| Osteoporosis(OP)is a systemic,skeletal disease that is characterized by decreased bone mass and disruption of bone fine structure,manifested as increased brittleness of the bone,which in turn greatly increases the risk of fracture,even if It is also prone to bone fractures when it is mildly traumatized or traumatized.However,there is currently no ideal method for the treatment of osteoporosis.Pulsed electromagnetic fields(PEMFs)as a non-invasive treatment are considered to be safe and effective methods for the treatment of osteoporosis.In this experimental group,various parameters of the pulsed electromagnetic field that can promote bone formation have been selected,including frequency,intensity,and action time.Under these parameters,the pulsed electromagnetic field significantly improved the mature mineralization of rat osteoblasts and increased bone mineral density in ovariectomized rats.However,the mechanism of action of pulsed electromagnetic fields in promoting bone formation is not yet clear,and it is a major constraint to the application of electromagnetic fields in the treatment of osteoporosis.The preliminary study of this experimental group showed that the 50 Hz 0.6 mT low-frequency pulsed electromagnetic field is one of the best parameters to promote bone formation.Under this condition,the pulsed electromagnetic field promotes bone formation,a special organelle that depends on the surface of mammalian cells—primary cilia.In view of the above research results,this paper carried out research on the relationship between pulsed electromagnetic field and bone morphogenesis,primary cilia and related signal pathways.First,in order to verify whether PEMFs can promote osteoclast maturation and mineralization,rat calvarial osteoblasts(ROBs)were treated different time with PEMFs,the alkaline phosphatase activity was significantly increased,the osteogenesis-related factors RUNX2,OSX,BMP-2 gene transcription and protein expression were significantly increased,indicating that 50 Hz 0.6 mT pulsed electromagnetic field can promote bone formation.Second,in order to investigate whether PEMFs promote osteoclast maturation and mineralization depend on the cAMP/PKA/CREB signaling pathway,rat osteoblasts intracellular cAMP rapidly increased were treat at different times after PEMFs.The expression of p-PKA and p-CREB protein increased significantly,and resulted in the nucleartranslocation of intracellular CREB,indicating that the promotion of bone formation may be related to the cAMP/PKA/CREB signaling pathway.The ability of PEMFs to promote the maturation of osteoblasts is inhibited after the cAMP-PKA signaling pathway is blocked by adenylate cyclase(AC)and protein kinase A(PKA)inhibitors.This suggests that PEMFs activate and depend on the cAMP/PKA/CREB signaling pathway in promoting bone formation.We further examined the expression of ten isomers of adenylyl cyclase upstream of cAMP and found that the expression levels of various AC genes and proteins were significantly increased after PEMFs treatment,whereas AC7 was not expressed in osteoblasts.Finally,in order to investigate whether the primary cilia and the cAMP/PKA/CREB signaling pathway are involved,the immunofluorescence was used to co-localize PKA,pPKA and adenylate cyclase with primary cilia.It was found that both p-PKA and soluble adenylyl cyclase(sAC)were located in the primary cilia.After treatment of osteoblasts with the sAC-specific inhibitor KH7,it was found that the cAMP-PKA-CREB signaling pathway was blocked,and the phenomenon of promoting the expression of bone formation-related factors and calcification nodule formation was inhibited.In summary,this study demonstrated that 50 Hz 0.6 mT PEMFs promote bone formation by activating the sAC/cAMP/PKA/CREB signaling pathway,which is dependent on primary cilia,suggesting that PEMFs through primary cilia/sAC/cAMP/PKA/CREB signaling pathways to promote bone formation. |
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