Metabolomic Analysis of Trehalose Alleviating Oxidative Stress in Myoblasts

Abstract

Trehalose, a naturally occurring non-toxic disaccharide, has attracted considerable attention for its potential in alleviating oxidative stress in skeletal muscle. In this study, our aim was to elucidate the metabolic mechanisms underlying the protective effects of trehalose against hydrogen peroxide (H₂O₂)-induced oxidative stress in C2C12 myoblasts. Our results show that both trehalose treatment and pretreatment effectively alleviate the H₂O₂-induced decrease in cell viability, reduce intracellular reactive oxygen species (ROS), and attenuate lipid peroxidation. Furthermore, using NMR-based metabolomics analysis, we observed that trehalose treatment and pretreatment modulate the metabolic profile of myoblasts, specifically regulating oxidant metabolism and amino acid metabolism, contributing to their protective effects against oxidative stress. Importantly, our results reveal that trehalose treatment and pretreatment upregulate the expression levels of P62 and Nrf2 proteins, thereby activating the Nrf2-NQO1 axis and effectively reducing oxidative stress. These significant findings highlight the potential of trehalose supplementation as a promising and effective strategy for alleviating oxidative stress in skeletal muscle and provide valuable insights into its potential therapeutic applications.

Keywords: Keap1-Nrf2; NMR-based metabolomics; oxidative stress; skeletal muscle; trehalose.

Figure 1

Trehalose treatment and pretreatment protected C2C12 cells against the H₂O₂-induced decrease in cell viability. (A) Cell viability after treatment with different concentrations of trehalose (n = 5). (B) Representative morphological images of the C, T, H, and HT cells. Scale bar, 100 μm. (C) Cell viability after treatment with H₂O₂ (200 μM) and trehalose at different concentrations (n = 5). (D) Cell viability after treatment with H₂O₂ (200 μM) and pretreatment with trehalose at 10 mM (n = 5). (E) Representative morphological images of the pC, pH, and pHT cells. Scale bar, 100 μm. The concentrations of H₂O₂ and trehalose were 200 μM and 10 mM, respectively. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2

Trehalose treatment and pretreatment promoted the scavenging of H₂O₂-induced ROS in C2C12 cells. (A) Representative confocal microscopy image of H₂DCFDA-stained C, T, H, and HT cells. Scale bar, 50 µm. (B) Intracellular ROS levels of the C, T, H, and HT cells detected by oxidized H₂DCFDA fluorescence signals (n = 4). (C) Intracellular MDA levels of the C, T, H, and HT cells measured by the MDA assay (n = 4). (D) Representative confocal microscopy image of the pC, pH, and pHT cells. Scale bar, 50 µm. (E) Intracellular ROS levels of the pC, pH, and pHT cells (n = 4). (F) Intracellular MDA levels of the pC, pH, and pHT cells (n = 3). p > 0.05, ns; p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.

Figure 3

Typical 1D ¹H-NMR spectra of C2C12 cells after treatment with H₂O₂ and trehalose. (A) NMR spectra of aqueous metabolites from the C, T, H, and HT cells. (B) NMR spectra of aqueous metabolites from the pC, pH, and pTH cells. In all the NMR spectra, the vertical scale remained constant, and the water peak signal was removed (4.70–4.90 ppm). Ten times magnification was performed in the 4.90–9.00 ppm region for the clarity. Abbreviations: GTP, guanosine triphosphate; AXP, adenine mono/di/triphosphate.

Figure 4

Multivariate statistical analyses for 1D ¹H-NMR spectral data of C2C12 cells after treatment with H₂O₂ and trehalose. (A) PCA scores plot of the C, H, T, and HT groups for the trehalose treatment experiment. (B,D,F) PCA scores plots for H vs. C, HT vs. H, and T vs. C. (C,E,G) OPLS-DA scores plots for H vs. C, HT vs. H, and T vs. C. (H) PCA scores plot of the pC, pH, and pTH groups for the trehalose pretreatment experiment. (I,K) PCA scores plots for pH vs. pC and pTH vs. pH. (J,L) OPLS-DA scores plots for pH vs. pC and pTH vs. pH.

Figure 5

Identification of significant and characteristic metabolites from pairwise comparisons between groups of C2C12 cells. (A–E) VIP score-ranking plots of significant metabolites identified from the OPLS-DA model for (A) H vs. C, (B) HT vs. H, (C) T vs. C, (D) pH vs. pC, and (E) pTH vs. pH. (F) Venn diagrams of characteristic metabolites identified from pairwise comparisons of H vs. C, T vs. C and HT vs. H. (G) Venn diagrams of characteristic metabolites identified from pairwise comparisons of pH vs. PC and pTH vs. pH. Metabolites in red were characteristic metabolites shared by the (F) three/(G) two pairwise comparisons. These metabolites were ranked in descending order according to the VIP scores (Tables S7 and S8). Statistical significance: ↓/↑, p < 0.05; ↓↓/↑↑, p < 0.01; ↓↓↓/↑↑↑, p < 0.001; ↓↓↓↓/↑↑↑↑, p < 0.0001.

Figure 6

Identification of significantly altered metabolic pathways from pairwise comparisons between groups of C2C12 cells. (A) H vs. C, (B) HT vs. H, (C) T vs. C, (D) pH vs. pC, and (E) pTH vs. pH. Significantly altered metabolic pathways were identified with two criteria of pathway impact values > 0.2 and p values < 0.05 using the pathway analysis module provided by MetaboAnalyst 5.0 webserver. The characters in the figure represent the following: P1: alanine, aspartate, and glutamate metabolism; P2: D-Glutamine and D-glutamate metabolism; P3: taurine and hypotaurine metabolism; P4: β-alanine metabolism; P5: glutathione metabolism; P6: glycine, serine, and threonine metabolism; P7: histidine metabolism; P8: phenylalanine, tyrosine, and tryptophan biosynthesis; P9: phenylalanine metabolism.

Figure 7

Trehalose treatment and pretreatment activated the Keap1-Nrf2 pathway. (A) Expression levels of Nrf2 in the C, T, H, and HT cells (n = 3). (B) Cell viability in the trehalose treatment experiment under short-term oxidative stress (n = 5). (C) Cell viability in the trehalose pretreatment experiment under prolonged oxidative stress (n = 5). (D) Expression levels of Nrf2 in the trehalose treatment experiment (t-Nrf2) and the trehalose pretreatment experiment (p-Nrf2) (n = 3). (E) Expression levels of NQO1 in the trehalose treatment experiment (t-NQO1) and the trehalose pretreatment experiment (p-NQO1) (n = 3). p < 0.05, *; p < 0.01, **; p < 0.001, ***.

Figure 8

Trehalose treatment and pretreatment enhanced the expression levels of p62 and Nrf2 in cell nuclei. (A,B) Expression levels of Nrf2 in cell nuclei relative to cytoplasm in the trehalose treatment experiment (A) (n = 3) and the trehalose pretreatment experiment (B) (n = 3). (C,D) Expression levels of p62 in the pC, pH, and pTH cells (C) (n = 3) and the pH and pTH cells (D) (n = 3). Statistical significance: p < 0.05, *; p < 0.01, **.