摘要
目的 探讨唑来膦酸(zoledronic acid,ZOL)通过调节沉默调节蛋白3(sirtuin 3,SIRT3)/P53的表达影响成骨分化与骨形成的机制.方法 诱导骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨分化,检测细胞中SIRT3表达,分析SIRT3与P53的靶向调控关系.干预SIRT3、P53在细胞内的表达并使用ZOL处理细胞,分别采用MTT法、Western blot法、试剂盒检测细胞活性、成骨相关基因Osteo-protegerin(Osteoprotegerin,OPG)、Runt相关转录因子2(runt-related transcription factor 2,Runx2)表达、碱性磷酸酶(alkaline phosphatase,ALP)活性与茜素红S(alizarin red S,ARS)染色情况.使用卵巢切除术(ovariecto-my,OVX)构建大鼠模型,探讨ZOL对体内骨质疏松症(osteoporosis,OP)进展的影响.结果 ZOL能促进BMSCs成骨分化.与健康人血清相比,SIRT3在OP患者血清中表达下调(1.00±0.26 vs.0.78±0.23.t=3.85,P＜0.001).在BMSCs成骨分化过程中,SIRT3表达水平随着诱导成骨时间延长而逐渐升高.与对照组p53蛋白表达和BMSCs活性相比,敲减SIRT3能提高P53蛋白表达水平,分别为0.59±0.05 vs.1.01±0.11(t=6.02,P=0.004),但抑制BMSCs的活性,分别为 100.00±8.41 vs.51.26±5.59(t=8.36,P=0.001).ZOL处理后,敲减SIRT3后细胞活性,分别为49.61±5.11vs.87.61±7.31(t=7.38,P=0.002),对成骨的抑制作用被解除,且P53水平被抑制分别为1.10±0.10vs.0.69±0.04(t=6.59,P=0.003).P53过表达部分抵消ZOL对细胞活性的促进作用,分别为84.61±6.52 vs.66.54±5.47(t=3.68,P=0.021).与假手术组相比,OVX组大鼠成骨受到抑制,ZOL治疗后显著改善成骨抑制.ZOL治疗提高OVX大鼠骨组织中SIRT3蛋白表达水平,但抑制P53的表达水平.结论 ZOL通过促进SIRT3对P53泛素化降解作用来促进BMSCs成骨分化与骨形成.
Abstract
Objective To explore the mechanism of zoledronic acid(ZOL)affects osteogenic differentia-tion and bone formation through the regulation of sirtuin 3(SIRT3)/P53 expression.Methods Bone marrow mes-enchymal stem cells(BMSCs)were induced to differentiate into osteogenic cells,the expression of SIRT3 in the cells was detected,and the targeting regulation relationship between SIRT3 and P53 was analyzed.The intracellu-lar expressions of SIRT3 and P53 were intervened and ZOL was used to treat the cells.MTT method,Western blot method and kit were used to detect cell viability,osteogenesis-related genes Osteoprotegerin(OPG),runt-related transcription factor 2(Runx2)expression,alkaline phosphatase(ALP)activity and alizarin red S(ARS)staining,respectively.Ovariectomy(OVX)was used to construct a rat model and explore the effect of ZOL on the progres-sion of osteoporosis(OP)in vivo.Results ZOL promoted osteogenic differentiation of BMSCs.The expression of SIRT3 was down-regulated in the serum of OP patients(0.78±0.23)compared with that of healthy subjects(1.00± 0.26 vs.0.78±0.23.t=3.85,P＜0.001).During the osteogenic differentiation of BMSCs,the expression level of SIRT3 gradually increased with the prolonged induction of osteogenesis.Compared with the p53 protein expression and BMSCs activity in the control group,SIRT3 knockout could increase the expression level of p53 protein(0.59± 0.05 vs.1.01±0.11.t=6.02,P=0.004)but inhibited the activity of BMSCs(100.00±8.41 vs.51.26±5.59.t=8.36,P=0.001).After ZOL treatment,the inhibitory effect of SIRT3 on cell viability(49.61±5.11 vs.87.61±7.31.t=7.38,P=0.002)and osteogenesis was relieved,and the level of P53 was inhibited(1.10±0.10 vs.0.69±0.04.t=6.59,P=0.003).P53 overexpression partially offseted the effects of ZOL on cell viability(84.61±6.52 vs.66.54±5.47.t=3.68,P=0.021)and osteogenesis.Compared with the sham surgery group,the OVX group showed inhibition of os-teogenesis in rats,and ZOL treatment significantly improved osteogenic inhibition.ZOL treatment increased the ex-pression level of SIRT3 protein in bone tissue of OVX rats,but inhibited the expression level of P53.Conclusion ZOL promoted osteogenic differentiation and bone formation of BMSCs by promoting the ubiquitination and degra-dation of P53 by SIRT3.
基金项目
河南省医学科技攻关计划项目(LHGJ20210984)
维普
万方数据