Basic parameters in ApoE−/− mice

As shown in Table 2, there was no statistically significant difference in body weight gain and daily food intake among the three groups (p > 0.05). Similarly, there was no significant difference in the weight of the heart or aorta among the three groups. According to the daily food intake of each mouse and 8 mice per group, the mean of daily calorie intake per group was about 150.4 kcal. In addition, lycopene was incorporated in the high-fat diet at a concentration of 1 g/kg of food (0.1% w/w), the mean of daily lycopene intake in HFD + lycopene group was about 32 mg.

Table 2 Basic parameters and biochemical markers measured in ApoE−/− miceLycopene lowered blood lipids and serum atherogenic inflammatory factors

Serum TC, TG, LDL-C, TNF-α, MCP-1, IL-6 and IL-1β levels were the highest in the HFD group, while the HFD + lycopene group showed significantly reduced levels of serum TC, LDL-C, TNF-α, MCP-1, IL-6 and IL-1β (p < 0.05, p = 0.02, 0.03, 0.02, 0.01, 0.01, 0.02), but there was no difference in serum TG levels between HFD group and HFD + lycopene group. At the same time, the HFD group had the lowest serum HDL-C concentration, while the HDL-C concentration in HFD + lycopene group was markedly increased (p < 0.05, p = 0.03) (Table 2).

Lycopene suppressed Atherosclerosis progression in the aortic sinus

Oil Red O staining was applied to reveal the atherosclerotic lesions in the three groups. As shown in Fig. 1A, the percentage of atherosclerotic lesions was the lowest in the HFD + lycopene group, while the atherosclerotic lesions area in HFD group was larger than that in control group (p < 0.05, p = 0.01). However, the atherosclerotic lesions area was reduced by 84% in HFD + lycopene group compared to HFD group (Fig. 1B).

Fig. 1

Lycopene supplementation inhibited the atherosclerosis of aortic sinus in HFD-fed ApoE−/− mice. ApoE−/− mice were fed a low-fat diet, a high-fat diet supplemented with or without 0.1% lycopene (w/w) for 19 weeks. (A) Oil red O staining of the aortic sinus is presented graphically (40×magnification), as described in Materials and Methods. The larger the area of red plaque, the deeper the atherosclerosis. (B) The ratio of the area of atherosclerotic plaque to the area of the aortic sinus was analyzed by Image J software. Data are presented as the mean ± SD (n = 8 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. HFD, high-fat diet; LY, lycopene

Lycopene modulated the diversity and composition of gut microbiota

In the α-diversity analysis, both the Observed species and the Shannon index were reduced in HFD group, while the Simpson index was increased in HFD group. However, lycopene administration improved the diversity of gut microbiota, the Observed species and Shannon index were elevated and the Simpson index was reduced after lycopene treatment (Fig. 2A-C). In the β-diversity analysis, the weighted PCoA diagrams of the three groups were shown in Fig. 2D, which reflected that the composition of gut microbiota in the HFD group was significantly different from the other two groups. Furthermore, a partial overlap was found between HFD + lycopene group and control group, indicating that lycopene supplementation could improve gut microbiota composition induced by HFD. In the heatmap of sample-sample distances, a similar effect was observed (Fig. 2E).

Fig. 2

Lycopene supplementation improved the diversity and composition of gut microbiota in HFD-fed ApoE−/− mice. ApoE−/− mice were fed a low-fat diet, a high-fat diet supplemented with or without 0.1% lycopene (w/w) for 19 weeks. (A, B, C) The data of the alpha diversity was analyzed by the ggplot2, vegan, and ggpubr packages of R software. Data are presented as the mean ± SD (n = 6 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. (D, E) The data of the beta diversity was analyzed by the phyloseq package of R software. Figure D showed the specificity among three groups. Figure E showed the sample distance of three groups in heatmap; The redder the relationship was closer, while the bluer the relationship was more distant

Lycopene modulated the abundance of gut microbiota

To further analyze the alterations in gut microbiota composition, we analyzed the relative abundance of gut microbiota at the phylum and genus levels (Fig. 3A, B). At the phylum level, Firmicutes and Bacteroidetes occupied the largest proportion. Compared to the control group, the relative abundance of Firmicutes was significantly increased and the relative abundance of Bacteroidetes was reduced in HFD group, which was reversed by lycopene (Fig. 3C, D). The Firmicutes/Bacteroides ratio (F/B ratio) was considerably elevated than 1 in HFD group, whereas F/B ratio in the other two groups was significantly less than 1 (p < 0.05, p = 0.02) (Fig. 3E). Verrucomicrobia was less prevalent in HFD group, whereas its abundance was statistically enhanced in HFD-fed mice supplemented with lycopene (p < 0.05, p = 0.02) (Fig. 3F). At the genus level, Akkermansia and Alloprevotella were the lowest in the HFD group while being significantly more prevalent in the HFD + lycopene group (p < 0.05, p = 0.02) (Fig. 3G, H).

