Zhang Zitao, Hu Polu, Wang Zhen, Qiu Xusheng, Chen Yixin
Department of Orthopedics, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China.
Nanjing Red Cross Blood Center, Nanjing, China.
J Cell Mol Med. 2020 Sep;24(18):10792-10802. doi: 10.1111/jcmm.15704. Epub 2020 Aug 16.
Brain-derived neurotrophic factor (BDNF) has been reported to participate in fracture healing, whereas the mechanism is still unclear. Since osteoblast migration is important for fracture healing, investigating effects of BDNF on osteoblasts migration may help to reveal its mechanism. Here, MC3T3-E1 cells were used in vitro while closed femur fracture mice were applied in vivo. Cells migration was assessed with Transwell assay. The protein expression was analysed by immunoblotting. X-ray and Micro-CT were performed at different time after fracture. Our results showed that BDNF promoted MC3T3-E1 cells migration, integrin β1 expression and ERK1/2 and AKT phosphorylation. K252a, a specific inhibitor for TrkB, suppressed BDNF-induced migration, integrin β1 expression and activation of ERK1/2 and AKT. PD98059 (an ERK1/2 inhibitor) and LY294002 (an AKT inhibitor) both inhibited BDNF-induced migration and integrin β1 expression while integrin β1 blocking antibody only suppressed cell migration. X-ray and Micro-CT analyses showed that the adenoviral carried integrin β1 shRNA group had slower fracture healing at 7 and 21 days, but not 35 days compared to the control group. Thus, we proposed that BDNF stimulated MC3T3-E1 cells migration by up-regulating integrin β1 via TrkB mediated ERK1/2 and AKT signalling, and this may help to enhance the fracture healing.
据报道，脑源性神经营养因子（BDNF）参与骨折愈合，但其机制仍不清楚。由于成骨细胞迁移对骨折愈合很重要，研究BDNF对成骨细胞迁移的影响可能有助于揭示其机制。在此，体外实验使用MC3T3-E1细胞，体内实验采用闭合性股骨骨折小鼠。通过Transwell实验评估细胞迁移。通过免疫印迹分析蛋白质表达。在骨折后的不同时间进行X射线和显微CT检查。我们的结果表明，BDNF促进MC3T3-E1细胞迁移、整合素β1表达以及ERK1/2和AKT磷酸化。TrkB的特异性抑制剂K252a抑制BDNF诱导的迁移、整合素β1表达以及ERK1/2和AKT的激活。PD98059（一种ERK1/2抑制剂）和LY294002（一种AKT抑制剂）均抑制BDNF诱导的迁移和整合素β1表达，而整合素β1阻断抗体仅抑制细胞迁移。X射线和显微CT分析表明，与对照组相比，携带整合素β1 shRNA的腺病毒组在第7天和第21天骨折愈合较慢，但在第35天并非如此。因此，我们提出BDNF通过TrkB介导的ERK1/2和AKT信号上调整合素β1来刺激MC3T3-E1细胞迁移，这可能有助于促进骨折愈合。
