Study On The Molecular Mechanism Of Low-Magnitude High-Frequency Vibration(LMHFV) On Diabetic Osteoporosis

Background:Diabetes mellitus(DM)is a common chronic metabolic disease characterized by elevated blood glucose in the internal environment.It has dozens of complications,including osteoporosis.Hyperglycemia can affect the biological behavior of osteoblasts and osteoclasts,destroy the dynamic balance between bone formation and resorption,thereby reducing bone mass,increasing bone fragility,eventually leading to the occurrence of osteoporosis,which is called DOP(Diabetic Osteoporosis).At present,the treatment strategy of DOP is based on the treatment of hyperglycemia,combined with other treatment methods such as anti-osteoporosis,diet management and exercise.However,hyperglycemia therapies(insulin,thiazolidinediones,and sulfonylureas drugs)may decrease bone mineral density and increase the risk of fractures.Teriparatide,an anti-osteoporosis drug,has been reported to be associated with elevated fasting blood glucose.Although exercise can regulate blood glucose and antiosteoporosis,it increases the risk of falls and some obese patients have a low willingness to exercise.Low-magnitude High-frequency Vibration(LMHFV)is a "passive" form of exercise.Patients only need to stand on the mechanical vibration table with handrail,which is safer than traditional exercise.Due to the vibration characteristics of high frequency and low load(20-90 Hz,acceleration <1g),LMHFV can cause mechanical effects on the body and avoid the damage to human health.Our group recently found that LMHFV could decrease blood glucose level,improve bone mineral density,bone microstructure and mechanical properties of the femur in diabetic rats.However,the underlying mechanisms of LMHFV in treating diabetic osteoporosis remain unclear,the effects of LMHFV on osteoblasts and osteoclasts under high glucose environment and the underlying molecular mechanisms need to be further investigated.Methods:In vitro: 1.Osteoblasts:(1)MC3T3-E1 cells were cultured in high glucose medium to establish a cell model of osteoblasts in high glucose environment;LMHFV is applied to construct a LMHFV treatment model of osteoblasts in high glucose environment.(2)CCK8 assay was used to detect the cell viability of MC3T3-E1 cells.The cytoskeleton of MC3T3-E1 cells was observed using phalloidin staining.(3)Western Blot(WB)and Enzyme-linked Immunosorbent Assay(ELISA)were used to detect the expression of osteogenesis related proteins in MC3T3-E1 cells.(4)Alkaline Phosphatase(ALP)staining and Alizarin Red S(ARS)staining were used to analyze the differentiation and mineralization of MC3T3-E1 cells.(5)The m RNA sequencing technology was used to detect the gene expression of MC3T3-E1 cells.The limma package of R language was used to screen Differentially Expressed Genes(DEGs)between groups.Function and pathway enrichment analysis and protein-protein interaction analysis of the DEGs were performed to explore the key signaling pathways and genes which is sensitive to LMHFV.(6)Immunofluorescence assay was used to observe the morphology,positive rate and length of primary cilia in MC3T3-E1 cells.IFT88 sh RNA was used to construct the MC3T3-E1 cell model with primary cilia knockout.2.Osteoclasts:(1)RAW264.7cells were cultured in high glucose medium to construct a cell model of osteoclasts in high glucose environment;LMHFV is applied to construct a LMHFV treatment model of osteoclasts in high glucose environment.(2)RANKL was used to induce RAW264.7cells to differentiate into osteoclasts.Mature osteoclasts were detected by Tartrateresistant Acid Phosphatase(TRAP)staining.(3)WB and ELISA were used to detect the expression of osteoclasts-related proteins.Bone resorption test was used to detect the bone resorption ability of osteoclasts.Transwell assay was used to detect the migration ability of RAW264.7 cells.(4)The RAW264.7 cell model with MMP9 overexpression was constructed by lentivirus transfection.(5)Discovery Studio was used to evaluate the crystal structure of MMP9 protein,and pharmacophore analysis was performed on highly selective antagonists of MMP9.Shordinger was used for virtual docking analysis of MMP9 crystal structure and MMP9’s highly selective antagonist.Molecular dynamics simulation analysis was performed using GROMACS software.In vivo:(1)Streptozocin(STZ)was used to establish a diabetic rat model and LMHFV was applied to treat the diabetic rat.