南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 39-45.doi: 10.12122/j.issn.1673-4254.2023.01.05

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特立帕肽通过cAMP/PKA/CREB信号通路调控高糖微环境下的成骨细胞分化

侯 甜，秦雅芝，张 妍，温国琛，戚孟春，董 伟   

  1. 华北理工大学口腔医学院，河北 唐山 063210
  • 出版日期:2023-01-20 发布日期:2023-02-22

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway

HOU Tian, QIN Yazhi, ZHANG Yan, WEN Guochen, QI Mengchun, DONG Wei   

  1. School of Stomatology, North China University of Science and Technology, Tangshan 063210, China
  • Online:2023-01-20 Published:2023-02-22

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摘要： 目的 探讨特立帕肽对高糖微环境下小鼠胚胎成骨细胞（MC3T3-E1）分化的影响及作用机制。方法 将MC3T3-E1细胞分为正常糖组（NG，5.5 mmol/L葡萄糖）、NG+特立帕肽组（5.5 mmol/L葡萄糖+10 nmol/L特立帕肽）、高糖组（HG，25 mmol/L葡萄糖）、HG+特立帕肽组（25 mmol/L葡萄糖+10 nmol/L特立帕肽）和HG+特立帕肽+PKA抑制剂组（25 mmol/L葡萄糖+10 nmol/L特立帕肽+20 μmol/L H-89）。CCK-8法检测细胞增殖；ELISA实验检测各组cAMP水平；试剂盒检测碱性磷酸酶（ALP）活性；茜素红染色检测细胞矿化结节生成情况；鬼笔环肽染色后观察细胞骨架；Real-time PCR检测细胞中PKA、CREB、RUNX2和Osx的mRNA表达水平。结果 各组细胞增殖能力差异无统计学意义（P>0.05）。与NG组相比，NG+特立帕肽组细胞cAMP水平升高（P<0.05），ALP活性增强（P<0.05），茜素红矿化结节生成能力增强（P<0.05），细胞骨架清晰程度有所提升，PKA、CREB、RUNX2和Osx的mRNA表达上调（P<0.05）；与NG组相比，HG组的上述检测结果则呈减弱趋势。与HG组相比，HG+特立帕肽组细胞cAMP水平上升（P<0.05），ALP活性增强（P<0.05），茜素红矿化结节生成能力增强（P<0.05），细胞骨架清晰程度有所提升，PKA、CREB、RUNX2和Osx的mRNA表达上调（P<0.05）。与HG+特立帕肽组相比，HG+特立帕肽+H-89组细胞cAMP水平下降（P<0.05），ALP活性减弱（P<0.05），茜素红矿化结节生成能力减弱（P<0.05），细胞骨架清晰程度下降，PKA、CREB、RUNX2和Osx的mRNA表达下调（P<0.05）。结论 特立帕肽可通过激活cAMP/PKA/CREB信号通路有效改善高糖微环境对MC3T3-E1细胞分化的抑制作用。

关键词: 成骨细胞；细胞分化；特立帕肽；骨质疏松；cAMP/PKA/CREB信号通路；高糖微环境

Abstract: Objective To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism. Methods MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells. Results The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P>0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P<0.05) with enhanced ALP activity and increased area of mineralized nodules (P<0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P<0.05). The opposite changes wereobserved in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P<0.05), ALP activity and the area of mineralized nodules (P<0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P<0.05). Conclusion Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Key words: osteoblasts; cell differentiation; teriparatide; osteoporosis; cAMP/PKA/CREB signaling pathways; high-glucose microenvironment