Experimental Study Of Pulsed Electromagnetic Field Against Radiation Bone Injury Through Primary Cilia-Ku70 Signal Cascade In Osteoblasts

Tumor is one of the most common diseases that threatens human life for a long time.Radiotherapy is the main clinical treatment of tumor(especially malignant tumor).About 7million cancer patients worldwide receive radiation treatment each year.Although the development of technology has improved the precision and targeting of radiotherapy,it is remains difficult to avoid the adverse impact on the surrounding tissues and consequently reduce the quality of life of patients.Bone tissue is one of the most common sites of injury in radiotherapy.Radiation can lead to continuous loss of bone mass and deterioration of bone mechanical properties,increasing the risk of osteoporotic fractures and osteonecrosis.With the development of tumor diagnosis and treatment technology,the cure rate of tumor is significantly improved,and the survival period of patients is significantly prolonged,which also makes the radiation bone injury(an important long-term complication of radiation therapy)attract more and more attention of scientists and clinical workers.However,the mechanisms of radiation bone injury remain unclear,and there is still a lack of effective and safe methods to prevent and treat radiation bone injury.Pulsed electromagnetic field(PEMF)plays an important role in maintaining the physiological functions of bone tissue and bone cells,and exogenous PEMF stimulation can significantly accelerate fracture healing.PEMF,with its unique advantages of safety and simplicity,has become an important means for the treatment of clinical orthopedic diseases(osteoporosis,fracture healing,etc.).Our previous study found that PEMF stimulation can repair bone damage in type 1 diabetes mellitus.However,whether PEMF stimulation can attenuate radiation-induced bone injury and the underlying mechanisms against radiationinduced bone injury still remain unclear.In this study,an animal model of local radiationinduced bone injury(total radiation dose of 8 Gy,1.6 Gy/min irradiation for 5 min)was constructed to investigate the effects of PEMF stimulation on bone anabolism and bone injury repair in radiation-induced bone injury animals.Then,in vitro experiments were conducted to investigate the effects of PEMF stimulation on the proliferation,apoptosis,differentiation and mineralization of primary osteoblasts(OB)after radiation injury,as well as the expression of osteogenic genes and proteins.The effects of PEMF stimulation on the activity and function of osteocytes(OCY),osteoclasts(OC)and tumor cells(MCF7,SW620,A375,143B)after radiation were systematically studied.Finally,the associated molecular mechanism of the attenuating effects of PEMF stimulation on radiation-impaired OB was clarified by gene silencing technique.This study proposes that PEMF stimulation can promote bone anabolism and accelerate bone repair to resist radiation bone injury,and explores the relevant transduction mechanism.This study lays a critical scientific foundation for the extensive clinical application of PEMF stimulation in the treatment of patients with radiation bone injury in the future,and has important clinical and scientific value.The whole study is mainly divided into the following four parts:Part Ⅰ: In vivo experimental study of PEMF against radiation-induced bone loss Background: Radiation therapy of tumor patients increases the risk of osteoporotic fractures and may lead to osteonecrosis.Therefore,it is of great clinical value to explore safe,effective and highly targeted interventions for radiation bone injury based on the promotion of osteogenesis.Low-intensity PEMF treatment can prevent and treat osteoporosis,nonunion and delayed fracture union,suggesting that PEMF plays a key role in maintaining the physiological functions of bone tissue and cells.However,whether PEMF stimulation can counter the bone injury caused by radiotherapy has not been reported at home and abroad.Methods: The rat model of radiation bone injury was established by applying local radiation to the right hind limb of rats with radiotherapy equipment.The rats in PEMF group were subjected to systemic PEMF stimulation for 45 days.The bone mass,bone microstructure,bone histomorphology,bone anabolism,the activities and functions of OB,OC and OCY were analyzed by micro-CT,H&E staining,immunofluorescence staining,calcein fluorescence double standard,immunohistochemistry,TUNEL staining,scanning electron microscopy,TRAP staining,serum ELISA and nanoindentation test.Results: After radiation injury,the number of trabecular bone,bone