(2)Body weight was measured using a weight scale,and blood glucose was measured using a glucometer.(3)The bone mineral density and microstructure of proximal tibia of rats were analyzed by micro-CT scanning.(4)Hematoxylin-Eosin(HE)and Masson staining were used to observe the bone histomorphology of the proximal tibia of rats.(5)Immunohistochemistry(IHC)staining was used to detect the expression of PI3 K,AKT,TRAP and MMP9 in tibial tissue of rats.Results:In vitro: 1.Osteoblasts:(1)High glucose environment could inhibit the activity of MC3T3-E1 cells,reduced the expression level of osteogenic related proteins and inhibited cell differentiation,and the effect of 25.5mmol/L glucose concentration was significant.LMHFV could improve the differentiation,maturation and mineralization of MC3T3-E1 cells under high glucose environment,and the effect was best after 30 minutes of LMHFV.(2)Differential expression analysis showed that 150 and 147 DEGs were regulated by LMHFV loading in normal and high glucose environment,respectively.LMHFV was closely related to PI3K/AKT signaling pathway.MMP9 expression was sensitive to LMHFV.PI3K/AKT signaling pathway was involved in the development of diabetes and osteoporosis.(3)Primary cilia could be observed on MC3T3-E1 cells,and high glucose environment can reduce the length of primary cilia.LMHFV could activate PI3K/AKT signaling pathway,which was inhibited in MC3T3-E1 cells under high glucose environment.(4)IFT88sh RNA could knock out the primary cilia of MC3T3-E1 cells,and the primary cilia did not affect the biological behavior and PI3K/AKT signaling pathway of MC3T3-E1 cells without mechanical loading.(5)LMHFV activates of PI3K/AKT signaling pathway under high glucose required the presence of primary cilia.LMHFV could promote the differentiation,maturation and mineralization of MC3T3-E1 cells under high glucose environment through the primary cilia-PI3K/AKT signaling pathway.2.Osteoclasts:(1)RANKL could induce RAW264.7 cells to differentiate into mature osteoclasts.(2)Osteoclasts differentiation and maturation,bone resorption and pre-osteoclasts migration were inhibited by LMHFV.LMHFV reduced the protein expression of MMP9 and regulated the TRAF6-ERK/p38 MAPK signaling pathway which is upstream of MMP9.(3)MMP9overexpression could promot osteoclasts differentiation and maturation,enhance bone resorption,while LMHFV inhibited osteoclasts differentiation by reducing MMP9 expression.(4)JNJ0966 and MMP9-IN-1,the highly selective antagonists of MMP9,could successfully dock with the crystal structure of MMP9 protein with docking scores of-6.629 and-8.618.Molecular dynamics simulations showed that the complex formed by MMP9 and its highly selective antagonist was stable.(5)JNJ0966 and MMP9-IN-1,highly selective antagonists of MMP9,inhibited osteoclasts differentiation and maturation,bone resorption and pre-osteoclasts migration.In vivo:(1)LMHFV could reduce blood glucose and alleviate the weight loss in diabetic rats.(2)The Bone Mineral Density(BMD),Bone Volume/Tissue Volume(BV/TV),Trabecular Number(Tb.N),and Trabecular Thickness(Tb.H)in proximal tibia of diabetic rats were decreased,the Trabecular Separation(Tb.Sp)was increased.LMHFV could improve the bone microstructure in the proximal tibia of diabetic rats.(3)HE staining and Masson staining showed that the morphology of bone in the proximal tibia of diabetic rats was damaged,and LMHFV could against this damage.(4)LMHFV increased the expression of PI3 K and AKT in the tibia of diabetic rats.(5)LMHFV inhibited the expression of TRAP and MMP9 in tibial tissue of diabetic rats.Conclusion:1.The high glucose environment of 25.5mmol/L glucose have a significant inhibitory effect on the differentiation,maturation and mineralization of osteoblasts.LMHFV could improve the biological behavior of osteoblasts under high glucose condition,and the effect was best when loaded for 30 minutes.2.Primary ciliaPI3K/AKT signaling pathway is involved in LMHFV to improve the biological behavior of osteoblasts under high glucose environment.3.LMHFV inhibite osteoclasts differentiation and maturation,bone resorption,and pre-osteoclasts migration.MMP9 is involved in the inhibitory effect of LMHFV on osteoclasts.4.LMHFV can reduce blood glucose,alleviate weight loss,and improve bone mineral density,bone microstructure and bone histomorphology in the tibia of diabetic rats.