Perturbations of Metabolomic Profiling of Spleen From Rats Infected With Clonorchis sinensis Determined by LC-MS/MS Method

Abstract

Clonorchiasis is an important zoonotic parasitic disease worldwide. In view of the fact that parasite infection affects host metabolism, and there is an intricate relationship between metabolism and immunity. Metabolic analysis of the spleen could be helpful for understanding the pathophysiological mechanisms in clonorchiasis. A non-targeted ultra high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF MS) approach was employed to investigate the metabolic profiles of spleen in rats at 4 and 8 weeks post infection with Clonorchis sinensis (C. sinensis). Then a targeted ultra-high performance liquid chromatography multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) approach was used to further quantify amino acid metabolism. Multivariate data analysis methods, such as principal components analysis and orthogonal partial least squares discriminant analysis, were used to identify differential metabolites. Finally, a total of 396 and 242 significant differential metabolites were identified in ESI+ and ESI- modes, respectively. These metabolites included amino acids, nucleotides, carboxylic acids, lipids and carbohydrates. There were 38 significantly different metabolites shared in the two infected groups compared with the control group through the Venn diagram. The metabolic pathways analysis revealed that pyrimidine metabolism, aminoacyl-tRNA biosynthesis, purine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis were significantly enriched in differential metabolites, which was speculated to be related to the disease progression of clonorchiasis. Furthermore, 15 amino acids screened using untargeted profiling can be accurately quantified and identifed by targeted metabolomics during clonrochiasis. These results preliminarily revealed the perturbations of spleen metabolism in clonorchiasis. Meanwhile, this present study supplied new insights into the molecular mechanisms of host-parasite interactions.

Keywords: Clonorchis sinensis; infection; metabolic pathway; non-targeted metabolomics; spleen.

FIGURE 1

Orthogonal partial least squares discriminant analysis (OPLS-DA) score scatter plots of splenic metabolites during C. sinensis infection in ESI+ mode. (A) OPLS-DA score scatter plot of 4 wpi vs control in ESI+ mode; (B) OPLS-DA score scatter plot of 8 wpi vs control in ESI+ mode; (C) OPLS-DA score scatter plot of 8 vs 4 wpi in ESI+ mode. Control, healthy control; 4 wpi, 4 weeks post infection; 8 wpi, 8 weeks post infection.

FIGURE 2

Permutation test of OPLS-DA model showing the stability of the model in ESI+ mode. (A) Permutation test of OPLS-DA model of 4 wpi vs control in ESI+ mode; (B) Permutation test of OPLS-DA model of 8 wpi vs control in ESI+ mode; (C) Permutation test of OPLS-DA model of 8 vs 4 wpi in ESI+ mode. The abscissa indicates the displacement retention of the permutation test, and the ordinate indicates the value of R2Y or Q2. The green dot indicates the R2Y value obtained by the displacement test, the blue square indicates the Q2 value obtained by the permutation test, and the two dotted lines indicate the regression lines of R2Y and Q2, respectively. The point where the displacement retention is 1 is R2Y and Q2 of the original model.

FIGURE 3

Volcano plot representation of the differential metabolites identified in ESI+ mode. (A) 4 wpi vs control; (B) 8 wpi vs control; (C) 8 vs 4 wpi. Each point in the map represents a metabolite. The size of the scatter represents the VIP value of the OPLS-DA model, and the larger the scatter, the larger the VIP value. Scatter color represents the final screening result, red represents significant up-regulation, blue represents significant down-regulation, and gray represents non-significant difference metabolites.

FIGURE 4

Heatmaps representation of the differential metabolites identified between different infection group vs control group in ESI+ mode. (A) 4 wpi vs control; (B) 8 wpi vs control; (C) 8 vs 4 wpi. The color blocks at different positions represent the relative expression of metabolites at corresponding positions, red represents up-regulated, and blue represents down-regulated.

FIGURE 5

Venn diagram displaying the common and unique metabolites among the two infection groups vs control group. There were 38 significantly differential metabolites were shared in two infected groups. 4 wpi vs con, 4 wpi group vs control group; 8 wpi vs con, 8 wpi group vs control group.

FIGURE 6

The distribution of amino acids were present by boxplot between 4 wpi vs control group. (A) L-Valine; (B) L-Asparagine; (C) L-Serine; (D) L-Methionine; (E) L-Phenylalanine; (F) L-Arginine; (G) L-Tryptophan; (H) L-Citrulline; (I) Glycine. Boxes represent the interquartile ranges (IQRs) between the first and third quartiles, and the line inside the box represents the median. The amino acid content was compared with the median value between the two groups. Circles represent outliers. *P < 0.05, ***P < 0.001.

FIGURE 7

The pathway analysis during C. sinensis infection in ESI+ mode. Plots depict the pathway impacts of the key metabolites (x-axis) and the computed metabolic pathway as a function of –log (P) (y-axis) that different between the 4 wpi vs control (A), 8 wpi vs control (B), 8 vs 4 wpi (C).

FIGURE 8

The integrated metabolic pathway shared in 4 and 8 wpi during C. sinensis infection. Beneath each metabolite, green and red represent 4 and 8 wpi compared with the control, respectively. The arrows indicate up and down-regulation of metabolites, respectively. Horizontal lines indicate that the metabolites are not significantly different in this infection stage.

FIGURE 9

Flow chart of metabolomics in spleen of rats infected with Clonorchis sinensis.