Study On Protective Effects And Mechanism Of Low-magnitude High-frequency Vibration On Diabetic Osteoporosis

Background:Osteoporosis is one of the chronic complications caused by diabetes in the bone.Osteoporosis induced by diabetes is also called diabetic osteoporosis(DOP).Patients with diabetes manifest bone mineral density reduction,bone microstructural destruction,increased bone fragility and increased fracture risk as diabetes progresses.The pathogenesis of DOP is closely related to bone formation reduction and bone resorption increasing.Meanwhile,a high glucose environment in vitro could significantly inhibit osteoblast mediated bone formation and eventually lead to bone loss.At present,the treatment of DOP is mainly based on hyperglycemia controlling and anti-osteoporosis.However,insulin injection and oral hypoglycemic agents have been reported to increase fracture risk in diabetics.The anti-osteoporosis drug teriparatide has also been found to increase fasting blood glucose.Therefore,finding new,safe and effective supplementary or alternative therapies for DOP is of great significance.As a physical therapy,low-magnitude high-frequency vibration(LMHFV)has been widely used in treating and rehabilitating patients with muscular atrophy,osteoporosis and paralysis.As a form of mechanical stimulation in vitro,LMHFV could promote bone formation by stimulating osteoblast proliferation and differentiation.LMHFV could also reduce the risk of falls and improve the bone mineral density of patients with osteoporosis.Recently,LMHFV has been reported to improve metabolism,reduce blood glucose,and relieve the symptoms of peripheral neuralgia in patients with diabetes.However,it is still unclear whether LMHFV could increase bone mineral density and improve bone microstructural damage and bone fragility caused by diabetes.The present study has investigated the protective effect of LMHFV on DOP and the underlying mechanism was also explored.Through the analysis and comparison of experimental results in vitro and in vivo,we want to clarify the protective effect and mechanism of LMHFV in treating diabetic osteoporosis and provide more laboratory evidence for further clinical application of LMHFV.Methods:In vitro: 1.MC3T3-E1 cells induced by a high glucose environment were used as a cell model of bone formation inhibited by diabetes.LMHFV applied to high glucoseinduced MC3T3-E1 cells acted as a treatment model in vitro.2.The DNA synthesis of cells was detected by a 5-Ethynyl-2'-deoxyuridine(EDU)assay,and the cell cycle was detected by flow cytometry.3.The early differentiation of MC3T3-E1 cells was analyzed by the alkaline phosphatase(ALP)staining and ALP activity semiquantitative tests.The late differentiation of MC3T3-E1 cells was analyzed by alizarin red S(ARS)staining and mineralized nodule semi-quantitative test.4.The differentially expressed genes(DEGs)were screened by RNA-sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze the biological function and signal pathway of DEGs to clarify the biological behavior involved in the effect of LMHFV on MC3T3-E1 cells induced by high glucose.The critical molecules of LMHFV's effect on high glucose-induced MC3T3-E1 cells were determined by taking the intersection of DEGs from different groups and combining the changes in expression amount.5.Real-time quantitative PCR(RT-PCR)and Western blot were used to verify the key molecules under different treatment conditions.The changes of related molecules,including p-?-Catenin,?-Catenin,Cyclin D1 and Osterix,were also analyzed.6.According to the changing trend of key molecules under different treatment conditions,modified MC3T3-E1 cell lines with key molecules overexpression or knockdown were constructed.Specifically,the overexpression cell line would be constructed if the key molecule were increased under high glucose and decreased by LMHFV application.At the same time,if the molecule were decreased by high glucose and increased by LMHFV application,a knockdown cell line would be constructed.Then,previous experiments were performed to clarify the role of key molecules on LMHFV's effect in high glucose-induced MC3T3-E1 cells.In vivo: 1 Streptozotocin(STZ)induction was used to construct DOP rat models,and LMHFV application was used as treatment.2.During the treatment,the body weight and blood glucose of rats from different groups were measured.After treatment,the femurs of rats were collected for follow-up experiments.3.The bone mineral density and bone microstructure of