己酮可可碱对核因子-κB受体活化因子配基诱导的牙龈间充质干细胞破骨细胞分化功能的抑制作用

Inhibition effect of pentoxifylline on nuclear factor-κB receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation of gingival mesenchymal stem cells

目的研究己酮可可碱对核因子-κB受体活化因子配基(RANKL)诱导的牙龈间充质干细胞破骨细胞分化功能的抑制作用及其相关机制。方法将获得牙龈间充质干细胞(MSCs)用RANKL刺激后分为模型组和己酮可可碱(PTX)组,不进行RANKL刺激MSCs细胞作为空白对照组。4个质量浓度PTX组细胞分别用0.1,0.5,1.0和2.0 mg·mL^(-1)的PTX(命名为0.1-PTX组,0.5-PTX组,1.0-PTX组和2.0-PTX组)处理2 h,模型组和空白对照组用等渗DMSO处理。用细胞计数法8测定细胞活力,抗酒石酸磷酸酶(TRAP)检测破骨细胞(OC)含量,蛋白质印迹法检测抑制蛋白α(IκBα)、磷酸化抑制蛋白(p-IκB)、磷酸化抑制蛋白激酶α/β(p-IKKα/β)、蛋白激酶B(Akt)和磷酸化蛋白激酶B(p-Akt)蛋白表达水平(灰度值)。结果模型组和4个质量浓度PTX组的细胞活力分别为(100.00±2.57)%,(98.14±2.48)%,(98.07±2.79)%,(76.32±2.21)%和(60.45±2.92)%;空白对照组、模型组、0.1-PTX组和0.5-PTX组的牙龈间充质干细胞的TRAP细胞数量分别为(3.36±0.24),(97.61±2.39),(66.87±2.12)和(42.35±1.46)个;这4组的成骨干细胞的TRAP细胞数量分别为(100.21±2.45),(467.13±5.63),(245.48±3.38)和(141.67±2.72)个;这4组p-Akt/Akt蛋白表达百分率分别为(100.43±2.59)%,(432.25±4.68)%,(307.31±2.63)%和(135.47±1.41)%;这4组p-IκBα/IκBα蛋白表达百分率分别为(100.36±2.32)%,(224.27±2.15)%,(171.45±1.58)%和(124.61±1.29)%。上述这5个指标:模型组与空白对照组比较,差异均有统计学意义(均P<0.01);不同质量浓度PTX组与模型组比较,差异均有统计学意义(均P<0.01)。结论PTX通过抑制牙龈间充质干细胞破骨细胞谱系中RANKL诱导的核因子κB(NF-κB)和Akt活化,特异性地抑制破骨细胞的形成和存活。

Objective To study the inhibitory effect of pentoxifylline on the differentiation of gingival mesenchymal stem cells induced by nuclear factor-κB receptor activator of nuclear factor-κB ligand(RANKL)and its related mechanisms.Methods After obtaining gingival mesenchymal stem cells(MSCs),they were stimulated with RANKL and divided into two groups:model group and pentoxifylline(PTX)treatment group;MSCs were not stimulated by RANKL as blank control group.Cells in the treatment group were treated with 0.1,0.5,1.0,2.0 mg·mL^(-1)PTX for 2 h,respectively,called 0.1-PTX group,0.5-PTX group,1.0-PTX group and 2.0-PTX group;the model group and blank control group was treated with isotonic DMSO.After the treatment,cell counting method 8 was used to determine the cell viability.Anti-tartrate phosphatase(TRAP)was used to detect the content of osteoclasts(OC).Western blotting was used to determine the expression levels(gray value)of the inhibitory proteinα(IκBα),phosphorylation inhibitor(P-IκB),phosphorylation inhibits protein kinaseα/β(phosphorylation inhibits protein kinaseα/β,p-IKKα/β),protein kinase B(Akt),and phosphorylated protein kinase B(p-Akt)protein.Results The cell viability of the model group,0.1-PTX group,0.5-PTX group,1.0-PTX group and 2.0-PTX group were(100.00±2.57)%,(98.14±2.48)%,(98.07±2.79)%,(76.32±2.21)%and(60.45±2.92)%;the number of TRAP cells in gingival mesenchymal stem cells of blank control group,model group,0.1-PTX group and 0.5-PTX group were(3.36±0.24),(97.61±2.39),(66.87±2.12)and(42.35±1.46);the number of TRAP cells in the bone stem cells of the 4 groups were(100.21±2.45),(467.13±5.63),(245.48±3.38)and(141.67±2.72)cell;the p-Akt/Akt protein expression percentage in the 4 groups were(100.43±2.59)%,(432.25±4.68)%,(307.31±2.63)%and(135.47±1.41)%;the p-IκBα/IκBαprotein expression percentage in the 4 groups were(100.36±2.32)%,(224.27±2.15)%,(171.45±1.58)%and(124.61±1.29)%.The above five indicators:the difference between the model group and the blank control group were statistically significant(all P<0.01);the difference between the different mass concentrations of PTX groups and the model group were statistically significant(all P<0.01).Conclusion Pentoxifylline specifically inhibits the formation and survival of osteoclasts by inhibiting RANKL-induced nuclear factor kappa-B and Akt activation in the gingival mesenchymal stem cells.