Pulsed Electromagnetic Fields Promote The Differentiation And Maturation Of Osteoblasts Via PC2/sAC/PKA/CREB Signaling Pathway

Osteoporosis is a major public health problem in today’s society.At present,the clinical drugs for the treatment of osteoporosis generally have some problems such as large side effects and high prices.Therefore,people have been striving to find alternative therapies for osteoporosis with small side effects and low prices.The significant efficacy of pulsed electromagnetic fields(PEMFs)in the treatment of osteoporosis has been discovered for a long time,and it has the advantages of convenient use,process safety,and few adverse reactions.However,there are still some problems in this therapy,such as unstable efficacy and poor repeatability.The reason for this is the inconsistent treatment parameters caused by the unclear mechanism of action.In our previous study,50 Hz 0.6 m T PEMFs were selected as the optimal parameter to promote bone formation.The PEMFs can promote the differentiation and mineralization of rat calvarial osteoblasts(ROBs)through a variety of signaling pathways including s AC/PKA/CREB signaling pathway,and depend on the presence of primary cilia.However,the electromagnetic field receptors responsible for receiving physical signals of PEMFs and converting them into intracellular chemical signals remains unknown.In response to this problem,this paper investigated whether the role of PEMFs in promoting the differentiation and maturation of osteoblasts is related to the primary cilia,polycystin2(PC2)located in the primary cilia and s AC/PKA/CREB signaling pathways to lay a foundation for elucidating the mechanism of PEMFs in promoting bone formation.The main experimental methods of this paper are as follows: First,the expression of PC2,s AC,PKA,CREB and their phosphorylated proteins in ROBs were detected after the treatment with 50 Hz 0.6 m T PEMFs for different time;then immunofluorescence staining was used to detected the co-localization of the above proteins with primary cilia.Subsequently,we detected the activity changes of s AC/PKA/CREB signal pathway and wehther the differentiation and maturation of ROBs were affected after blocking the PC2 function by amiloride hydrochloride(AMI).Moreover,the above indexes were detected after specifically knocking down the expression of PC2 using RNA interference.Finally,we detected the expression of PC2 and signaling proteins of s AC/PKA/CREB pathway and the differentiation and maturation of ROBs after inhibiting primary ciliogenesis by RNA interference.The results of this experiment are as follows: the expression levels of PC2,s AC,pPKA and p-CREB were significantly increased after ROBs were treated with PEMFs for different times.PC2,s AC,p-PKA and p-CREB could be located in the whole primary cilia or its roots,but PKA and CREB were not distributed in the primary cilia.After PC2 was blocked by AMI,PEMFs could no longer increase the expression of PC2 and signaling proteins of s AC/PKA/CREB pathway,nor alkline phosphatase(ALP)activity,and the promoting effect of PEMFs on osteogenic protein and gene expression was also offset,and the differentiation and maturation of osteoblasts were blocked.The results of knocking down PC2 expression level were similar to blocking PC2 function.After interfering with the primary cilia,PEMFs treatment could not increase the expression of PC2,activate the s AC/PKA/CREB signal pathway,no longer increase ALP activity,and no longer promote the differentiation and maturation of osteoblasts.Conclusion: PC2,located on the surface of primary cilia of osteoblasts,can perceive and transmit the physical signals from PEMFs,and promote the differentiation and maturation of osteoblasts by activating the PC2/s AC/PKA/CREB signaling pathway.The promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2.This study lays a foundation to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.