Inulin alleviates atherosclerosis through improving lipid metabolism, inflammation, and gut microbiota in ApoE-knockout mice: the short-chain is more efficacious

Abstract

Introduction: Atherosclerosis (AS) is considered the underlying cause of many diseases, particularly cardiovascular and cerebrovascular diseases. Inulin, a type of fructan, has shown potential in improving atherosclerosis, although there are conflicting findings. It is hypothesized that the polymerization degree of inulin may largely influence its therapeutic effectiveness. Therefore, this study aimed to investigate the effects and mechanisms of short-chain and long-chain inulin in AS.

Methods: ApoE^-/- mice fed a high fat diet (HFD) were used to establish an atherosclerosis model. These mice received daily oral administration of either short-chain or long-chain inulin for 12 weeks. Plasma lipid metabolism-related indices were measured using biochemical analysis, and plasma immunological indices were analyzed via ELISA. The aorta, aortic root regions, liver tissue, adipose tissue, and colon tissue were examined through various staining techniques, including ORO staining, hematoxylin and eosin staining, Alcian blue staining, and immunofluorescent or immunohistochemical assays. Microbiome analysis was conducted in the cecal content.

Results: The results indicated that both short-chain and long-chain inulin substantially reduced the formation of atherosclerotic plaques. Inulin also improved plasma lipid concentrations and hepatic lipid metabolism, and partially alleviated both localized (atherosclerotic lesions) and systemic inflammation. Short-chain inulin was more effective than long-chain inulin in reducing atherosclerotic plaques formation, enhancing lipid metabolism and reducing inflammation. Additionally, both types of inulin showed similar effectiveness in enhancing intestinal epithelial barrier integrity, gut microbiota composition and functionality.

Conclusion: These findings suggest that inulin has a protective role against atherosclerosis by enhancing lipid metabolism, reducing inflammation, and improving intestinal barrier and gut microbiota. As a dietary intervention, short-chain inulin is more effective than long-chain inulin, offering clinical implications for using inulin as a therapeutic agent for atherosclerosis.

Keywords: atherosclerosis; gut microbiota; inflammation; intestinal barrier; inulin; lipid metabolism.

FIGURE 1

Inulin administration alleviated the progression of atherosclerosis in ApoE ^−/− mice. (A) Schematic illustration of the experimental design. (B) Representative images of plaque lesions in the whole area of the aorta stained with Oil Red O (ORO) staining. (C) Representative images of ORO staining of the aorta root sections. (D) Quantitative analysis of lesion area in aorta, aortic arch, thoracic aorta, abdominal aorta, and aorta root sections. Data were shown as mean ± SD. n = 9. Significances were determined by one-way analysis of variance (ANOVA), followed by post hoc pairwise comparisons with the Tukey honest significant difference. ns (no significance), P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

FIGURE 2

Inulin supplementation could improve lipid metabolism in ApoE ^−/− mice. (A) Plasma total triglyceride. (B) Plasma low-density lipoprotein (LDL). (C) Plasma total cholesterol. (D) Plasma High-density lipoprotein (HDL). (E) Representative images of Oil Red O (ORO) staining of liver sections. (F) Representative images of H&E staining of adipocytes. (G) Percentage of ORO positive area was calculated from (E) by ImageJ software. (H) Quantitative analysis of adipocyte size from (F) by ImageJ software. (I–N) Representative gene expression levels for Cyp7a1, SREBP1, PCSK9, ABCA1, ABCG1, and ABCG5 in the liver were quantified. Data were shown as mean ± SD. n = 5 to 9. Significances were determined by one-way analysis of variance (ANOVA), followed by post hoc pairwise comparisons with the Tukey honest significant difference. ns (no significance), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

FIGURE 3

Inulin administration ameliorate localized and systemic inflammation in ApoE ^−/− mice. (A) Representative immunofluorescence staining for ICAM-1 (red) in atherosclerotic lesions. (B) Representative immunohistochemical staining for VCAM-1 (brown) in atherosclerotic lesions. (C) The fluorescence intensity of ICAM-1 was quantified by ImageJ software. (D) The positive area of VCAM-1 was quantified by ImageJ software and calculated as the percentage of total lesion area. (E–G) The circulating levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. Data were shown as mean ± SD. n = 6 to 9 Significances were determined by one-way analysis of variance (ANOVA), followed by post hoc pairwise comparisons with the Tukey honest significant difference. ns (no significance), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).

FIGURE 4

Inulin modulate intestinal barrier integrity in ApoE ^−/− mice. (A) Representative immunohistochemical staining for ZO-1 (red) in the colon. (B) Representative immunohistochemical staining for occludin (red) in the colon. (C) Representative Alcian blue staining of colon sections. The mucin layer and the goblet cells (Gc) were visualized under the microscope. (D, E) Quantitative analysis of images from (A) and (B). (F, G) Quantitative analysis of the images from (C). (H) Intestinal permeability was determined by measuring the 4,000 Da fluorescent dextran-FITC (DX-4000-FITC) level in serum.

FIGURE 5

Inulin regulate gut microbial community in ApoE ^−/− mice. (A) Observed species. (B) Chao1 richness estimator. (C) Shannon diversity index. (D) Simpson diversity index. (E) Mean relative abundance of top 10 phyla. (F) Firmicutes/Bacteroidetes ratio. (G) Top 20 genera with mean relative abundance. The relative abundance of (H) Bilophila, (I) Akkermansia, (J) Tyzzerella, (K) Faecalibaculum. (L) The principal components analysis (PCA). (M) The principal coordinate analysis (PCoA). (N) The nonmetric multidimensional scaling (NMDS) index. (O) LEfSe histograms. Bacterial taxa that met the criterion of an LDA score > 4 were considered biomarker taxa. Data were shown as mean ± SD. n = 8. Significances were determined by one-way analysis of variance (ANOVA), followed by post hoc pairwise comparisons with the Tukey honest significant difference. ns (no significance), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).