摘要
目的探讨胡椒碱对成骨细胞成骨分化的影响以及机制。方法分离、培养胎鼠颅骨成骨细胞,分别给予不同浓度的胡椒碱处理,采用MTT法检测胡椒碱对成骨细胞毒性的影响;采用碱性磷酸酶(ALP)染色和ALP活性检测试剂盒检测ALP活性;采用茜素红S染色评估成骨细胞成骨及矿化水平;采用实时荧光定量聚合酶链反应(RT-qPCR)检测ALP、I型胶原蛋白α1(COL1A1)、成骨相关转录因子蛋白(OSX)、骨桥蛋白(OPN)、runt相关转录因子2(Runx2)的mRNA表达水平;采用Western blot检测Wnt 1和β-catenin蛋白水平;采用免疫荧光检测β-catenin分布与表达;采用Wnt/β-catenin信号途径特异性拮抗剂IWR-1验证胡椒碱对成骨细胞成骨分化的信号通路。结果在0~60μmol·L^-1浓度范围内,胡椒碱对成骨细胞活力无明显影响(P>0.05)。在0~40μmol·L^-1浓度范围内,胡椒碱呈剂量依赖性促进成骨细胞ALP活性(P<0.01或P<0.001)和矿化结节形成(P<0.05或P<0.01),促进ALP、COL1A1、OSX、OPN和Runx2的mRNA表达水平(P<0.05或P<0.01或P<0.001),促进Wnt 1和β-catenin蛋白的表达水平(P<0.05或P<0.01或P<0.001)。用IWR-1预处理后,由胡椒碱诱导的成骨细胞的基质矿化受到明显抑制(P<0.01)。结论胡椒碱可通过增强Wnt/β-catenin信号促进成骨细胞的成骨分化。
Objective To investigate the effect of piperine on osteogenic differentiation of osteoblasts and its mechanism.Methods Fetal rat calvarial osteoblasts were isolated,and then treated with different concentrations of piperine.The effect of piperine on cytotoxicity was observed by MTT assay.Alkaline phosphatase(ALP)activity was measured by ALP staining and ALP activity detection kit.The osteogenesis and mineralization were evaluated by alizarin red S staining.The mRNA levels of ALP,collagen 1A1(COL1A1),osterix(OSX),osteopontin(OPN)and runt-related transcription factor 2(Runx2)were detected by RT-qPCR.The protein levels of Wnt 1 andβ-catenin were detected by Western blot.The distribution and expression ofβ-catenin were determined by immunofluorescence.The signaling pathway of piperine for osteogenic differentiation of osteoblasts was validated by IWR-1,a specific antagonist of the Wnt/β-catenin signaling pathway.Results Piperine had no significant effect on cell viability of osteoblasts in the range of 0-60μmol·L^-1(P>0.05).In the range of 0-40μmol·L^-1,piperine enhanced ALP activity,promoted mineralized nodule formation,and increased mRNA expression of ALP,COL1A1,OSX,OPN and Runx2 and protein expression of Wnt 1 andβ-catenin in a dose-dependent manner(P<0.05,P<0.01 or P<0.001).After pretreatment with IWR-1,piperine-induced matrix mineralization was significantly inhibited in osteoblasts(P<0.01).Conclusion Piperine promotes osteogenic differentiation of osteoblasts by enhancing Wnt/β-catenin signaling.
作者
侯鸣夷
殷浩
贺学军
HOU Ming-yi;YIN Hao;HE Xue-jun(5th Department of Orthopedics,Hunan Provincial People’s Hospital,Changsha 410016,China)
出处
《南昌大学学报（医学版）》
CAS
2020年第4期15-22,共8页
Journal of Nanchang University：Medical Sciences
