN和OPN等成骨相关基因表达有关。

Objective To study the effect of procyanidin B2(PCB2)on the healing process of fracture injury in aged mice and its mechanism.Methods Totally,80 aged C57BL/6 mice were randomly divided into sham group,model group,and PCB2 group treated with different concentrations of 1 and 10 mg/ml(serving as PCB2-low dose group and PCB2-high dose group,respectively).The changes of serum bone gamma-carboxyglutamic-acid-containing proteins(BGP)and bone alkaline phosphatase(BALP)in each group were measured by enzyme-linked immunosorbent assay(ELISA).The number,maximum length and minimum length of trabecula in callus of mice in each group were measured by CT scanner.The changes of biomechanical strength in callus of mice in each group were measured by biomechanics.Quantitative reverse transcription polymerase chain reaction was used to detect the expression changes of cartilage polymerizable protein polysaccharide(aggrecan),collagen type X alpha 1 chain(col10a),bone morphogenetic protein(BMP-2),osteocalcin(OCN)and osteopontin(OPN)genes in bone tissue of mice in each group.Results The BGP and BALP concentrations at 7th,14th and 21st d in the model mice were significantly lower than in sham mice with statistically significant difference(t=16.235,7.482,12.276,20.388,15.221,8.912,all P<0.05).The trabecular number and mechanical strength of trabecular bone after fracture injury in PCB2-high dose group were significantly greater than those in PCB2-low dose group(6.59±1.51 vs.5.23±1.18,3.56±0.29 vs.2.83±0.27,respectively).The maximum scab length[(2.11±0.19)mm vs.(2.74±0.26)mm]and minimum length[(1.86±0.12)mm vs.(1.99±0.15)mm]in PCB2-high dose group were significantly lower than in PCB2-low dose group(t=16.238,14.009,10.562,13.682,All P<0.05).The mRNA expression of Aggrecan and Col10a genes after fracture injury in PCB2-high dose group was significantly lower than PCB2-low dose group(0.58±0.07 vs.0.83±0.18,0.43±0.05 vs.0.74±0.16),and the expression of BMP-2,OCN and OPN mRNA in PCB2-high dose group was significantly higher than that in PCB2-low dose group(2.78±0.18 vs.1.93±0.11,3.24±0.21 vs.1.88±0.12,2.86±0.17 vs.1.99±0.15)(t=6.811,10.932,9.209,13.477,15.761,P<0.05).Conclusion PCB2 can promote healing after fracture injury in aged mice,and its mechanism may be related to the regulation of osteogenesis-related genes such as Aggrecan,Col10a,BMP-2,OCN,and OPN.