Objective: The purpose of this study is to investigate the potential effects of sclerostin (SOST) on the biological funtions and related mechanisms of cementoblasts under mechanical stress.
Methods: OCCM-30 cells were treated with varying doses of SOST (0, 25, 50, and 100 ng·mL⁻¹) and were loaded with uniaxial compressive stress (2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase (ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), bone sialoproteins (BSP), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA.
Results: The expression of p-smad 1/5/8 was significantly downregulated with increasing SOST. β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under mechanical compressive stress with increasing SOST concentrations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased.
Conclusions: Under mechanical comprehensive stress, SOST can adjust the bone morphogenetic protein (BMP) /smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors (Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors (RANKL/OPG) through BMP signal pathways.
目的 探究骨硬化蛋白（SOST）对处于机械压应力中的永生化成牙骨质细胞（OCCM-30）的功能影响及其相关的机制。方法 用不同浓度SOST培养液（0、25、50、100 ng·mL⁻¹）处理细胞后依靠四点弯曲细胞力学加载器对细胞加载大小为2 000 μstrain、频率是0.5 Hz的单轴压应力6 h，用免疫印迹法检测β-连环蛋白（β-catenin）、磷酸化的细胞信号转导分子p-smad1/5/8、细胞信号转导分子smad1/5/8的蛋白水平；用碱性磷酸酶（ALP）活性检测法检测ALP活性；用荧光实时定量PCR检测核心结合蛋白因子2（Runx-2）、骨钙素（OCN）、骨涎蛋白（BSP）以及细胞核因子κB受体活化因子（RANKL）、骨保护因子（OPG）的表达。结果 p-smad1/5/8随着SOST浓度增加，呈减低的趋势，而β-catenin、smad1/5/8未有显著差异性。在仅对细胞加力时ALP活性降低，随SOST浓度升高，ALP活性逐渐发生下降，Runx-2、OCN和BSP的表达也都呈现降低趋势，RANKL的表达随着SOST增加而升高，OPG则随之下降。结论 压应力下，SOST的升高会对骨形态发生蛋白（BMP）/smad 通路产生抑制作用，对β-catenin表达未产生明显改变。外源性SOST对于BMP存在反馈性的负向调节作用。压应力下SOST对OCCM-30的矿化功能是抑制的，其机制或许是一方面利用BMP信号通路对成骨相关因子Runx2、OCN、BSP等实现下调，另一方面提高了与破牙骨质有关分子 RANKL/OPG的比率。.
Keywords: cementoblast; mechanical stress; mineralization; sclerostin; signal pathway.