Biomechanical Mechanisms Underlying The Improvement Of Obesity-induced Osteoporosis By Low-magnitude High-frequency Vibration

Background:In recent years,unhealthy lifestyles have become prevalent among residents in China,with dietary fat intake gradually increasing.More than half of the adult population is overweight or obese,with overweight and obesity rates reaching 34.3%and 16.4%,respectively.A high-fat diet(HFD)is identified as a primary factor contributing to obesity.Previous studies suggested that obesity could increase mechanical loading on bones,thus promoting bone formation and increasing bone mass.However,recent research indicates that obesity induced by a high-fat diet leads to fat accumulation and lipid abnormalities,resulting in decreased bone density,increased risk of fractures,and the onset of osteoporosis in obese individuals.Obesity also promotes differentiation of bone marrow mesenchymal stem cells into adipocytes,thereby reducing the number of osteoblasts and inhibiting bone formation.Currently,anti-osteoporosis medications are the main treatment for obesity-induced osteoporosis,but they come with certain side effects.For instance,the use of bisphosphonates such as zoledronic acid may cause fever,generalized bone and muscle pain,and in severe cases,kidney damage or osteonecrosis of the jaw.Exercise and dietary management are the primary non-pharmacological approaches for treating obesity-related osteoporosis.However,due to the increased risk of falls associated with weight gain in obese patients,motivation for exercise is often low.Therefore,there is an urgent need for novel,safe,effective,and minimally invasive non-pharmacological treatment modalities.Mechanical stimulation is a critical regulatory factor in bone growth and remodeling processes.Low-magnitude high-frequency vibration(LMHFV),as a form of mechanical stimulation,has become a current research hotspot due to its low cost,ease of implementation,and minimal side effects.Clinical studies have shown that LMHFV can increase bone density in the femoral neck and lumbar spine.Currently,LMHFV has been found to improve insulin resistance and lipid metabolism disorders in obese patients.However,whether LMHFV can improve obesity-induced osteoporosis has not been clearly reported.Therefore,LMHFV was used as a treatment for obesity-induced osteoporosis in the present study.By exploring the mechanical and biological mechanisms underlying the improvement of obesity-induced osteoporosis by LMHFV,this research aims to provide theoretical and experimental foundations for the clinical treatment of obesity-induced osteoporosis.Methods:In vitro experiments: 1.Palmitic acid(PA)was added to the complete culture medium of MC3T3-E1 cells to simulate the high-fat environment in obese patients.An in vitro treatment model was constructed by subjecting PA-treated MC3T3-E1 cells to high-frequency low-magnitude vibration(LMHFV).2.Cell viability and proliferation of MC3T3-E1 cells were determined using the CCK-8 assay.Apoptosis was detected using flow cytometry.3.Alkaline phosphatase(ALP)staining was performed to detect ALP activity in MC3T3-E1 cells.Alizarin Red S staining was used to assess the number and formation ability of mineralized nodules in MC3T3-E1 cells.5.The expression levels of proteins associated to osteogenesis in MC3T3-E1 cells were measured using Western blot(WB)analysis.6.RNA sequencing was performed on different groups of the experiment to screen Differentially expressed genes(DEGs).Functional enrichment analysis was conducted to identify core pathways closely related to DEGs.The intersection of DEGs between groups was determined,and core molecules affected by LMHFV under high-fat conditions were screened using protein-protein interaction methods.7.Small interfering RNA(si RNA)was used to construct an ITGβ1-inhibited cell model.In vivo experiments: 1.The obese rat model was established in rats through highfat diet feeding,followed by LMHFV application.2.Regular monitoring of rat body weight and blood lipid indicators was conducted during high-fat diet feeding and LMHFV application.3.Rats were anesthetized and their femurs removed following the end of their LMHFV therapy.Rat femur morphometric parameters and bone tissue morphology were evaluated using Masson’s trichrome staining,Micro-Computed Tomography(Micro-CT),and Hematoxylin-Eosin(HE).Rat femurs were stained with immunohistochemistry(IHC)to look for alterations in ITGβ1,p-FAK,p-PI3 K,and pAKT.Results:In vitro experiments: 1.In MC3T3-E1 cells,high-fat environments built with varying concentrations of PA all cause apoptosis,suppress the cells’ ability to proliferate,differentiate,and mineralize,and lower the expression of proteins linked to osteogenesis.The greatest impact was noted at 0.5 m M PA.2.LMHFV can suppress apoptosis in MC3T3-E1 cells under high-fat conditions and improve their proliferation,differentiation,and mineralization.3.After exposure to high-fat environments and LMHFV,some characteristic gene expression changes were observed in MC3T3-E1 cells,and DEGs were selected through bioinformatics analysis.4.The GO enrichment results of DEGs under high-fat conditions mainly include cell cycle regulation,integrin binding,regulation of apoptosis,and inflammatory response.The KEGG enrichment results mainly include the PI3K/AKT signaling pathway,JAK-STAT signaling pathway,and apoptosis-related pathways.After LMHFV application,the GO enrichment results of DEGs mainly relate to cell cycle,cell differentiation,integrin binding,and regulation of apoptosis.KEGG enrichment analysis shows that the PI3K/AKT signaling pathway is sensitive to LMHFV and closely associated with the treatment of obesity-induced osteoporosis by LMHFV.5.High-fat environments inhibit the ITGβ1-FAK/PI3K/AKT signaling pathway in MC3T3-E1 cells,and this inhibition is reduced after LMHFV application.6.In the high-fat environment,the mechanoreceptor ITGβ1 can sense the stimulation of LMHFV and activate its downstream FAK/PI3K/AKT signaling pathway to improve the proliferation,differentiation and mineralization of osteoblasts.In vivo experiments: 1.A high-fat diet induces weight gain and dyslipidemia in rats,whereas LMHFV improves both weight and lipid abnormalities in obese rats.2.An obesity-inducing high-fat diet results in a decline in the distal femoral metaphysis of rats’ trabecular bone density,microstructure,and morphology,but has no appreciable effect on cortical bone growth.Low-Magnitude,High-Frequency Vibration(LMHFV)has the potential to improve the morphology,microstructure,and trabecular bone density in the distal femoral metaphysis of obese rats.3.In skeletal tissue,LMHFV can activate the ITGβ1-FAK/PI3K/AKT signaling pathway.Conclusions:1.In a high-fat environment,osteoblast apoptosis significantly increases,and the biological behaviors of differentiation,proliferation,and mineralization are markedly inhibited.LMHFV can reduce the apoptosis rate of osteoblasts,increase their differentiation,proliferation,and mineralization,thereby promoting bone formation.2.In a high-fat environment,the ITGβ1-FAK/PI3K/AKT signaling pathway of osteoblasts is inhibited.ITGβ1 can sense the mechanical stimulation of LMHFV and activate the FAK/PI3K/AKT signaling pathway,thereby improving the biological behavior of osteoblasts.3.A high-fat diet induces weight gain and lipid abnormalities in rats,while simultaneously reducing trabecular bone density and disrupting trabecular bone microstructure and morphology,with no apparent effect on cortical bone.LMHFV can ameliorate obesity-induced weight gain and lipid abnormalities in rats,while also increasing trabecular bone density and improving trabecular bone microstructure and morphology.In rat bone tissue,LMHFV is capable of activating the ITGβ1-FAK/PI3K/AKT signaling pathway,significantly increasing the expression levels of key proteins within this signaling cascade.