The Potential Role of Pyrroloquinoline Quinone to Regulate Thyroid Function and Gut Microbiota Composition of Graves' Disease in Mice

Abstract

Graves' disease (GD) is an autoimmune disorder disease, and its prevalence continues to increase worldwide. Pyrroloquinoline quinone (PQQ) is a naturally antioxidant compound in milk, vegetables, and meat. We aim to identify the treatment efficacy of PQQ on GD and its regulatory effect on intestinal microbiota. The GD mice model was built by an adenovirus expressing autoantigen thyroid-stimulating hormone receptor (Ad-TSHR289). Fecal samples were collected for 16S rDNA sequencing after PQQ pretreatments (20, 40, or 60 mg/kg BW/day) for 4 weeks. Thyroid and intestine functions were measured. The levels of serum TSHR and T4 were significantly raised, and the thyroid gland size was typically enlarged in the GD group than in controls, reversed by PQQ therapy. After PQQ replenishment, IL6 and TNFα levels in small intestine tissues were lower than those in the GD group, with Nrf2 and HO1 levels improved. Also, the PQQ supplement could maintain the mucosal epithelial barrier impaired by GD. In microbial analyses, PQQ treatment could prompt the diversity recovery of gut microbiota and reconstruct the microbiota composition injured by GD. Lactobacillus served as the most abundant genus in all groups, and the abundance of Lactobacillus was increased in the GD group than in control and PQQ groups. Besides, Lactobacillus was highly correlative with all samples and the top 50 genera. PQQ supplementation regulates thyroid function and relieves intestine injury. PQQ changes the primary composition and abundance of GD's intestine microbiota by moderating Lactobacillus, which may exert in the pathogenesis and progression of GD.

Keywords: Graves’ disease; Lactobacillus; gut microbiota; pyrroloquinoline quinone; thyroid function.

Fig. 1.

PQQ supplement modulates thyroid function and ameliorates thyroid injury. A) The concentrations of T4 and B) TRAb levels were determined inserum samples using Mouse ELISA Kit, C) the representative pictures of H&E staining of thyroids (200 × magnification with 100 μm of scale). Data were represented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 between groups. Con – control group, GD – Graves’ disease group, GD + PQQ/low – GD mice treated with 20 mg PQQ/kg BW/day, GD + PQQ/mid – GD mice treated with 40 mg PQQ/kg BW/day, GD + PQQ/high – GD mice treated with 60 mg PQQ/kg BW/day, GD + MMI – GD mice treated with 2.5 mg MMI/kg/BW/day, PQQ – pyrroloquinoline quinone, MMI – methimazole

Fig. 2.

PQQ supplement decreases inflammation and oxidative stress response and alleviates small intestine epithelial injury. Relative expression of mRNA in colons of A) IL6, TNFα, B) Nrf2, HO1 was detected by PCR, C) the representative pictures of the hematoxylin and eosin (H&E) staining of colons (200 × magnification with 100 μm of scale). Data are represented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 between groups

Fig. 3.

Oral gavage of PQQ restores the diversity of gut microbiota in the GD group. A) Rank-abundance curves of the gut microbiota at the OTU level, B) rarefaction curves (SOBs index) of the gut microbiota at the OTU level. Comparison of a diversity in C) Sobs, D) Ace, E) Chao indices in Con, GD and PQQ/mid groups at the OTU level. Analysis of β diversity using F) PCA, G) PCoA based on bray_curtis, H) binary_jaccard and I) unweighted_unifrac distance and J) NMDS on the genus level. **p < 0.01; ***p < 0.001. OTUs – operational taxonomic units, PCA – principal component analysis, PCoA – principal coordinate analysis, NMDS – non-metric multidimensional scaling

Fig. 4.

PQQ alters the composition and abundance of intestine flora in the GD group. A) The species Venn diagram at the genus level. Overlapping parts in the figure indicate common species. On the genus level, percent of community abundance unique to B) the Con group, C) GD group and E) PQQ group. D) Percent of community abundance shared by 3 groups.

Fig. 4.

PQQ alters the composition and abundance of intestine flora in the GD group. F) Percent of community abundance of 3 groups on the Phylum level. G) Percent of community abundance of 3 groups on the Genus level. On the genus level, percent of community abundance in H) the Con group, I) GD group and J) PQQ group.

Fig. 4.

PQQ alters the composition and abundance of intestine flora in the GD group. I) GD group and J) PQQ group. K) The heatmap on the abundance of top 30 genera among 3 groups. L) The Circos analysis of the abundance association between the sample and the top 10 genera.

Fig. 5.

Spearman correlation analysis and PICRUSt prediction under PQQ supplement in the GD group. A) Kruskal-Wallis H test validated the differential distributed genera and C) the abundance of Lactobacillus among all 3 groups. B) LEfSe analysis showed that the relative abundance of 14 genera was significantly different among all groups (LDA score > 3.0 or < –3.0, p < 0.05). LEfSe – linear discriminant analysis effect size, LDA – linear discriminant analysis, KEGG – Kyoto Encyclopedia of Genes and Genomes, COG – Clusters of Orthologous Groups, RDA – redundancy analysis

Fig. 5.

Spearman correlation analysis and PICRUSt prediction under PQQ supplement in the GD group. D) The Heatmap of KEGG pathway, and E) the COG functional abundance box distribution based on the PICRUSt prediction among 3 groups. LEfSe – linear discriminant analysis effect size, LDA – linear discriminant analysis, KEGG – Kyoto Encyclopedia of Genes and Genomes, COG – Clusters of Orthologous Groups, RDA – redundancy analysis

Fig. 5.

Spearman correlation analysis and PICRUSt prediction under PQQ supplement in the GD group. D) The Heatmap of KEGG pathway, and E) the COG functional abundance box distribution based on the PICRUSt prediction among 3 groups. F) RDA results of gut microbiota and environmental factors (T4, TRAB, IL6, TNFα, Nrf2 and HO1) of mice in 3 groups. G) Network Diagram based on the Collinearity Analysis in 3 groups. LEfSe – linear discriminant analysis effect size, LDA – linear discriminant analysis, KEGG – Kyoto Encyclopedia of Genes and Genomes, COG – Clusters of Orthologous Groups, RDA – redundancy analysis

Fig. 5.

Spearman correlation analysis and PICRUSt prediction under PQQ supplement in the GD group. H) Spearman Correlation Analysis between samples and top 50 genera. LEfSe – linear discriminant analysis effect size, LDA – linear discriminant analysis, KEGG – Kyoto Encyclopedia of Genes and Genomes, COG – Clusters of Orthologous Groups, RDA – redundancy analysis