Q Ren1, J Zhou1, M G Wang2, K M Chen1. 1. Institute of Orthopaedics, Lanzhou General Hospital of PLA, Lanzhou 730050, China. 2. College of Life Science and Engineering,Lanzhou University of Technology, Lanzhou 730050, China.
Abstract
OBJECTIVE: To study whether the pulsed electromagnetic fields (PEMF) promoting rat osteoblasts differentiation and maturation is related to the primary cilia and PI3K/AKT pathway, and to explore the mechanism of PEMF in promoting bone differentiation. METHODS: Enzyme solution was used to obtain newborn SD rats calvarial osteoblasts (ROB), which were processed by 50 Hz 0.6 mT PEMF for 0, 0.5, 1, 1.5 and 2 h, detecting PI3K and AKT protein expression and changes in primary cilia length and incidence; with LY294002 blocking PI3K/AKT signaling pathways we observed whether PEMF promoted osteogenic differentiation of ROB was affected; by interfering IFT88 gene expression by RNAi to inhibit primary cilia we observed whether PI3K/AKT signaling pathway and osteogenic differentiation of ROB was affected. Osteogenic differentiation indexes included alkaline phosphatase (ALP) activity, Real-time PCR and Western blot detection of osteogenic related genes of BMP-2, COL-1 and OSX and calcified nodules number, etc.. RESULTS: After exposure to PEMF for 0, 0.5, 1, 1.5, and 2 h, the protein expression of PI3K and AKT in ROB were increased significantly (P<0.01) and the primary cilia became longer; and the protein expression of PI3K reached the highest level at 0.5 h, as the treatment time of PEMF increased, the PI3K protein expression decreased. AKT showed higher protein expression at 0.5 h and 1.5 h. After blocking the PI3K/AKT signaling pathway with the PI3K blocker LY294002, PEMF could no longer increase ALP activity and the gene expressions of BMP-2, COL-1, OSX which were osteogenically related. However, PEMF could increase the ALP activity and the osteogenically related gene expression in ROB before blocking. After RNAi interfered the primary cilia, PEMF could no longer increase the protein expression of PI3K, which indicated that PEMF could not activate the PI3K/AKT signaling pathway after primary cilia interfering; secondly, the effect of PEMF on enhancing ALP activity disappeared, it also decrease the gene expressions of BMP-2, COL-1, and OSX, and the ability of increasing the calcification nodule formation also disappeared, indicating that the ability of PEMF to promote osteoblast maturation and mineralization disappeared after primary cilia interference. CONCLUSION: PEMF activated the PI3K/AKT signaling pathway through primary cilia on the surface of osteoblasts, then promoted bone formation activity and differentiation.
OBJECTIVE: To study whether the pulsed electromagnetic fields (PEMF) promoting rat osteoblasts differentiation and maturation is related to the primary cilia and PI3K/AKT pathway, and to explore the mechanism of PEMF in promoting bone differentiation. METHODS: Enzyme solution was used to obtain newborn SD rats calvarial osteoblasts (ROB), which were processed by 50 Hz 0.6 mT PEMF for 0, 0.5, 1, 1.5 and 2 h, detecting PI3K and AKT protein expression and changes in primary cilia length and incidence; with LY294002 blocking PI3K/AKT signaling pathways we observed whether PEMF promoted osteogenic differentiation of ROB was affected; by interfering IFT88 gene expression by RNAi to inhibit primary cilia we observed whether PI3K/AKT signaling pathway and osteogenic differentiation of ROB was affected. Osteogenic differentiation indexes included alkaline phosphatase (ALP) activity, Real-time PCR and Western blot detection of osteogenic related genes of BMP-2, COL-1 and OSX and calcified nodules number, etc.. RESULTS: After exposure to PEMF for 0, 0.5, 1, 1.5, and 2 h, the protein expression of PI3K and AKT in ROB were increased significantly (P<0.01) and the primary cilia became longer; and the protein expression of PI3K reached the highest level at 0.5 h, as the treatment time of PEMF increased, the PI3K protein expression decreased. AKT showed higher protein expression at 0.5 h and 1.5 h. After blocking the PI3K/AKT signaling pathway with the PI3K blocker LY294002, PEMF could no longer increase ALP activity and the gene expressions of BMP-2, COL-1, OSX which were osteogenically related. However, PEMF could increase the ALP activity and the osteogenically related gene expression in ROB before blocking. After RNAi interfered the primary cilia, PEMF could no longer increase the protein expression of PI3K, which indicated that PEMF could not activate the PI3K/AKT signaling pathway after primary cilia interfering; secondly, the effect of PEMF on enhancing ALP activity disappeared, it also decrease the gene expressions of BMP-2, COL-1, and OSX, and the ability of increasing the calcification nodule formation also disappeared, indicating that the ability of PEMF to promote osteoblast maturation and mineralization disappeared after primary cilia interference. CONCLUSION:PEMF activated the PI3K/AKT signaling pathway through primary cilia on the surface of osteoblasts, then promoted bone formation activity and differentiation.