Serum Metabolomics Uncovers the Mechanisms of Inulin in Preventing Non-Alcoholic Fatty Liver Disease

Abstract

Inulin may be a promising therapeutic molecule for treating non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms of its therapeutic activity remain unclear. To address this issue, a high-fat-diet-induced NAFLD mouse model was developed and treated with inulin. The NAFLD phenotype was evaluated via histopathological analysis and biochemical parameters, including serum levels of alanine aminotransferase, aspartate aminotransferase, liver triglycerides, etc. A serum metabolomics study was conducted using ultra-performance liquid chromatography coupled with tandem mass spectrometry. The results revealed that inulin mitigated NAFLD symptoms such as histopathological changes and liver cholesterol levels. Through the serum metabolomics study, 347 differential metabolites were identified between the model and control groups, and 139 differential metabolites were identified between the inulin and model groups. Additionally, 48 differential metabolites (such as phosphatidylserine, dihomo-γ-linolenic acid, L-carnitine, and 13-HODE) were identified as candidate targets of inulin and subjected to pathway enrichment analysis. The results revealed that these 48 differential metabolites were enriched in several metabolic pathways such as fatty acid biosynthesis and cardiolipin biosynthesis. Taken together, our results suggest that inulin might attenuate NAFLD partially by modulating 48 differential metabolites and their correlated metabolic pathways, constituting information that might help us find novel therapies for NAFLD.

Keywords: inulin; metabolites; metabolomics; non-alcoholic fatty liver disease.

Figure 1

Biochemical indices of mice in each group. (A) Body weight, (B) liver index, (C) serum TG, (D) serum TC, (E) serum LDL-C, (F) serum HDL-C, (G) serum AST, and (H) serum ALT. Data are expressed as means ± SD (n = 9; * p < 0.05).

Figure 2

Histopathological changes in liver tissues of mice in each group. (A) H&E staining of liver tissues (magnification, ×200). (B) ORO staining of liver tissues (magnification, ×200). (C) NAFLD activity score (NAS Score), including steatosis, inflammation, and ballooning scores (* p < 0.05).

Figure 3

PCA and OPLS-DA plots among the three groups. (A) PCA score plots of the control, model, and inulin groups. Blue, red, and yellow dots represent the control, model, and inulin groups, respectively. (B) OPLS-DA score plots of the control, model, and inulin groups. Blue, red, and yellow dots represent the control, model, and inulin groups, respectively.

Figure 4

PCA and OPLS-DA plots between pairwise groups. (A) PCA score plots of the control and model groups. Blue and red dots represent the control and model groups, respectively. (B) OPLS-DA score plots of the control and model groups. Blue and red dots represent the control and model groups, respectively. (C) PCA score plots of the inulin and model groups. Red and yellow dots represent the model and inulin groups, respectively. (D) OPLS-DA score plots of the inulin and model groups. Red and yellow dots represent the model and inulin groups, respectively.

Figure 5

Validation models of OPLS-DA and VIP values. (A) The validation model of OPLS-DA for the model and control groups. Green dots and blue squares represent R² and Q², respectively. (B) The validation model of OPLS-DA for the inulin and model groups. Green dots and blue squares represent R² and Q², respectively. (C) VIP values of OPLS-DA for the model and control groups. X-axis represents metabolite names, and Y-axis represents VIP values. (D) VIP values of OPLS-DA for the inulin and model groups. X-axis represents metabolite names, and Y-axis represents VIP values.

Figure 6

Venn diagram and hierarchical cluster heatmap. (A) Venn diagram comparing the two pairwise groups (model vs. control and inulin vs. model) revealed 73 overlapped metabolites. (B) A hierarchical cluster heatmap of 48 metabolites whose changes were reversed following inulin treatment. Red and green indicate an increase and decrease in the levels of metabolites.

Figure 7

Overview of metabolic pathway enrichment analysis. (A) Overview of top 25 enriched pathways. The X-axis represents the enrichment ratio, and the Y-axis represents pathway names; the color represents the different p values. (B) Bubble plots of the enrichment analysis. The X-axis represents −log10 (p value), and the Y-axis represents pathway names; the size and color of the dots represent different enrichment ratios and p values, respectively.