Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis
Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis
复制标题
杆菌肽通过刺激骨形态发生蛋白-2/Smad轴促进人骨髓间充质干细胞成骨分化
DOI：
10.1016/j.biopha.2018.04.084
复制
发表时间：
2018-07-01
影响因子：
7.5
通讯作者：
Yue, Bing
中科院分区：
文献类型：
--
作者：
Li, Hui;Nie, Bin'en;Yue, Bing
杆菌肽是一种广泛使用的金属多肽抗生素，已被报道用于局部治疗伤口，没有全身不良反应。我们的初步研究表明杆菌肽可促进人骨髓间充质干细胞向成骨细胞分化。本研究探讨杆菌肽是否影响牛骨髓间充质干细胞的成骨分化，并探讨其分子机制。用细胞计数试剂盒-8(CCK-8)检测杆菌肽对HBMSCs增殖的影响。采用流式细胞仪检测杆菌肽对HBMSCs细胞周期和细胞凋亡率的影响。采用碱性磷酸酶(ALP)染色、胶原沉积(天狼星红)和矿化(茜素红)染色和定量检测方法研究HBMSCs的成骨分化。用定量逆转录聚合酶链式反应(RT-qPCR)检测成骨分化标志物的表达。用荧光素酶报告实验和Western blotting检测相关信号通路的激活情况。杆菌肽治疗增加了HBMSCs的成骨分化，没有细胞毒性，也没有对细胞周期进展或细胞凋亡产生不利影响。荧光素酶报告实验表明杆菌肽激活了骨形态发生蛋白2(BMP2)基因的转录，BMP2基因是BMP2/Smad信号轴的关键基因。Western blotting显示，杆菌肽刺激成骨可明显激活该轴。此外，转化生长因子-β/Smad抑制剂(LDN-193189)和小干扰核糖核酸(SiRNA)基因沉默(si-BMP2)可抑制杆状杆菌蛋白对Smad磷酸化的激活和成骨分化。综上所述，我们的结果提示杆菌肽可以通过激活BMP2/Smad信号轴来促进HBMSCs的成骨。
Bacitracin, a widely used metallopeptide antibiotic, has been reported to be locally used in treating wounds without systemic adverse reactions. Our preliminary study showed that bacitracin might enhance the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). The present study investigated whether bacitracin affects the osteogenic differentiation of HBMSCs and the molecular mechanisms involved. The proliferation of HBMSCs in the presence of bacitracin was examined using a cell counting kit-8 (CCK-8) assay. The effects of bacitracin on the cell cycle and apoptosis of HBMSCs were observed using flow cytometry assay. Staining and quantitative assays for alkaline phosphatase (ALP) staining, collagen deposition (Sirius Red), and mineralization (Alizarin Red) were used to study osteogenic differentiation of HBMSCs. The expression of osteogenic differentiation markers was detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. The activation of related signaling pathways was examined using a luciferase reporter assay and western blotting. Bacitracin treatment increased osteogenic differentiation of HBMSCs without cytotoxicity and did not adversely affect cell cycle progression or apoptosis. The luciferase reporter assay showed that bacitracin activated the transcription of bone morphogenetic protein-2 (BMP2) gene, a key gene in the BMP2/Smad signaling axis. Western blotting indicated that this axis was markedly activated by bacitracin stimulation of osteogenesis. Moreover, the activation of Smad phosphorylation and osteogenic differentiation by bacitracin was inhibited by a transforming growth factor (TGF)-beta/Smad inhibitor (LDN-193189 HCl) and small interfering RNA (siRNA) gene silencing (si-BMP2). In conclusion, our results suggest that bacitracin can promote osteogenesis of HBMSCs by activating the BMP2/Smad signaling axis.