核因子-κB配体的受体在Wnt/β-连环蛋白通路促成骨样细胞转化中的作用及意义

Implication of nuclear factor kappa-B ligand-receptor in promoting osteoid cell transformation in Wingless-related integration site/β-catenin pathway

目的:探讨核因子(NF-κB)配体的受体在Wnt/β-连环蛋白(β-catenin)通路促成骨样细胞转化中的作用及其意义。方法:分离健康雄性远交群(SD)大鼠主动脉平滑肌细胞(SMC)并诱导为成骨样细胞,分别用LiCl、LiCl+OPG(0.1 ng/ml)、LiCl+OPG(1.0 ng/ml)、LiCl+OPG(10.0 ng/ml)、LiCl+OPG(100.0 ng/ml)处理成骨样细胞,对照组未行LiCl及OPG处理,LiCl浓度均为20 mmol/L。RT-qPCR检测各组细胞OPG、RANKL转录水平;Von Kossa染色,钙离子含量测定,碱性磷酸酶(ALP)活性测定,骨钙素蛋白质印迹法(Western blot)检测各组细胞钙化程度;免疫组织化学和Western blot检测Wnt/β-catenin通路激活情况。采用单因素方差分析,组内进一步两两比较采用LSD-t检验,两两比较采用t检验。结果:LiCl组OPG的表达(4.35±0.88)稍高于成骨样细胞组(4.12±0.82,t=0.603,P>0.05),RANKL表达则LiCl组(2.61±0.42)显著高于成骨样细胞组(1.52±0.35,t=2.317,P<0.05);OPG/RANKL的比值LiCl(1.67±0.41)组显著低于成骨样细胞组(2.71±0.48,t=2.542,P<0.05)。成骨样细胞的转化实验中,成骨样细胞组及加LiCl+OPG的4组钙离子含量、ALP活性、骨钙素的水平(25.2±5.8、68.5±13.3、61.1±12.8、57.6±11.3、56.3±11.4)、(80.4±10.2、141.2±17.6、125.7±15.3、123.5±15.9、122.7±15.1)、(0.61±0.10、0.82±0.14、0.79±0.13、0.72±0.12、0.73±0.13)均低于LiCl组(99.5±16.5)、(186.0±39.3)、(0.98±0.15),(t=5.971、2.795、2.810、2.829、2.831,P<0.05)、(t=5.623、2.597、2.765、2.791、2.793,P<0.05)(t=4.931、2.379、2.384、2.391、2.392,P<0.05);加LiCl+OPG的4组中钙离子含量、ALP活性、骨钙素的水平差异无统计学意义(P>0.05),均高于成骨样细胞组水平(t=5.362、2.769、2.635、2.633,P<0.05)、(t=5.105、2.758、2.732、2.733,P<0.05)、(t=2.762、2.569、2.566、2.563,P<0.05)。Wnt/β-catenin通路的激活程度检测中,β-catenin的表达水平β-catenin表达水平LiCl组(1.75±0.26)高于成骨样细胞组及加LiCl+OPG的4组(1.32±0.18、1.25±0.17、1.25±0.16、1.22±0.16),(t=4.442、2.650、2.654、2.712、2.709,P<0.05)、加LiCl+OPG的4组高于成骨样细胞组(t=2.492、2.455、2.439、2.437,P<0.05),加LiCl+OPG的4组之间比较差异无统计学意义(P>0.05)。结论:激活Wnt/β-catenin通路促进成骨样细胞转化的机制,有RANKL依赖性,也有非RANKL依赖性。

Objective To investigate the role and significance of nuclear factor-kappa B(NF-κB)ligand receptor in the transformation of osteoid cells through the Wingless-related integration site(Wnt)/β-catenin pathway.Methods Aortic smooth muscle cells(SMC)were isolated from SD rats and induced into osteoblast like cells.Osteoblast like cells were treated with LiCl,LiCl+bone protection element(OPG,0.1 ng/ml),LiCl+OPG(1.0 ng/ml),LiCl+OPG(10.0 ng/ml)and LiCl+OPG(100.0 ng/ml)respectively.The control group was not treated with LiCl or OPG,and the concentration of LiCl was 20 mmol/L.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the transcription levels of OPG and RANKL.The von Kossa staining,calcium content,alkaline phosphatase(ALP)activity,osteocalcin and Western blotting were used to detect the degree of calcification.Immunohistochemistry and Western blotting were used to detect the activation of Wnt/β-catenin pathway.One-way ANOVA was used to compare the differences between the experimental groups and control group,LSD-t test was used for multiple comparison.Results The expression of OPG in LiCl group(4.35±0.88)was slightly higher than that in osteoblast like cell group(4.12±0.82,t=0.603,P>0.05).The expression of RANKL in LiCl group(2.61±0.42)was significantly higher than that in osteoblast like cell group(1.52±0.35,t=2.317,P<0.05).The ratio of OPG/RANKL in LiCl group(1.67±0.41)was significantly lower than that in osteoblast like cell group(2.71±0.48,t=2.542,P<0.05).In the transformation experiment of osteoblast like cells,the calcium content,ALP activity and osteocalcin level in osteoblast like cells group and the LiCl+OPG groups[(25.2±5.8,68.5±13.3,61.1±12.8,57.6±11.3,56.3±11.4),(80.4±10.2,141.2±17.6,125.7±15.3,123.5±15.9,122.7±15.1),(0.61±0.10,0.82±0.14,0.79±0.13,0.72±0.12,0.73±0.13)]were significantly reduced as compared with those in the LiCl group[(99.5±16.5),(186.0±39.3),(0.98±0.15),(t=5.971,2.795,2.810,2.829,2.831,P<0.05),(t=5.623,2.597,2.765,2.791,2.793,P<0.05),(t=4.931,2.379,2.384,2.391,2.392,P<0.05)].There was no significant difference in the calcium content,ALP activity and osteocalcin level among the LiCl+OPG groups(P>0.05),but the calcium content,ALP activity and osteocalcin level in LiCl+OPG groups were significantly higher than those in osteoblast like cells group[(t=5.362,2.769,2.635,2.633,P<0.05),(t=5.105,2.758,2.732,2.733,P<0.05),(t=2.762,2.569,2.566,2.563,P<0.05)].The expression level ofβ-catenin in LiCl group(1.75±0.26)was significantly higher than that in osteoblast like cell group and LiCl+OPG groups(1.32±0.18,1.25±0.17,1.25±0.16,1.22±0.16),(t=4.442,2.650,2.654,2.712,2.709,P<0.05),and that in LiCl+OPG groups was significantly higher than that in osteoblast like cell group(t=2.492,2.455,2.439,2.437,P<0.05).There was no significant difference among the LiCl+OPG groups(P>0.05).Conclusion Activation of Wnt/β-catenin pathway promotes the transformation of osteoid cells by RANKL-dependent and non-RANKL-dependent mechanisms.