Untargeted Metabolomic Profiling of Cuprizone‐Induced Demyelination in Mouse Corpus Callosum by UPLC‐Orbitrap/MS Reveals Potential Metabolic Biomarkers of CNS Demyelination Disorders

Abstract

Multiple sclerosis (MS) is a neurodegenerative disorder characterized by periodic neuronal demyelination, which leads to a range of symptoms and eventually to disability. The goal of this research was to use UPLC-Orbitrap/MS to identify validated biomarkers and explore the metabolic mechanisms of MS in mice. Thirty-two C57BL/6 male mice were randomized into two groups that were fed either normal food or 0.2% CPZ for 11 weeks. The mouse demyelination model was assessed by LFB and the expression of MBP by immunofluorescence and immunohistochemistry. The metabolites of the corpus callosum were quantified using UPLC-Orbitrap/MS. The mouse pole climbing experiment was used to assess coordination ability. Multivariate statistical analysis was adopted for screening differential metabolites, and the ingenuity pathway analysis (IPA) was used to reveal the metabolite interaction network. We successfully established the demyelination model. The CPZ group slowly lost weight and showed an increased pole climbing time during feeding compared to the CON group. A total of 81 metabolites ( VIP > 1 and P < 0.05 ) were determined to be enriched in 24 metabolic pathways; 41 metabolites were markedly increased, while 40 metabolites were markedly decreased in the CPZ group. The IPA results revealed that these 81 biomarker metabolites were associated with neuregulin signaling, PI3K-AKT signaling, mTOR signaling, and ERK/MAPK signaling. KEGG pathway analysis showed that two significantly different metabolic pathways were enriched, namely, the glycerophospholipid and sphingolipid metabolic pathways, comprising a total of nine biomarkers. Receiver operating characteristic analysis showed that the metabolites (e.g., PE (16 : 0/22 : 6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)), PC (18 : 0/22 : 4(7Z, 10Z, 13Z, 16Z)), cytidine 5 ′ -diphosphocholine, PS (18 : 0/22 : 6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)), glycerol 3-phosphate, SM (d18 : 0/16 : 1(9Z)), Cer (d18:1/18 : 0), galabiosylceramide (d18:1/18 : 0), and GlcCer (d18:1/18 : 0)) have good discrimination ability for the CPZ group. In conclusion, the differential metabolites have great potential to serve as biomarkers of demyelinating diseases. In addition, we identified metabolic pathways associated with CPZ-induced demyelination pathogenesis, which provided a new perspective for understanding the relationship between metabolites and CNS demyelination pathogenesis.

Integrating Lipidomics and Transcriptomics Reveals the Crosstalk Between Oxidative Stress and Neuroinflammation in Central Nervous System Demyelination

“…Male C57BL/6 mice (8 weeks old) were purchased from Spelford (Beijing) Biotechnology Company. Mice were housed in an air-conditioned room at 22 ± 1 ° C with a 12-h light-dark cycle (lights on from 7:00 a.m. to 7:00 p.m.) ( Zhao et al, 2021 ). Food and tap water were freely available.…”

“…Twenty male C57BL/6 mice (8 weeks old) were randomly divided into the control group ( n = 10) and the cuprizone (CPZ) group ( n = 10), and mice in the CPZ group were fed by a diet containing 0.2% cuprizone (reagent purchased from Sigma, custom-made feed from Jiangsu Hershey Feeds). By referring to the literature ( Wellman et al, 2020 ), C57BL/6 male mice were selected to be fed 0.2% cuprizone chow to construct the demyelination model, and we chose continuous feeding (11 weeks) for the CPZ group ( Zhao et al, 2021 ). Then, mice were executed, and the corpus callosum was immediately stored at −80°C for subsequent detection of the targeted lipidomics and transcriptomics.…”

“…MS is a progressive, incurable disease that affects more than 2.5 million individuals globally ( Zhao et al, 2020 ). Patients with MS have a variety of neurological symptoms such as motor impairment, cognitive impairment, depressive tendencies, visual impairment, sleep disturbances, and anxiety tendencies, all of which negatively affect the quality of life and lead to a significant impact on medical and healthcare systems ( Shao et al, 2021 ; Zhao et al, 2021 ). However, to date, the comprehensive pathogenesis of MS remains unknown, and there is no effective therapy ( Meng-Ru et al, 2021 ).…”

“…Metabolomics widens our knowledge of small molecule metabolites, which are often considered as biomarkers, end products of cellular metabolism, while their regulatory roles in the genome, transcriptome, and proteome are often overlooked. Our prior investigation discovered an abnormal lipid metabolism in demyelinated central nerve tissue ( Zhao et al, 2021 ). At present, there is no obvious association between abnormal lipid metabolism and mitochondrial dysfunction, oxidative stress, and neuroinflammation in CNS demyelinated tissues.…”

Identifying Oxidized Lipid Metabolism-Related LncRNAs as Prognostic Biomarkers of Head and Neck Squamous Cell Carcinoma

“…ROS have physiological roles in normal quantities but harmful effects at high concentrations on cells. Oxidative stress is the harm that occurs when a living organism produces an excessive amount of ROS [ 13 , 14 , 15 ]. Increased levels of ROS can harm macromolecules, such as DNA, proteins, and lipids in a way that is “non-specific”.…”