microstructure and mechanical properties were significantly damaged,the expression of serum OCN and P1 NPwere significantly decreased and the expression of serum CTX-1 and TRACP-5B were significantly increased.In addition,the levels of γ-H2 AX and Cbfα1 in femur OB,and the number of OB was significantly decreased.The activity of OC was increased,and the morphology of OCY was significantly damaged.PEMF stimulation significantly reduced the expression of γ-H2 AX,and improved the number,microstructure and mechanical properties of trabecular bone in rats after radiation injury.However,PEMF stimulation not only promoted the expression of Cbfα1 in OB and inhibited OB apoptosis after radiation injury,but also significantly increased the expression of OCN and P1 NP in serum.However,PEMF stimulation had no significant effect on OC activity and OCY morphology of rat femur after radiation.No significant changes were observed in serum CTX-1 and TRACP-5B.Conclusion: Radiation injury destroys bone microstructure and material properties by inhibiting bone formation and promoting bone absorption.Through inhibiting DSB,promoting osteogenesis and inhibiting the apoptosis of OB,PEMF stimulation promotes bone formation and improves bone microstructure and material properties to resist radiation bone injury.This study provides a basis for the clinical application of PEMF stimulation for osteopenia and bone injury induced by radiation.Part Ⅱ: In vitro experimental study on the regulation of PEMF against radiation in bone cells Background: When bone resorption and bone formation are in dynamic balance,bone can maintain its structural and functional integrity.When the bone homeostasis is destroyed,bone microstructure will be impaired.The results of the first part showed that PEMF stimulation could significantly increase the rate of bone formation to resist radiation bone injury,and had no significant inhibitory effect on bone resorption.Whether PEMF antagonizes the development of radiation bone injury by activating OB has not been reported yet.Methods: PEMF generator developed by our research group was used to apply PEMF stimulation to the radiation-impaired bone cell model.OB for in vitro experimental studies was obtained from tissue blocks.Immunofluorescence staining,ALP staining,ALP activity analysis,Alizarin red S staining,qRT-PCR and Western Blotting were used to analyze the expression of γ-H2 AX,differentiation,mineralization and osteogenesis of OB in each group.The expression levels of OCY cytokines in each group were analyzed by qRT-PCR,and the activity of OC was analyzed by TRAP staining.Results: The results of in vitro experiments showed that radiation significantly increased the expression of γ-H2 AX,inhibited the differentiation,mineralization and osteogenic related gene and protein expression of OB,and significantly increased OC activity,whereas radiation had no significant effect on the OCY cytokine secretion.After PEMF stimulation,the expression of γ-H2 AX in radiation-injured OB decreased,and the differentiation,mineralization and expression of osteogenic related genes and proteins increased significantly,but OC activity and OCY cytokine secretion levels remained unchanged.Conclusion: PEMF stimulation can specifically inhibit DSB in OB after radiation bone injury,thus improving the ability of differentiation and mineralization,and promoting the expression of genes and proteins related to bone formation in OB.However,PEMF stimulation has no significant effect on the activities of OCY and OC after radiation,indicating that OB is highly sensitive to PEMF.It is revealed that PEMF can specifically regulate OB to attenuate radiation bone injury.This study not only enriches the understanding about PEMF against radiation bone injury,but also prompts PEMF as a safe,non-invasive,accessible and low-cost method,in addition to being countermeasures against bone loss during space flight,may also be a promising method against bone loss and increased risk of bone fracture induced by clinical radiotherapy.Part Ⅲ: The experimental study of differential expression of primary cilia between osteoblasts and tumor cells mediated PEMF specificity against radiation bone injury Background: Primary cilia are sensory organelles on the surface of OB,capable of sensing and transducing external mechanical and electromagnetic stimuli to regulate intracellular signal transduction.In addition,primary cilia are specifically expressed in OB,which is lacking in many tumor cells.The results of the previous part prove that PEMF,as a physical factor,can specifically activate OB to promote bone formation and alleviate radiation bone