Structural Characterization of Peptides From Huangjiu and Their Regulation of Hepatic Steatosis and Gut Microbiota Dysbiosis in Hyperlipidemia Mice

Abstract

Hyperlipidemia is a chronic disorder that is difficult to cure and usually treated with long-term lipid-reducing drugs. Recent trends have led to the use of diet therapies or food-derived strategies in the treatment of such long-term diseases. The Chinese rice wine (huangjiu) contains a wide range of bioactive peptides that are produced during the multi-species fermentation process. To clarify the regulation effects of lipid metabolism and gut microbiota by huangjiu bioactive peptides, three huangjiu peptides were isolated, purified and characterized by hyper-filtration, macroporous resin, gel filtration separation and structural identification. Meanwhile, a mouse model of high-fat diet-induced hyperlipidemia was established to study the effects of huangjiu peptides on serum biomarker, hepatic metabolism and gut microbiota dysbiosis. Experimental results showed that huangjiu peptides T1 and T2 (HpT1, HpT2) treatment alleviated the increase in serum total cholesterol, triglyceride, low-density lipoprotein cholesterol levels and aberrant hepatic lipid accumulation in the high-fat diet-induced hyperlipidemia mice. Furthermore, HpT2 and HpT1 restored the α-diversity and structure of gut microbial community after hyperlipidemia-induced microbiota disturbance compared with simvastatin and HpT3. The administration of HpT2 and HpT1 regulated the microbiota-mediated gut ecology through alterations of characteristic taxa including Lactobacillus, Ileibacterium, Faecalibaculum and Alloprevotella by linear discriminant analysis effect size analysis. Collectively, our results offer new insights into the abilities of food-derived peptides on alleviation of high-fat diet-induced hyperlipidemia, hepatic steatosis and gut dysbiosis in mice.

Keywords: dietary interventions; food-derived peptides; gut dysbiosis; hepatic steatosis; hyperlipidemia; microbe interactions.

FIGURE 1

Huangjiu peptide T1 and T2 treatment alleviated aberrant lipid metabolism in the liver induced by high-fat diet. Mice were fed with high-fat diet for 8 weeks except control (Con) group were treated with normal diet; Mice of the high-fat diet group (Hfd) were treated with saline and fed with high-fat diet for 8 weeks; the simvastatin treatment group (SiT) were treated with simvastatin (6 mg kg⁻¹); and huangjiu peptide T1 group (HpT1), huangjiu peptide T2 group (HpT2), huangjiu peptide T3 group (HpT3) were administrated with huangjiu peptide T1, T2 or T3 (0.3 g kg⁻¹) by gavage, respectively. These group names were used throughout this research paper. Levels of (A) ALT and (B) AST in serum were determined by biochemical kits, and these samples were obtained from Con, Hfd, SiT, HpT1, HpT2, HpT3 mice groups. Levels of (C) TC, (D) TG and (E) MDA in liver were measured thought kits and calculated according to hepatic protein content. (F) Representative liver tissue sections were stained with H&E to indicate lipid contents and bars = 50 μm. Data were presented as mean ± SEM of six to nine mice per group. Unpaired two-tailed t tests were used between Con and Hfd groups, and the black * was labelled on column with *p < 0.05, **p < 0.01, ****p < 0.0001. One-way ANOVA analyses were applied followed by Dunnett’s multiple comparisons test, and the significant p value of each group was compared with Hfd group. The blue # label indicated #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, and ns means no significant differences.

FIGURE 2

Huangjiu peptides T1 and T2 reduced epididymal fat weight gain and decreased serum TC and LDL-C in high-fat induced mice. (A) Liver index and (B) food intake were examined among mice groups. (C) Level of epididymal fat was measured. (D) TC, (E) TG (F) LDL-C and (G) HDL-C of serum samples were assayed by biochemical kits. Data were presented as mean ± SEM of six to nine mice per group. Unpaired two-tailed t tests were used between Con and Hfd groups, and the black * was labelled on column with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA analyses were applied followed by Dunnett’s multiple comparisons test, and the significant p value of each group was compared with Hfd group. The blue # label indicated #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, and ns means no significant differences.

FIGURE 3

Microbial α, β-diversity and relative abundance in fecal samples indicated a restorative effect of huangjiu peptides following high fat diet-induced gut dysbiosis. (A) α-diversity as measured using Shannon index and (B) Simpson index from different groups. Data were presented as mean ± SEM of six to nine mice per group. Two-tailed t-test was used to determine statistical significance, *p < 0.05, **p < 0.01, ns means no significant differences. (C) Principal coordinates analysis (PCoA) clarified differences in microbial community structure between different groups. The first principal component (PC1) and second principal component (PC2) explained 28.17 and 16.31% of the variance in the unweighted UniFrac metrics, respectively. Each point represents the fecal microbiome in a single sample. (D) Pairwise distance based on unweighted Unifrac distance measurements and Euclidean distance calculation of microbiota in fecal samples from different mice groups. Data were presented as mean ± SEM of the distances. Unpaired two-tailed t tests were used to determine statistical significance between groups, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Alterations of top 10 microbial community at a family level in different groups.

FIGURE 4

Comparison of differential microbial abundance, characteristic taxa and predicted functions from different mice groups. (A) Histogram of taxa with differential abundance in Con and Hfd groups using computed LDA scores. The LDA scores represent the degree of difference abundance in characteristic microbial communities between Con and Hfd groups. (B) Circular cladogram of statistically and biologically differences in fecal samples between Con and Hfd groups. Each of circle diameter was proportional to the abundance of taxon. Green = taxon significantly enriched in Con, red = taxon significantly enriched in Hfd and yellow = non-significant. (C) Histogram of taxa with differential abundance among HpT1, HpT2 and HpT3 groups using computed LDA scores. The LDA scores represent the degree of difference abundance in characteristic microbial communities among HpT1, HpT2 and HpT3 groups. (D) Circular cladogram of statistically and biologically differences in fecal samples among HpT1, HpT2 and HpT3 groups. (E) The heatmap of functional comparison and clustering based on Tax4Fun at level 2 among mice groups. The heat map was formed based on top 35 abundant taxa features of samples according to functional annotations and abundance information in the database, and clustered from the level of functional differences. (F) Box charts represented relative abundance of Bacteroides, Allobaculum, Lactobacillus, Faecalibaculum, Ileibacterium, Alloprevotella, and Fusobacterium in fecal samples from different groups of mice. Boxes represent the interquartile range (IQR), outliers (crosses) (>1.5∗IQR), range (whiskers) and median (horizontal line within the box).