Effects of lactoferrin on intestinal flora of metabolic disorder mice

Abstract

To study the mechanism of lactoferrin (LF) regulating metabolic disorders in nutritionally obese mice through intestinal microflora. Twenty-one male C57BL/6 mice were randomly divided into 3 groups: control group, model group and LF treatment group. The mice in control group were fed with maintenance diet and drank freely. The mice in model group were fed with high fat diet and drank freely. The mice in LF treatment group were fed with high fat diet and drinking water containing 2% LF freely. Body weight was recorded every week. Visceral fat ratio was measured at week 12. Blood glucose and serum lipid level were detected by automatic biochemical analyzer. The gut microbiota of mice was examined using 16 s rRNA sequencing method. LF treatment significantly reduced the levels of visceral adipose ratio, blood glucose, triglyceride, total cholesterol and low-density lipoprotein cholesterol (LDL-C) in high-fat diet mice (p < 0.05). It can be seen that drinking water with 2% LF had a significant impact on metabolic disorders. At the same time, the Firmicutes/Bacteroidetes ratio(F/B) of LF treated mice was decreased. The abundance of Deferribacteres, Oscillibacter, Butyricicoccus, Acinetobacter and Mucispirillum in LF treatment group were significantly decreased, and the abundance of Dubosiella was significantly increased (p < 0.05). In the LF-treated group, the expression levels of glucose metabolism genes in gut microbiota were increased, and the expression levels of pyruvate metabolism genes were decreased. It can be seen that metabolic disorders were related to intestinal flora. In conclusion, LF regulates metabolic disorders by regulating intestinal flora.

Keywords: Intestinal flora; Lactoferrin; Metabolic disorders.

Fig. 1

a Weight differences in mice at week 12. Data are presented as mean ± SD (n = 7) (SPSS 11.0). Abbreviations: K, the control group; M, the model group; Y2, the LF-treated group. * p < 0.05, ** p < 0.01. b Visceral adipose tissue differences in mice at week 12. Data are presented as mean ± SD (n = 7) (SPSS 11.0). Abbreviations: K, the control group; M, the model group; Y2, the LF-treated group. * p < 0.05, ** p < 0.01

Fig. 2

Effects of LF on energy intake in mice

Fig. 3

Rarefaction curves of fecal sample. The number of OTU tended to be stable after the sequence was over 40,000. Abbreviations: K1-K7, control group; M1-M7, model group; Y1-Y7, LF-treated group

Fig. 4

Comparison of the flora alpha diversity indices. a Indices of Chao 1, b Indices of Shannon c Indices of Simpson. There were not significant differences among the groups. The results showed that the diversity of intestinal microflora of mice in group M was higher than that in group K, and the diversity of intestinal microflora of mice in group Y2 was closer to that in group K. Abbreviations: K, the control group; M, the model group; Y2, the LF-treated group

Fig. 5

Beta diversity of P CoA. There were significant differences in the composition and structure of intestinal flora among the three groups. Group K was clustered in the upper left part of the main horizontal axis. The Y2 group was clustered in the upper right part of the main horizontal axis. Group M gathered in the lower part of the main horizontal axis. Abbreviations: square, control group; round, model group; triangle, LF-treated group. Note: K control group， M model group，Y experimental group

Fig. 6

Effects of LF on the abundance of ileal microbiota in mice, n = 7. a Phylum-level composition. The ratio of Firmicutes and Bacteroidetes (F/B) decreased in Y2 group relative to M group. b t-test analysis at the phylum level, the abundance of Deferribacteres in group M was significantly higher than that in group K(P = 0.022). c t-test analysis at the phylum level, the abundance of Deferribacteres in group M was significantly higher than that in group Y2(P = 0.030). d Genus-level composition. e t-test analysis at the genus level, the abundance of Dunaliella in group M was significantly lower than that in group K (P = 0.022). f t-test analysis at the genus level, the abundance of Dunaliella in group Y2 was significantly higher than that in group M(P = 0.034). Abbreviations: K, the control group; M, the model group; Y2, the LF-treated group

Fig. 7

High-dimensional biomarkers (LDA value distribution histogram revealed by LEfSe software). a Histogram of LDA analysis. When species with LDA Score > 4 are statistically different, the length of the histogram (LDA Score) represent the impact size of the different species. b The distribution difference of flora was analyzed by LEfSe. Evolutionary branching trees from the inside out in a clade represent the level of phylum, class, order, family, genus (or species). Classification of species in which red/green nodes play an important role in healthy controls. The yellow nodes indicate species that are not significantly different. Abbreviations: K, the control group; Y2, the LF-treated group

Fig. 8

The KEGG prediction of intestinal flora. a Annotations the functional gene. b Expression levels analysis by t-test. Significant difference when p < 0.05. Abbreviations: K, the control group; M, the model group; Y2, the LF-treated group