Low-frequency Pulsed Electromagnetic Fields Promote Bone Formation By Activating Primary Cilia-regulated IGF-1R/NO Signaling Pathway

Osteoporosis(OP),a metabolic bone disease commonly found in middle-aged women and the elderly,is threatening their health.In addition to causing bone pain,cervical and lumbar spine deformation,height shorter,hunchback and lung function reduced,because of the destruction of bone micro structure,increase bone fragility and particularly easy to cause fracture,the fracture treatment difficult,high mortality and disability rate,will give patients and their families to bring heavy mental pressure and economic burden.Currently,there is no ideal treatment for osteoporosis,and drug therapy is mainly used in clinical practice.Commonly used drugs include bisphosphonates and calcitonin,etc.People have been trying to find alternative therapies due to their expensive price and large side effects caused by longterm use,among which electromagnetic therapy is one of the hot spots in current research.PEMFs(Pulsed electromagnetic field)has the advantages of non-trauma,simple operation and low price,but its mechanism of action is not yet clear,and the "biological window effect" of treatment parameters has resulted in poor reproducibility of efficacy,greatly limiting its application.In this study,a group of optimal PEMFs parameter combinations(50 Hz,0.6mt,50%duty cycle)were selected and determined by the research group,and it was found that primary cilia were required to participate in PEMFs to promote bone formation activity,and further studies showed that PEMFs promoted bone formation through IGF-1R and NO signal pathway regulated by primary cilia.The main methods,results and conclusions are as follows.Methods: 1.Osteoblasts(ROBs)were isolated and cultured from newborn rat skulls;2.The activity of alkaline phosphatase(ALP),the expression of IGF-1R and the content of nitric oxide(NO)were detected after ROBs was treated with PEMFs with optimal parameters for different times;3.Pretreatment of ROBs by BEZ with IGF-1R blocker GSK and PI3 K blocker BEZ was used to analyze the changes of bone formation activity promoted by PEMFs.;4.Co-localization of signal proteins and primary cilia;5.Interfered with primary ciliary generation,analyzed the influence of PEMFs on promoting bone formation and activating IGF-1R related signal pathway.Experimental results:1.The protein expression level of IGF-1R was detected after ROBs was treated with the optimal parameter PEMFs for different times,and it was found that the protein expression level increased gradually after 0,0.5,1 and 1.5 hours,reached a peak at 1.5 hours,and was significantly higher than the expression level at 0 hours(P<0.01),after which the expressionlevels of 2,2.5,3,3.5 and 4h IGF-1R continued to decrease.2.After the pretreatment of ROBs by GSK with IGF-1R blocker,the mineralization and maturation activity of PEMFs was significantly inhibited,showing that the ALP activity of PEMFs group was significantly lower than that of PEMFs group(P<0.01),the number and area of ALP staining positive clones were reduced,and the number and area of calcified nodule showed the same change.It showed that inhibition of IGF-1R could antagonize the mineralization and maturation of PEMFs.3.Co-localization of IGF-1R and primary cilia was studied by immunofluorescence staining,and it was found that IGF-1R was distributed in all parts of primary cilia.After the inhibition of primary cilia by RNA interference,the mineralization and maturation activity of osteoblasts promoted by PEMFs was significantly decreased,and the effect of PEMFs on increasing the expression of IGF-1R was also disappeared,indicating that the expression level of IGF-1R was regulated by primary cilia.4.After pretreatment with GSK ROBs,PEMFs significantly reduced the expression of PI3 K and AKT and its phosphorylation level(P<0.05),indicating that the PI3K/AKT signaling pathway is located downstream of IGF-1R.At the same time,it was also found that the NO signal pathway was inhibited after blocking IGF-1R with GSK,indicating that the NO signal pathway was also located in the downstream of IGF-1R.5.ROBs pretreated with BEZ of different concentrations was used to screen the optimal site for the inhibition of PI3 K by BEZ,and the results showed that the inhibition effect of ATR site was the most significant.After the pretreatment of ROBs with ATR concentration,ALP activity,ALP staining positive clones and the number of calcified nodules all decreased significantly,while the expressions of eNOS,iNOS and PKG were also significantly inhibited,indicating that the NO signaling pathway is located in the downstream of PI3K/AKT.The research results show that 50 Hz 0.6 mT PEMFs could significantly promote the rats skull osteoblasts into osseous differentiation and mineralization of mature,PEMFs can activate the IGF-1R signaling pathways,and IGF-1R in primary cilia roots and its expression by primary cilia regulation,so the PEMFs is through activation of primary cilia regulation of IGF-1R/PI3K/AKT/NO signaling pathways promote bone formation.This study provided experimental basis for further screening the best therapeutic parameters of PEMFs and improving the clinical efficacy of PEMFs.