The Mechanism Of Low-frequency Pulsed Electromagnetic Fields Promoting Bone Formation Through Primary Cilia Regulated ERα/IGF-1R Signaling Pathwayby

Osteoporosis(OP)is a degenerative bone disease.With the advent of aging society,osteoporosis has become a common and frequently-occurring disease.Osteoporosis is a systemic,systemic skeletal disorder characterized by reduced bone mass,disrupted fine structure of bone tissue,increased bone fragility,and increased fracture risk.Occurs in the elderly and postmenopausal women,accompanied by pain after the onset of more,more serious fractures and other serious harm to people’s health.At present,clinical therapy mainly uses drug therapy,but it lacks ideal prevention measures.Current research finds that lowfrequency electromagnetic fields have the effect of preventing and treating osteoporosis.It has a non-invasive,non-invasive treatment method,but there is clinical The poor reproducibility and unclear mechanism of action have limited the large-scale clinical applications.The analysis literature found that the main reason for poor reproducibility is that there is no uniform standard and mechanism for the treatment of osteoporosis parameters with the electromagnetic field.It is unclear on the basis of these problems.After many years of research,the research team has obtained some parameters.The 50 Hz,0.6 mT pulsed electromagnetic field was effective in promoting the maturation and mineralization of ROBs cultured in vitro,but its mechanism of action is not yet fully understood.Based on the problem of unclear mechanism of action,this dissertation studies.The current study suggests that the relationship between the estrogen signaling pathway and osteoporosis is closely related.Can the electromagnetic field activate the estrogen signaling pathway? Our earliest studies have found that EMFs have the ability to increase the expression of estrogen receptors,but its specific mechanism is not yet fully understood.This paper has conducted a detailed study on this point.METHODS: Primary rat calvarial osteoblasts(ROBs)were isolated from the skull of neonatal SD rats.ROBs were randomly divided into groups and pretreated with different methods.ROBs cells were pretreated by different methods:(1)10-8 mol /I estrogen receptor blocker ICI182780 pretreatment of osteoblast estrogen receptor;(2)siRNA inhibition of ROBs estrogen receptor alpha expression;(3)25 nmol/L of IGF-1R Blocker GSK1904529 A pretreated osteoblasts;(4)Osteoblasts were pretreated with a 25 nmol/L ERK1/2 blocker SCH772984;(5)siRNA inhibited primary cilia on the surface of ROBs.50 Hz 0.6 mT pulsed electromagnetic field treatment of pre-treated ROBs cells to observe osteogenic differentiation indicators and osteogenic related signal transduction mechanisms,including determination of alkaline phosphatase(ALP)activity,alizarin red calcified nodule staining,real time RT-PCR and western blot were used to analyze the expression level of osteogenic related signals and their proteins and genes.Immunofluorescence staining and laser confocal microscopy were used to observe the morphology and number of primary cilia and the related nuclear translocation.PEMFs treatment resulted in a significant increase in osteogenic differentiation of ROBs,but this activity disappeared after pretreatment with estrogen receptor blocker ICI182780.After RNAi inhibition of ERα gene expression,the activity of PEMFs to promote bone formation disappeared.PEMFs promote bone formation through ER is dependent on ERα but not ERβ;after inhibiting IGF-1R gene expression with IGF-1R blocker GSK1904529 A,PEMFs can significantly inhibit the osteogenic differentiation of ROBs,and the expression of ERα is no longer significant.Elevated,the expression level of downstream signaling protein ERK1/2 was also significantly inhibited;after pretreatment of ROBs with ERK1/2 blockers,PEMFs promoted osteogenic differentiation of ROBs was significantly inhibited,and ERα and IGF-1R genes were Protein expression was also inhibited;immunofluorescence staining and confocal laser scanning microscopy revealed that IGF-1R can be localized in primary cilia,but pIGF-1R produced after treatment with PEMFs is not located in primary cilia.After RNAi inhibition of IFT88 expression,primary cilia were significantly inhibited.The activity of PEMFs to increase osteogenic differentiation of ROBs disappeared,and the expression of IFG-1R and ERα increased.Through the above experimental results,it was found that 50 Hz and 0.6 mT PEMFs required the participation of primary cilia,ERα,IGF-1R/ERK1/2,and other signaling pathways in the process of maturation and mineralization of primary rat osteoblasts cultured in vitro.Therefore,the experimental results further demonstrate that the electromagnetic field receives the electromagnetic signal and activates the IGF-1R and ERα signaling pathways to further activate the downstream ERK1/2 signaling pathway to promote bone formation.This result laid the foundation for the primary cilia to EMFs to promote bone formation receptors,and also laid the foundation for the interpretation of EMFs in the treatment of osteoporosis.