晚期糖基化终产物通过调控F-actin/YAP抑制小鼠胚胎成骨细胞成骨分化

Advanced glycation end products inhibit osteogenic differentiation of mouse embryonic osteoblasts by regulating F-actin/YAP

目的考察晚期糖基化终产物(AGE)对小鼠胚胎成骨细胞系MC3T3-E1细胞增殖和分化的影响及其作用机制。方法用不同质量浓度(100、200、300 mg/L)AGE作用于MC3T3-E1细胞,采用CCK-8法检测细胞增殖活性,采用流式细胞术检测细胞凋亡率,采用碱性磷酸酶(ALP)染色检测细胞成骨能力,采用qPCR检测成骨相关基因(骨钙素、ALP和Runx2)及Yes相关蛋白(YAP)和β-联蛋白的mRNA表达,采用蛋白质印迹法检测YAP和β-联蛋白的蛋白质表达,采用免疫荧光法观察YAP和β-联蛋白的细胞核内含量及细胞骨架蛋白纤丝状肌动蛋白(F-actin)的表达。结果MC3T3-E1细胞在200、300 mg/L AGE处理后增殖活性降低、凋亡率增加(P均<0.05),100 mg/L AGE对细胞增殖和凋亡无明显影响,故选取100 mg/L AGE进行实验。在成骨诱导培养条件下,与对照组相比,MC3T3-E1细胞经100 mg/L AGE处理后ALP染色较浅,骨钙素、ALP和Runx2的mRNA表达均较低(P均<0.05)。在常规培养条件下,与对照组相比,MC3T3-E1细胞经100 mg/L AGE处理后F-actin形态和分布发生明显改变;YAP的mRNA和蛋白质表达均无明显变化,但其细胞核内含量减少;β-联蛋白的mRNA和蛋白质表达均降低(P均<0.05),但其细胞核内含量无明显变化。结论AGE能抑制MC3T3-E1细胞的增殖活性,诱导细胞凋亡,降低成骨分化能力,F-actin、YAP和β-联蛋白参与其调控过程。

Objective To explore the effects of advanced glycation end product(AGE)on proliferation and differentiation of mouse embryonic osteoblast line MC3T3-E1 cells and its mechanisms.Methods MC3T3-E1 cells were treated with different mass concentrations of AGE(100,200,and 300 mg/L).Cell proliferation activity and apoptosis rate were detected by cell counting kit 8 and flow cytometry,respectively.Osteogenic ability was detected by alkaline phosphatase(ALP)staining,and the mRNA expression of osteogenesis-related genes(osteocalcin,ALP,and Runx2),Yes-associated protein(YAP),andβ-catenin was detected by quantitative polymerase chain reaction.The protein expression of YAP andβ-catenin was detected by Western blotting,and the nuclear contents of YAP andβ-catenin and the expression of cytoskeleton protein filamentous actin(F-actin)were observed by immunofluorescence assay.Results The proliferation activity of MC3T3-E1 cells was significantly decreased and the apoptosis rates were significantly increased after treatment with 200 or 300 mg/L AGE(all P<0.05),and 100 mg/L AGE had no significant effect on cell proliferation or apoptosis and was selected for the experiment.Under osteogenic induction culture condition,compared with the control group,the ALP staining was shallow in MC3T3-E1 cells after treatment with 100 mg/L AGE,and the mRNA expression levels of osteocalcin,ALP and Runx2 were significantly lower(all P<0.05).Under conventional culture condition,compared with the control group,the morphology and distribution of F-actin were significantly changed in MC3T3-E1 cells after treatment with 100 mg/L AGE;there were no significant changes in the mRNA or protein expression of YAP,but the nuclear contents were decreased;and the mRNA and protein expression levels ofβ-catenin were significantly decreased(both P<0.05),but the nuclear contents had no significant change.Conclusion AGE can inhibit proliferation activity,induce apoptosis,and inhibit osteogenic differentiation ability of MC3T3-E1 cells.F-actin,YAP,andβ-catenin participate in the regulation process.