低频脉冲电磁场通过PI3K/AKT/GSK3β/β-catenin信号途径促进成骨细胞矿化成熟  

Effect of Low-frequency Pulsed Electromagnetic Fields on Osteoblastic Differentiation Through PI3K/AKT/GSK3β/β-catenin Signaling Pathway

作　　者：何文芳 李雪雁[1] 任莉 吴思敏 刘菁 高玉海 陈克明 HE Wen-Fang;LI Xue-Yan;REN Li;WU Si-Min;LIU Jing;GAO Yu-Hai;CHEN Ke-Ming(College of Life Science and Engineering,LanZhou University of Tenchnolog,Lanzhou 730050,China;The 940th Hospital of Joint Logistics Support force of Chinese People’s Liberation Army,Lanzhou 730050,China)

机构地区：[1]兰州理工大学生命科学与工程学院,兰州730050 [2]解放军联勤保障部队第940医院骨科研究所,兰州730050

出　　处：《中国生物化学与分子生物学报》2020年第12期1464-1472,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81770879)资助。

摘　　要：低频脉冲电磁场(pulsed electromagnetic fields,PEMFs)可以促进体外培养大鼠颅骨成骨细胞(rat calarial osteoblasts,ROB)的矿化成熟,但其具体机制尚不清楚。本实验主要研究PEMFs促进大鼠成骨细胞成熟矿化与PI3K/AKT/GSK3β/β-catenin信号通路的关系,以PEMFs促进成骨细胞成熟矿化的机制。首先,将成骨细胞经不同强度PEMFs处理后检测胞内碱性磷酸酶(ALP)活性的变化,以探明PEMFs促进成骨分化的最佳磁场强度;接着,采用蛋白质印迹法检测PEMFs处理不同时间后细胞内PI3K、p-PI3K、AKT、p-AKT、GSK3β、p-GSK3β、β-联蛋白的变化情况;最后,细胞经PI3K特异性阻断剂处理后,检测ALP活性变化、钙盐沉积情况、成骨性相关基因RUNX2、BMP2表达量,以及BMP2/Smad1/5/8信号途径的变化情况,同时也检测了该条信号途径下游蛋白质表达量及相关转录因子的核易位情况。结果发现,磁场强度为0.6 mT时,ALP活性最高;PEMFs处理成骨细胞不同时间后,PI3K、AKT和GSK3β总蛋白质无明显变化,但是p-PI3K、p-AKT、p-GSK3β、β-联蛋白的表达量较对照组显著升高,同时β-联蛋白发生核易位;经过PEMFs处理的成骨细胞的ALP活性显著升高,成骨性相关基因表达量明显增加,钙盐沉积能力增强,BMP2/Smad1/5/8信号途径被激活;在加入PI3K特异性阻断剂后,PEMFs促进成骨细胞矿化成熟的能力消失,并且不再提高该条信号途径下游蛋白质的表达和相关转录因子的核转位。上述结果提示,0.6 mT PEMFs促进成骨分化的此过程中激活了PI3K/AKT/GSK3β/β-catenin信号通路,若该条信号通路被抑制,则电磁场促成骨作用被抵消,表明低频脉冲电磁场促进成骨细胞矿化成熟依赖于PI3K/AKT/GSK3β/β-catenin信号途径,这为采用脉冲电磁场治疗骨质疏松症提供了理论基础。Effect of Low-Frequency Pulsed Electromagnetic Fields(PEMFs)has been considered as a stimulator for rat osteoblasts(ROBs)maturation and mineralization.However,the mechanism of the PEMFs-induced osteogenesis is still unknown.In this study,we examined the role of the PI3K/AKT/GSK3β/β-catenin signaling pathway in PEMFs-induced maturation and mineralization of ROBs.First,the ROBs were exposed at the PEMFs in different magnetic field strengths,then intracellular,alkaline phosphatase(ALP)activity was detected to find outthe best magnetic field strength for promotion of osteogenic differentiation;Second,protein expression levels of PI3K,AKT,GSK3β,p-GSK3β,and β-catenin were detected by Western blot after PEMFs treatment for different times;Finally,we used the PI3K specific inhibitors to block the PI3K signaling pathway,and then detected the bone-related indicators.We also detected ALP activity,calcium salt deposition and RUNX2,BMP2 gene expression of bone-related indicator,as well as downstream protein expression levels of the signaling pathway and related the nuclear translocation of transcription factors.The results showed that when the magnetic field strength of PEMFs was 0.6 mT,the ALP activity was the highest;by PEMFs treatment for different times,there was no change in the total protein levels of PI3K and AKT,but p-PI3K,p-AKT,p-GSK3β and β-catenin expression was improved,as well as nuclear translocation of β-catenin was detected.With the effect of PEMFs,ALP activity and the bone-related indicators were increased.The calcium salt deposition increased capacity and the BMP2/Smad1/5/8 signaling pathway was activated.When PI3K specific inhibitors were applied,all the above phenomena disappeared.Collectively,we demonstrated that the magnetic field strength of PEMFs exposed to 0.6 mT has the most obvious effect of promoting osteogenic differentiation and activating PI3K/AKT/GSK3β/β-catenin signaling;If the pathway was inhibited,the osteogenesis of PEMFs was offset.The results suggested that 0.6 mT PEMFs can prom

关 键 词：骨质疏松症 脉冲电磁场 成骨细胞 PI3K/AKT/GSK3β/β-catenin信号通路 

分 类 号：Q257[生物学—细胞生物学]