基于超高效液相色谱-串联质谱的股骨头坏死组织外泌体脂质代谢组学分析

Lipid metabolomic analysis in exosomes of osteonecrosis of the femoral head based on ultra performance liquid chromatography-tandem mass spectrometry

股骨头坏死(ONFH)是一种可导致股骨头塌陷进而需要接受全髋关节置换的疾病。外泌体作为一种细胞间交流的方式,在一系列生理和病理过程中起着至关重要的作用,已在疾病的诊断和治疗中发挥独特作用。该研究利用非靶向代谢组学方法,探讨股骨头坏死组织外泌体内的脂质代谢特征,阐释股骨头坏死时机体发生的脂质代谢变化。该研究采用超速离心的方法,对股骨头坏死组织的外泌体进行了分离富集,并使用动态光散射(DLS)、蛋白质免疫印迹和透射电子显微镜(TEM)3种方法鉴定外泌体。采用超高效液相色谱-串联质谱(UPLC-MS/MS)结合多变量统计分析识别股骨头坏死外泌体的脂质代谢谱。采用主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)对差异表达的外泌体脂质代谢物进行多变量统计分析。在外泌体中检测到18种明显改变的脂质代谢物,包括丙烯醇酯类、脂肪酸酯类、甘油酯类及其衍生物。通过代谢分析网站进行通路分析,从而确定受影响的代谢通路并进行可视化。代谢通路分析显示外泌体内的甘油磷脂代谢和鞘脂代谢改变最为明显,鞘脂和甘油磷脂之间的不平衡导致脂肪毒性损伤,这与常见代谢性疾病的病理生理学有关。同时,甘油磷脂与细胞增殖、分化和凋亡之间具有相关性,甘油磷脂比例的变化可以反映脂质代谢的紊乱。外泌体内的脂质代谢变化可能在一定程度上反映了ONFH疾病的代谢变化。ONFH外泌体脂质代谢组学分析可有助于探索坏死骨组织外泌体中的脂质代谢变化和受影响的脂质代谢通路。

Osteonecrosis of the femoral head(ONFH)can lead to its collapse which requires total hip arthroplasty.Exosomes,which are important for intercellular communication are involved in a series of physiological and pathological processes,and therefore play a unique role in disease diagnosis and treatment.In this study,untargeted metabolomics was used to investigate the metabolic characteristics of lipids in exosomes of femoral head tissue with osteonecrosis and to explain the metabolic changes that occur in the body during this disease.Ultracentrifugation was used to separate and enrich exosomes from femoral head tissue with osteonecrosis.Exosomes were identified using dynamic light scattering(DLS),Western blotting,and transmission electron microscopy(TEM).Gradient elution was performed with ultrapure water and acetonitrile as mobile phases using a Kinetex XB-C18 column(100 mm×2.1 mm,2.6μm).The column oven temperature,flow rate of the mobile phase,and duration were 30℃,300μL/min,and 15 min,respectively.A triple TOF 4600 high resolution mass spectrometry system was used,and the mass scan range of m/z was set at 100-1000.Other conditions were as follows:sheath gas,380 kPa;auxiliary gas,380 kPa;curtain gas,170 kPa;and atomization temperature,600℃.Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)combined with multivariate statistical analysis was used to identify the lipid metabolic profile of ONFH-derived exosomes.The exosome metabolites were characterized in detail,which enables their identification and provided a reliable method for quality evaluation.After transforming the obtained original data using MarkView software,peak identification,peak alignment,subtraction of solvent peak,impurity peak,noise filtering,and other treatments,a three-dimensional matrix was obtained from the exported data table.Principal component analysis(PCA)and orthogonal partial least squares discrimination analysis(OPLS-DA)in the SIMCA-P14.1 software were used for multivariate statistical analysis of differentially expressed exosome lipid metabolites.This strategy was validated using lipid metabolites from patients with ONFH and healthy controls.The correlation distribution was shown according to the point dispersion of the PCA score plot,and lipid metabolites from the same disease showed ideal clustering.This result indicates a small difference between the groups.A good clustering effect is also obtained using OPLS-DA,and the statistical model has high reliability.A total of 18 significantly altered lipid metabolites were detected in the exosomes,including acrylolipids,fatty acid esters,glycerides,and their derivatives.The pathway analysis was conducted with MetaboAnalyst(https://www.metaboanalyst.ca/)via database source including the HMDB(http://www.hmdb.ca/)and MMCD(http://mmcd.nmrfam.wisc.edu/)for confirming the impacted metabolic pathways and visualization.Metabolic pathway analysis showed that glycerophospholipid and sphingolipid metabolism were the most significantly altered in exosomes.An imbalance between sphingolipids and glycerophospholipids leads to lipotoxic damage,which is implicated in the pathophysiology of common metabolic diseases.Furthermore,glycerophospholipids are correlated with cell proliferation,differentiation,and apoptosis,and the change in glycerophospholipid ratio can reflect the disturbance in lipid metabolism.The metabolic changes in exosomes may reflect the metabolic changes in ONFH.In this study,lipid metabolomics analysis based on UPLC-MS/MS was used to determine metabolic differences between exosomes extracted from ONFN and femoral neck fracture(FNF).Metabolomic analysis of necrotic femoral head tissue-derived exosomes can help explore the most relevant pathways for assessing the changes in exosome metabolism that affect exosome metabolism in necrotic bone tissue.