injury.However,whether OB is stimulated by external PEMF through the differential expression of primary cilia between osteoblasts and tumor cells to resist radiation bone injury needs further investigation.Methods: PEMF generator was used to apply PEMF stimulation to the in vitro model of radiation-damaged bone cells.OB was extracted by tissue block method,and the expression of primary cilia in OB and tumor cells was detected by immunofluorescence.The proliferation and apoptosis of OB and tumor cells(MCF7,SW620,A375,143B)were analyzed by MTT and flow cytometry.shRNA inhibited the expression of primary cilia,and ALP staining,ALP activity analysis,Alizarin red S staining,qRT-PCR and Western Blotting were used to analyze the expression ability of differentiation,mineralization and osteogenic factors of OB.Results: Results showed that primary cilia were specifically expressed in OB,and there was no significant expression of primary cilia in tumor cells.The results also showed that radiation significantly inhibited the activity of OB and tumor cells and promoted the apoptosis of OB and tumor cells.After PEMF stimulation,the activity of OB was increased and the apoptosis of OB was decreased,whereas there was no significant effect on tumor cells.PEMF stimulation significantly inhibited the apoptosis of OB,increased the proliferation,differentiation,mineralization and osteogenic related gene and protein expression of OB after radiation.After primary cilia gene silence,PEMF stimulation had no significant effect on the proliferation,differentiation,mineralization and gene and protein expression of osteogenesis in OB.Conclusion: OB regulates the function and activity of OB through the differential expression of primary cilia between OB and tumor cells,and specifically responds to PEMF stimulation to attenuate radiation bone injury.This study enriches the understanding that primary cilia play an important role in PEMF against radiation bone injury,lays a foundation for exploring the related mechanisms,and provides a key scientific basis for the extensive clinical application of PEMF stimulation in the treatment of radiation bone injury in the future.Part Ⅳ: Experimental study of PEMF specifically against radiation bone injury through cascade of “primary cilia-NF-κB-Ku70” signaling in osteoblasts Background: The above results have confirmed that PEMF stimulation can mediate OB activity and inhibit DSB to resist radiation bone injury.Because OB specifically express primary cilia,OB can sense external PEMF stimulation to alleviate radiation bone injury.However,how OB induces DSB repair through primary cilia sensing external PEMF stimulation has not been systematically elucidated.Ku70 is a major protein in DSB repair,and its expression is increased under stress(e.g.,radiation injury).In addition,it has been found that the expression of Ku70 is regulated by NF-κB.Therefore,we hypothesized that PEMF regulates Ku70 by stimulating primary cilia in OB to activate NF-κB entering into the nucleus and thus repair DNA damage to counter radiation bone injury.Methods: Immunofluorescence was used to analyze the effects of PEMF stimulation on the expression of Ku70 in OB after radiation bone injury.Flow cytometry,ALP staining,ALP activity assay,Alizarin red S staining,qRT-PCR and Western Blotting were used to analyze the changes of differentiation,mineralization and apoptosis of OB after IFT88 gene inhibition,as well as the expression levels of osteogenic genes and proteins.The expression of NF-κB in OB after IFT88 gene inhibition was analyzed by immunofluorescence technique.Western Blotting was used to analyze the expression of Ku70 in OB after NF-κB inhibition.Results: Firstly,it was determined that PEMF stimulation activated Ku70 in OB after radiation injury.When Ku70 was inhibited,the positive effects of PEMF stimulation on osteoblastic differentiation,mineralization and osteogenic related protein expression was also significantly inhibited.After silencing primary cilia,the nuclear NF-κB expression in OB was also inhibited.After NF-κB inhibtion,PEMF stimulation significantly inhibited Ku70 expression in OB after radiation injury.Conclusions: We found that PEMF stimulation regulates the function and activity of OB through primary cilia-NF-κB-Ku70 signal cascade in osteoblasts to resist radiation bone injury.This study can further enrich understanding of radiation bone injury and mechanisms of bone electromagnetic signal transduction,provides a key scientific basis for the wide application of PEMF stimulation in the treatment of radiation bone injury,which has important clinical and scientific research value.