the femur were detected by Micro-Compute Tomography(Micro-CT).4.Hematoxylin-eosin(HE)and Masson staining were used to analyze the morphology of femur tissue.5.Immunohistochemical stain(IHC)investigated the expression of key molecules and Osteocalcin(OCN)within bone tissue.6.Three points bending test was used to detect the mechanical properties of femurs.7.The matrix composition of cortical bone was detected by Raman spectroscopy.8.The surface microstructure of the cross-section of femur shafts was observed by scanning electron microscope(SEM)and atomic force microscope(AFM).Results:In vitro: 1.LMHFV alleviated the inhibitory effect of high glucose on the proliferation of MC3T3-E1 cells by promoting DNA synthesis and the transformation of the cell cycle from the G0/G1 phase to the S phase.2.LMHFV reduced the inhibitory effect of high glucose on the early and late differentiation of MC3T3-E1 cells by promoting the expression of ALP and the ability of mineralized nodule formation.3.After high glucose induction or LMHFV application,the mRNA of MC3T3-E1 cells changed,and DEGs were screened.4.Go enrichment analysis showed that the DEGs caused by high glucose were related to the regulation of osteoblast differentiation,ossification and endochondral ossification,while DEGs caused by LMHFV application were related to positive regulation of ossification and regulation of osteoblast differentiation.KEGG enrichment analysis showed that the DEGs caused by LMHFV application was mainly related to fluid shear stress and atherosclerosis and the Wnt signaling pathway.Dickkopf family 2(Dkk2)has been reported to be associated with fluid shear stress,atherosclerosis and the Wnt signaling pathway.The Dkk2 mRNA increased significantly by high glucose and decreased significantly after LMHFV application.Dkk2 acted as the potential target of the protective effect of LMHFV on MC3T3-E1 cells proliferation and differentiation inhibited by high glucose.5.High glucose resulted in the increased protein level of Dkk2 and p-?-Catenin and decreasing protein level of ?-Catenin,Cyclin D1 and Osterix in MC3T3-E1 cells,which was attenuated by the LMHFV application.6.On the one hand,Dkk2 overexpression inhibited the DNA synthesis and led to the transformation of the cell cycle from S and G2/M phase to G0/G1 phase.inhibiting the proliferation of MC3T3-E1 cells.On the other hand,Dkk2 overexpression inhibited the expression of ALP and the formation of mineralized nodules,which decreased the early and late differentiation of MC3T3-E1 cells.Moreover,Dkk2 overexpression increased p-?-Catenin while decreasing ?-Catenin,Cyclin D1 and Osterix protein levels.7.Dkk2 overexpression inhibited the promoting effect of LMHFV on the proliferation and differentiation of MC3T3-E1 cells in a high glucose environment.It also weakened its effects on Dkk2 and p-?-Catenin,?-Catenin,Cyclin D1 and Osterix protein expression.In vivo: 1.LMHFV treatment reduced blood glucose and increased body weight in STZ induced diabetic rats.2.LMHFV treatment increased bone mineral density,trabecular volume,number,thickness,and cortical bone thickness while reducing trabecular separation.3.LMHFV treatment promoted the recovery of the trabecular morphology in the femur of diabetic rats and increased collagen distribution within the trabecular.4.LMHFV treatment reduced the expression of Dkk2 while promoting the expression of OCN in femur tissue of diabetic rats.5.LMHFV treatment increased the maximum load and stiffness of the femoral shaft in diabetic rats.6.LMHFV treatment changed the composition of the bone matrix and increased the mineral content.7.LMHFV treatment influenced the cross-section of femur shafts' surface morphology,which change from coarse particles to fine particles.Conclusions:1.LMHFV could alleviate the inhibitory effect of high glucose on osteoblast mediated bone formation by promoting osteoblast proliferation and differentiation.2.The mechanism of the protective effect of LMHFV on bone formation inhibited by high glucose was related to Dkk2.LMHFV attenuated the increase of Dkk2 expression caused by high glucose,then increased ?-Catenin,Cyclin D1 and Osterix expression to promote the proliferation and differentiation of osteoblasts.3.LMHFV treatment had therapeutical effects on DOP by increasing bone mineral density,improving bone microstructure,enhancing bone mechanical properties and regulating bone matrix composition.LMHFV could also reduce the increase of Dkk2 expression induced by diabetes in femur tissue.