Study On The Pulsed Electromagnetic Field Promotes Bone Formation Through PC1/NO Signaling Pathway In Primary Cilia

Osteoporosis(OP)is a systemic bone metabolism disease characterized by deterioration of bone tissue structure and decreased bone density,which leads to increased bone fragility.The pain caused by osteoporosis has brought a serious negative impact on the lives of patients,and the resulting osteoporotic fractures greatly increase the risk of death.At the same time,osteoporosis was caused significant economic losses to humans.However,there is no ideal control method.Pulsed electromagnetic field(EMF),as an non-invasive,safe and widely used physical therapy method in research and diagnosis and treatment,is considered to be one of the effective methods to prevent and treat osteoporosis.Unfortunately,the mechanism of pulsed electromagnetic field is still unclear,which greatly restricts its promotion and application in clinical treatment.Our groups has done a lot of research foundation in the early stage,and screened out the best parameters of pulsed electromagnetic field that can promote the mineralization and maturation of rat osteoblasts and increase the bone density of ovariectomized rats,including frequency,intensity and duration of action.Therefore,this thesis uses 0.5 Hz0.6 m T pulsed electromagnetic field to study the relationship between its promotion of bone formation and the NO signal pathway,polycystin-1 and primary cilia,and provides some corresponding molecular biology for the pulsed electromagnetic field to treat osteoporosis in accordance with.First,in order to study the relationship between the promotion of bone formation by pulsed electromagnetic fields and the NO signaling pathway,in this experiment,after treating osteoblasts with 50 Hz 0.6 m T PEMFs for different periods of time,the NO content in the cell culture medium increased significantly,and i NOS,e NOS,pe NOS,s GC and PKG-1 protein expression also increased significantly,and caused the nuclear translocation of PKG-1,suggesting that PEMFs promote bone formation may be related to the NO signaling pathway.Subsequently,nitric oxide synthase inhibitor L-NAME was used to block the NO signaling pathway,and it was found that the ability of pulsed electromagnetic fields to promote bone formation was significantly inhibited.It was show that PEMFs activate and depend on the NO signaling pathway in the process of promoting bone formation.Secondly,in order to study whether PEMFs activates the NO signaling pathway and promotes bone formation whether there is a connection with polycystin-1.Experiments found that after PEMFs were treated with osteoblasts for different periods of time,the protein expression of PC1 increased significantly,suggesting that PEMFs can stimulate the expression of PC1 in osteoblasts.Then the PC1 sh RNA plasmid was used to interfere with the expression of PC1 in osteoblasts,and it was found that PEMFs not only impaired the ability to promote bone formation,but also could no longer activate the NO signaling pathway.It was show that PEMFs first stimulated the expression of PC1 in the process of promoting bone formation,and then activated the entire NO signal pathway.Again,in order to study whether there is a direct connection between the NO signaling pathway,PC1 and primary cilia in PEMFs promoting bone formation.In this experiment,IFT88 sh RNA plasmid was used to interfere with the discovery of primary cilia in osteoblasts.It was found that the ability of PEMFs to promote bone formation was impaired,indicating that the presence of primary cilia was required for PEMFs to promote bone formation.In addition,it was found that after interfering with primary cilia,PEMEFs can no longer increase the NO content in cell culture fluid,and the PC1/NO signaling pathway related proteins PC1,i NOS,e NOS,p-e NOS,s GC and PKG-1 protein expression no longer increase.The m RNA expression of i NOS,e NOS,s GC and PKG-1 was no longer increased,and the nuclear translocation ability of PKG-1 also disappears.It was show that PEMFs activates the PC1/NO signaling pathway and depends on the primary cilia.At the same time,in order to study whether there is a direct connection between the PC1/NO signal pathway and the primary cilia.The immunofluorescence staining method was used to observe before and after PEMFs process osteoblasts,the key proteins PC1,i NOS,e NOS,p-e NOS and s GC of the PC1/NO signaling pathway were all located in the primary cilia.Although PKG-1 was not in the primary cilia under normal circumstances,But when PEMFs were treated with osteoblasts for 90 min,some protein had enter the primary cilia.It was show that the primary cilia participate in the process of pulsed electromagnetic field to promote the mineralization and maturation of osteoblasts,and the activation of the PC1/NO signaling pathway cannot be separated from the primary cilia,and there was a direct connection between the two.Finally,in order to study whether PEMFs had an effect on the morphology of primary cilia.In this experiment,after treated osteoblasts with 50 Hz 0.6 m T PEMFs for different times,the immunofluorescence staining method showed that the length of primary cilia was significantly longer and the incidence increased significantly.The protein IFT88 and acetylation α-related to primary cilia were the protein expression of tubulin also increased significantly,indicating that the primary cilia have undergone dynamic changes during the process of PEMFs promoting bone formation.In order to investigate whether the change of cilia length was related to the NO signaling pathway,L-NAME was used to block the NO signaling pathway.It was found that PEMFs could no longer increase the length of primary cilia,although it has no effect on the expression of IFT88 protein,but acetylation the protein expression of α-tubulin no longer increases.It was suggested that the elongation of cilia induced by PEMFs may be closely related to the activation of NO signaling pathway.In summary,50 Hz 0.6m T PEMF promotes the mineralization and maturation of osteoblasts by activating the PC1/NO signaling pathway,and this process depends on the primary cilia,which indicates that PEMFs promote bone formation through the PC1/NO signaling pathway in the primary cilia.This result lays the foundation for the primary cilia as the receptors for PEMFs to promote bone formation,and also provides the corresponding molecular biological basis for PEMFs to treat osteoporosis.