Integrated fecal microbiota and metabolomics analysis of the orlistat intervention effect on polycystic ovary syndrome rats induced by letrozole combined with a high-fat diet

Abstract

Background: This study aimed to compare the characteristics of the gut microbiota and their metabolite profiles between polycystic ovary syndrome (PCOS) and orlistat-treated PCOS rats (ORL-PCOS), which could help to better understand the underlying mechanism of the effect of orlistat on PCOS.

Methods: PCOS rat models were established using letrozole combined with a high-fat diet. Ten rats were randomly selected as a PCOS control group (PCOS). The other three groups (n = 10/group) were additionally supplemented with different doses of orlistat (low, medium, high). Then, fecal samples of the PCOS and ORL-PCOS groups were analysed by 16S rRNA gene sequencing and untargeted metabolomics. Blood samples were collected to detect serum sex hormones and lipids.

Results: The results showed that orlistat attenuated the body weight gain, decreased the levels of T, LH, the LH/FSH ratio, TC, TG and LDL-C; increased the level of E2; and improved estrous cycle disorder in PCOS rats. The bacterial richness and diversity of the gut microbiota in the ORL-PCOS group were higher than those in the PCOS group. The ratio of Firmicutes to Bacteroidetes was decreased with orlistat treatment. Moreover, orlistat treatment led to a significant decrease in the relative abundance of Ruminococcaceae and Lactobacillaceae, and increases in the abundances of Muribaculaceae and Bacteroidaceae. Metabolic analysis identified 216 differential fecal metabolites in total and 6 enriched KEGG pathways between the two groups, including steroid hormone biosynthesis, neuroactive ligand-receptor interaction and vitamin digestion and absorption. Steroid hormone biosynthesis was the pathway with the most significant enrichment. The correlations between the gut microbiota and differential metabolites were calculated, which may provide a basis for understanding the composition and function of microbial communities.

Conclusions: Our data suggested that orlistat exerts a PCOS treatment effect, which may be mediated by modifying the structure and composition of the gut microbiota, as well as the metabolite profiles of PCOS rats.

Keywords: Gut microbiota; Metabolomics; Obesity; Orlistat; Polycystic ovary syndrome.

Fig. 1

Schematic diagram of the animal experiment design and timeline

Fig. 2

Effects of orlistat treatment on the estrous cycle. Three rats were randomly selected in each group and representative estrous cycles of the different groups are shown. P: Proestrus, E: Estrous, M: Metoestrus, D: Dioestrus

Fig. 3

Characteristics of the gut microbiota after orlistat treatment. A Venn diagram of OTUs. The overlapping section represents the shared OTUs. There was no significant difference in the Shannon index. B and Chao1 (C) index between the ORL-PCOS and PCOS groups. D PCoA of the gut microbiota of ORL-PCOS and PCOS group based on weighted UniFrac algorithms

Fig. 4

LEfSe of the gut microbiota of the ORL-PCOS and PCOS groups. A LEfSe taxonomic cladogram. B LDA chart (p = phylum, c = class, o = order, f = family, g = genus). LDA scores higher than 3.0 were used as a threshold for significance in LEfSe analyses

Fig. 5

Multivariate statistical analyses of fecal metabolites in the ORL-PCOS and PCOS groups. A OPLS-DA negative ion mode scatter diagram. B Corresponding validation plots of the OPLS-DA negative ion mode obtained from the permutation test. C OPLS-DA positive ion mode scatter diagram. D Corresponding validation plots of the OPLS-DA positive ion mode obtained from the permutation test

Fig. 6

Volcanic map analysis in negative (A) and positive (B) ion modes. According to the VIP value, p value and FC value, volcano maps were drawn to display the overall distribution of the differential metabolites. The blue dots represent significantly downregulated metabolites in the ORL-PCOS group, and the red dots represent significantly upregulated differential metabolites. The black dots represent nonsignificant differential metabolites

Fig. 7

KEGG pathway enrichment analysis of differential metabolites. A The ordinate represents the name of the pathway, and the abscissa represents the number of differentially expressed metabolites included in each KEGG metabolic pathway. The colour indicates the p value of enrichment analysis. The number on the column represents the rich factor. B Heatmap for steroid hormone biosynthesis, C Heatmap for vitamin digestion and absorption, D Heatmap for neuroactive ligand-receptor interaction

Fig. 8

Correlation heatmap analysis of the differential fecal metabolites and gut microbiota between the PCOS and ORL-PCOS groups. A significant positive correlation is shown in red; a significant negative correlation is shown in blue, with darker colours indicating stronger correlations. * p < 0.05, ** p < 0.01, *** p < 0.001