基于Wnt/β-连环蛋白通路探究金天格对肿瘤坏死因子-α诱导的小鼠MC3T3E1细胞生物学功能的影响实验研究

Effect of Jintiange on biological function of mouse MC3T3E1 cells induced by tumor necrosis factor-α based on Wnt/β-catenin pathway

目的:探讨金天格通过调节Wnt/β-连环蛋白(Wnt/β-catenin)通路对肿瘤坏死因子-α(TNF-α)诱导的小鼠成骨细胞(MC3T3E1)细胞生物学功能的影响。方法:体外培养MC3T3E1细胞,分为对照组、TNF-α组(50 ng/ml TNF-α)、L-金天格组(50 ng/ml TNF-α+10^(-6) g/L金天格)、M-金天格组(50 ng/ml TNF-α+10-5 g/L金天格)、H-金天格组(50 ng/ml TNF-α+10^(-4) g/L金天格)、Dickkopf-1(DKK-1)组(50 ng/ml TNF-α+10 ng/ml Wnt/β-catenin通路抑制剂DKK-1)、H-金天格+LiCl组(50 ng/ml TNF-α+10^(-4) g/L金天格+20μmol/L Wnt/β-catenin通路激活剂LiCl)。用CCK-8试剂盒对细胞活性进行检测,用流式细胞仪对细胞凋亡情况进行检测,用酶联免疫吸附试验对细胞白细胞介素-1β(IL-1β)和IL-6水平进行检测,用Western blot对细胞凋亡相关蛋白及Wnt/β-catenin信号通路蛋白表达情况进行检测。结果:与对照组比较,TNF-α组细胞活性、B淋巴细胞瘤-2(Bcl-2)、细胞程序性死亡配体-1(PD-L1)蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及B细胞淋巴瘤(Bax)、β-catenin、转录因子7样2(TCF7L2)、细胞周期蛋白D1(Cyclin D1)蛋白表达升高(均P<0.05)。与TNF-α组比较,L-金天格组、M-金天格组、H-金天格组、DKK-1组细胞活性及Bcl-2、PD-L1蛋白表达升高,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达降低(均P<0.05)。与H-金天格组比较,H-金天格+LiCl组细胞活性及Bcl-2、PD-L1蛋白表达降低,细胞凋亡率、IL-1β、IL-6水平以及Bax、β-catenin、TCF7L2、Cyclin D1蛋白表达升高(均P<0.05)。结论:金天格可能通过抑制Wnt/β-catenin通路减轻TNF-α诱导的MC3T3E1细胞损伤。

Objective:To investigate the effec of Jintiange on the biological function of mouse osteoblasts(MC3T3E1)induced by tumor necrosis factor-α(TNF-α)by regulating Wnt/β-catenin pathway.Methods:MC3T3E1 cells were cultured in vitro and divided into control group,TNF-αgroup(50 ng/ml TNF-α),L-Jintiange group(50 ng/ml TNF-α+10^(-6) g/L Jintiange),M-Jintiange group(50 ng/ml TNF-α+10-5 g/L Jintiange),H-Jintiange group(50 ng/ml TNF-α+10^(-4) g/L Jintiange),DKK-1 group(50 ng/ml TNF-α+10 ng/ml Wnt/β-catenin pathway inhibitor DKK-1),H-Jintiange+LiCl group(50 ng/ml TNF-α+10^(-4) g/L Jintiange+20μmol/L Wnt/β-catenin pathway activator LiCl).CCK-8 assay kit was applied to detect cell activity.Flow cytometry was applied to detect cell apoptosis.Cellular IL-1βand IL-6 levels were measured by ELISA.Western blot was applied to detect the expression of apoptosis related proteins and Wnt/β-catenin signaling pathway proteins.Results:Compared with the control group,the cell viability,the protein expression of Bcl-2,PD-L1 in TNF-αgroup were obviously reduced,and apoptosis rate,the levels of IL-1βand IL-6,the expression of Bax,β-catenin,TCF7L2 and Cyclin D1 proteins were significantly elevated(all P<0.05).Compared with the TNF-αgroup,the cell viability,the protein expression of Bcl-2 and PD-L1 in the L-Jintiange group,M-Jintiange group,H-Jintiange group,and DKK-1 group were obviously increased,and the apoptosis rate,the levels of IL-1βandIL-6,the expression of Bax,β-catenin,TCF7L2,and Cyclin D1 proteins were obviously reduced(all P<0.05).Compared with the H-Jintiange group,the cell viability,the expression of Bcl-2,and PD-L1 proteins in the H-Jintiange+LiCl group were obviously reduced,and the apoptosis rate,the levels of IL-1βand IL-6,the expression of Bax,β-catenin,TCF7L2 and Cyclin D1 proteins were obviously increased(all P<0.05).Conclusion:Jintiange may alleviate TNF-α-induced damage to MC3T3E1 cells by inhibiting the Wnt/β-catenin pathway.