Pulsed Electromagnetic Field Promotes Maturation Mineralization Of Osteoblasts By Inhibiting TRPV4 Expression

Osteoporosis（OP）is a kind of systemic metabolic bone disease,which can lead to brittleness fracture and make patients disabled for life or even death.With the aging of the population,osteoporosis is bringing heavy economic burden and mental pressure to more and more patients and their families.At present,in addition to being expensive,first-line osteoporosis drugs often have obvious side effects.Pulse electromagnetic fields（PEMFs）,as a non-invasive and safe alternative therapy for osteoporosis,has been widely concerned.However,the mechanism of PEMFs is still unknown,which has hindered its clinical application.In our previous studies,it was found that primary cilia was indispensable for promoting the differentiation and maturation activity of osteoblasts,and Transient receptor potential vanilloid 4（TRPV4）,as a Ca²⁺channel localized in primary cilia,In order to further study the role of TRPV4 and lay a foundation for elucidating the mechanism of PEMFs,the following studies are mainly carried out in this paper.1.Osteoblasts were isolated,cultured and amplified from the skull of newborn Wistar rats.Osteoblasts were treated with 50Hz 0.6m T PEMFs to study the changes of TRPV4 channel protein expression in the process of promoting bone formation by PEMFs.2.Osteoblasts were treated with TRPV4 agonist GSK1016790A and PEMFs at the same time to observe the effect of osteogenic differentiation and mineralization maturation.3.sh RNA targeting IFT88 interfered with primary cilia to observe the effect of osteogenic differentiation;Osteoblasts were treated with TRPV4 blocker HC-067047 at the same time to observe the effects on osteoblast differentiation and mineralization.4.To detect the relationship between oxidative stress of osteoblasts and changes in TRPV4 and Ca²⁺concentrations under the above conditions;5.In this paper,immunofluorescence staining,Western blot,Real time RT-PCR and other techniques and methods were adopted.The results of this study are as follows:1.In the process of promoting osteogenic differentiation of osteoblasts by PEMFs,the expression of TRPV4 decreased significantly;If GSK1016790A was used to activate TRPV4 function,PEMFs promoting osteogenic differentiation disappeared.2.TRPV4 can be localized in the whole primary cilia.When primary cilia were inhibited by RNA interference,the expression of TRPV4 was significantly increased,while the promotion of osteogenic differentiation by PEMFs was significantly decreased.When osteoblasts were treated with HC-067047,the promotion of osteogenic differentiation of PEMFs was restored.3.High expression of TRPV4 caused by interference with primary cilia can lead to Ca²⁺overload and oxidative stress in osteoblasts,while preconditioning osteoblasts with HC-067047 or maintaining intact primary cilia can inhibit TRPV4 expression,avoid oxidative stress,and improve osteogenic differentiation and mineralization maturation of osteoblasts.This paper draws the following conclusions:1.Treatment of osteoblasts by PEMFs resulted in decreased intracellular expression of TRPV4,while activation of TRPV4 resulted in impaired osteogenic differentiation activity of PEMFs,suggesting that low expression of TRPV4 was conducive to osteogenic differentiation.2.Primary cilia can control the expression of TRPV4 and keep it at a low expression level.Interference with primary cilia can disrupt the control of TRPV4 expression and cause overexpression of TRPV4,thus affecting the ability of PEMFs to promote osteogenic differentiation.3.High expression of TRPV4 can increase intracellular Ca²⁺concentration,lead to Ca²⁺overload,oxidative stress and damage osteogenic differentiation ability.PEMFs promotes osteogenic differentiation and mineralization of osteoblasts by inhibiting TRPV4 expression.This paper proposes the mechanism of PEMFs promoting bone formation from a new perspective,and provides an experimental basis for promoting the application of PEMFs therapy for osteoporosis.