Fig. 3

Lycopene supplementation modulated the relative abundance of gut microbiota in HFD-fed ApoE−/− mice. ApoE−/− mice were fed a low-fat diet, a high-fat diet supplemented with or without 0.1% lycopene (w/w) for 19 weeks. (A, C, D, F) Different expression of relative abundance of gut microbiota in three groups at phylum level. (E) The ratio of Firmicutes/Bacteroides in three groups. Data was analyzed by R software and presented as the mean ± SD (n = 6 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. (B, G, H) Different expressions of relative abundance of gut microbiota in three groups at genus level. Data was analyzed by R software and presented as the mean ± SD (n = 6 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. HFD, high-fat diet; LY, lycopene. For species difference analysis and abundance calculation, the reshape2, ggplot2, ggprism, and plyr packages of R software were used

Lycopene improved intestinal barrier function and reduced intestinal permeability

The intestinal barrier function can be reflected by the expression levels of epithelial tight junction protein ZO-1 and occludin. Immunohistochemical analysis and Western blotting revealed that the expression levels of intestinal ZO-1 and occludin in the HFD group were statistically decreased compared with the control group. In contrast, HFD + lycopene group showed higher levels of intestinal ZO-1 and occludin expression (Fig. 4A-F), reflecting an improved intestinal barrier function. Moreover, the concentrations of serum LPS, D-LA and DAO, which are the biomarkers of intestinal permeability, were noticeably increased in the HFD group, while the HFD + lycopene group showed decreased levels (p < 0.05, p = 0.02, 0.01, 0.03) (Fig. 4G-I), reflecting a reduced intestinal permeability.

Fig. 4

Lycopene supplementation increased intestinal ZO-1 and occludin expression and reduced intestinal permeability in HFD-fed ApoE−/− mice. ApoE−/− mice were fed a low-fat diet, a high-fat diet supplemented with or without 0.1% lycopene (w/w) for 19 weeks. (A, B) Immunohistochemical results of ZO-1 and occludin protein expression in the ileum were presented in the form of pictures (200×magnification), as described in Materials and Methods. The browner the color, the higher the protein expression. (C, D, E, F) The protein expression of ZO-1 and occludin in the ileum was determined by Western blotting. Results are representative of three independent experiments. It was analyzed by Image J software. Expression values were normalized to housekeeping gene GAPDH, and expression in the control group was set to 1. Data are presented as the mean ± SD (n = 8 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. (G, H, I) Intestinal permeability index such as LPS, D-LA and DAO was analyzed by SPSS software. One-Way ANOVA statistical method was used for calculation, and LSD and SNK methods were used for inter-group comparison. Data are presented as the mean ± SD (n = 8 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. HFD, high-fat diet; LY, lycopene

Lycopene inhibited inflammation in the aortic sinus

Elevated LPS enters the circulation and binds to TLR4, which in turn stimulates the TLR4/NF-κB signaling pathway, thereby activating various inflammatory factors and potentially resulting in atherosclerosis. As illustrated in Fig. 5A, B, the

immunohistochemical analysis showed that the expression of inflammatory protein TLR4 and p-NF-κB p65 in the aortic sinus was markedly increased in the HFD group, which was significantly reduced in HFD + lycopene group compared to the HFD group. There was little difference between the control group and HFD + lycopene group. In Western blotting analysis,

the expression levels of aortic TLR4, p-NF-κB p65 and NF-κB p65 were considerably higher in HFD group. Surprisingly, the expression levels of the three proteins in the HFD + lycopene group were decreased, while the expression levels of the control group were the lowest (Fig. 5C, D). Accordingly, the serum levels of inflammatory cytokines, such as TNF-α, MCP-1, IL-6 and IL-1β, were significantly reduced in HFD + lycopene group compared with HFD group (p < 0.05, p = 0.01, 0.02, 0.02, 0.03) (Table 2).

Fig. 5

Lycopene supplementation attenuated the inflammation of aortic sinus in HFD-fed ApoE−/− mice. ApoE−/− mice were fed a low-fat diet, a high-fat diet supplemented with or without 0.1% lycopene (w/w) for 19 weeks. (A, B) Immunohistochemical results of TLR4 and p-NF-κB P65 protein expression in the aortic sinus were presented graphically (40×magnification), as described in Materials and Methods. The browner the color, the higher the protein expression. (C, D) The protein expression levels of aortic TLR4, p-NF-ĸBp65 and NF-ĸBp65 were determined by Western blotting. Results are representative of three independent experiments. It was analyzed by Image J software. Expression values were normalized to housekeeping gene GAPDH, and expression in the control group was set to 1. Data are presented as the mean ± SD (n = 8 per group); ap < 0.05 compared with the control group; bp < 0.05 compared with the high-fat diet group. HFD, high-fat diet; LY, lycopene

Spearman’s correlations analysis between the gut microbiota and the biochemical markers

Spearman’s correlations analysis also showed that different gut microbiota was associated with serum lipid, inflammatory factors and intestinal permeability indexes. At the phylum level, Firmicutes were positively correlated with serum D-LA, DAO, MCP-1, IL-1β, IL-6, TC, TG and LDL-C levels, while Firmicutes were negatively correlated with serum HDL-C levels; Bacteroides were negatively correlated with serum LPS, DAO, D-LA, MCP-1, IL-6, TC and LDL-C levels, but Bacteroides were positively correlated with serum HDL-C levels. Similarly, Verrucomicrobia showed the same trend, but no negative correlation with TC was observed. The Firmicutes/Bacteroides ratio was positively correlated with serum LPS, D-LA, MCP-1, IL-6, TC, TG and LDL-C levels, while serum HDL-C levels were negatively correlated with the Firmicutes/Bacteroides ratio (Fig. 6A).

At the genus level, Akkermansia and Alloprevotella were negatively correlated with serum D-LA, DAO, MCP-1, IL-6, IL-1β, TNF-α, TC, TG and LDL-C levels, while the serum HDL-C levels showed a positive correlation (Fig. 6B).

Fig. 6

Spearman’s correlations between the gut microbiota and the biochemical markers. (A) The correlations between the gut microbiota and the biochemical markers at the phylum level. (B) The correlations between the gut microbiota and the biochemical markers at the genus level. The colour intensity represents the degree of the associations between the gut microbiota and the biochemical parameters, red represents positive correlation and blue indicates negative correlations. n = 6 per group, significant correlations are marked by

*p < 0.05 and **p < 0.01. HFD, high-fat diet; LY, lycopene