| BackgroundOsteonecrosis of the femoral head(ONFH)is a disease that can lead to collapse of the femoral head and then hip disability,of which,30%-70%of cases have necrosis,which seriously affects the lives of patients and brings great economic burden to society.However,the exact pathogenesis of ONFH remains unclear,and most patients eventually require total hip arthroplasty(THA).Many studies have confirmed that exosomes play a positive role in osteoarticular diseases.And all cells in bone tissue can secrete exosomes,and the loading of their contents is determined by the status of cells in bone tissue.Therefore,changes in the contents in exosomes can reflect physiological changes in bone tissue cells.Currently,there are no studies on metabolite changes in vivo in ONFH exosomes.Metabolomics is the most commonly used technique to analyze metabolite changes,which is based on analytical chemistry techniques to accurately and efficiently characterize or quantitatively analyze metabolites.Therefore,ultra-high performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS)was used to study the metabolic characteristics of bone tissue exosomes in osteonecrosis of the femoral head,which helped us to discover new metabolic changes in the disease.ObjectiveBy analyzing the metabolic characteristics of exosomes derived from osteonecrosis of the femoral head,the metabolic changes of ONFH disease were explained,and the metabolic pathways with important changes were explored to provide a theoretical basis for studying the pathogenesis and treatment of ONFH.Methods1.Thirty osteonecrosis of the femoral head(ONFH)bone tissue and30 femoral neck fracture(FNF)bone tissue samples were collected.And X-ray,magnetic resonance imaging(MRI),and histological examination were performed in each patient to confirm the diagnosis.2.After grinding the femoral head tissue samples,the exosomes were extracted using ultracentrifugation.The extracted exosomes were identified by dynamic light scattering(DLS),Western blot and transmission electron microscopy(TEM).3.The metabolic profiles of exosomes of ONFH were analyzed by ultra-high performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS).And principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)were performed on the metabolic data to analyze the metabolic profiles of exosomes and find the metabolites with significant changes.4.The metabolites with the most significant changes were selected as potential diagnostic markers,and receiver operating characteristic curve(ROC curve)analysis was performed to evaluate their diagnostic significance.5.Metabolites with obvious changes were analyzed by Metaboanalyst database,so as to determine their metabolic pathways and visualize them.Results1.30 ONFH bone tissue and 30 FNF bone tissue samples were successfully collected.The X-ray,magnetic resonance imaging(MRI)and histological findings of each patient were compared to confirm the correct diagnosis.2.The results of DLS showed that most of these exosomes were between 30 nm and 150 nm in size.TEM analysis showed that the exosomes were double-layered lipid-wrapped and disk-like in shape,with a size of about 100 nm.Western blot showed significant enrichment of CD9,TSG101,CD63,and ALIX,markers of exosomes.The above results indicate that exosomes in tissues have been successfully isolated.3.The data obtained from the exosome samples after analysis by the above conditions were processed to obtain PCA maps.The results showed that the components of exosomes from ONFH and those from FNF had a certain tendency to separate dimensionally.OPLS-DA uses partial least squares regression to establish a relationship model between metabolite expression and sample class to achieve prediction of sample class.The model evaluation parameters R~2Y and Q~2were 0.749 and 0.724,respectively,and both R~2Y and Q~2were greater than 0.5,indicating that the model was stable and reliable.4.Bioinformatics analysis of differential metabolites showed that 45differential metabolites were detected between exosomes derived from ONFH and FNF.By used the regression of binary logistic,we selected lysine,lysophospholipids,proline,and phosphatidic acid as potential biomarkers.5.Metabolic pathway analysis showed that riboflavin metabolism,sphingolipid metabolism,and glycerophospholipid metabolism were most significantly altered in exosomes from ONFH.ConclusionIn our study,PCA and OPLS-DA models were constructed by metabolomic analysis of exosomes extracted from tissues of ONFH patients;a total of 45 differential metabolites were identified;four potential biomarkers(lysine,lysophospholipids,proline,and phosphatidic acid)were identified;metabolic pathway analysis showed that riboflavin metabolism,Pantothenate and Co A biosynthesis,and glycerophospholipid metabolism were most significantly altered in exosomes from ONFH. |
