The Potential Role of Pyrroloquinoline Quinone to Regulate Thyroid Function and Gut Microbiota Composition of Graves’ Disease in Mice
Article Category: ORIGINAL PAPER
Publicado en línea: 16 dic 2023
Páginas: 443 - 460
Recibido: 02 ago 2023
Aceptado: 27 oct 2023
DOI: https://doi.org/10.33073/pjm-2023-042
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© 2023 Xiaoyan Liu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Graves’ disease (GD) is an autoimmune disorder characterized by an enlarged thyroid gland, Graves’ orbitopathy (GO), an accelerated heart rate, and hyperthyroidism (Wémeau et al. 2018; Davies et al. 2020; Hoang et al. 2022). Moreover, the upregulation of thyroid-stimulating hormone receptor antibodies (TRAB) and free thyroxine (fT4) is the main immunological feature in GD (Ehlers et al. 2019; Davies et al. 2020). In GD, the loss of immune tolerance to thyroid antigens is attributed to a complex interplay between genetic and environmental factors (Neag and Smith 2022). It is well known that T-cell-mediated immunity, TSHR, and mesenchymal stem cell properties in orbital fibroblasts have participated in the pathogenesis of GO (Taylor et al. 2020). However, the precise mechanism underlying Graves’ disease (GD) remains unclear. Recently, some studies have provided new insight into gut microbiota referring to the pathogenesis of GD (Knezevic et al. 2020; Moshkelgosha et al. 2021).
The human body harbors trillions of microorganisms on both mucosal and epidermal surfaces, with a significant concentration in the gastrointestinal tract. The intestinal microbiota is crucial for human health and has physiological functions such as regulating nutrient metabolism, immune regulation, and protecting the intestinal structure and function (Jandhyala et al. 2015). Once the intestinal microbiota is disrupted, it can cause various diseases, such as asthma, allergies, inflammatory bowel disease (Arrieta et al. 2015; Bunyavanich et al. 2016; Nishino et al. 2018). Currently, many studies have shown an association between gut microbiota and thyroid diseases. The gut microbiota has been reported to regulate thyroid hormone metabolism by controlling the absorption and degradation of thyroid hormone iodine and the enterohepatic circulation of thyroid hormone (Lin and Zhang 2017; Fröhlich and Wahl 2019). There are also studies indicating that the imbalance of Th17 and Treg cells caused by gut microbiota is related to the pathogenesis of GD (Smith et al. 2013). Thus, it can be seen that the gut microbiota is closely related to the occurrence and development of GD.
In 1964, pyrroloquinoline-quinone (PQQ) was initially identified as a bacterial dehydrogenase coenzyme and has since been associated with various biological processes such as mitochondriogenesis, growth reproduction, and aging (Hauge 1964; Mohamad Ishak and Ikemoto 2023). Due to its quinone structure, PQQ can undergo reduction by numerous substances and can be restored to its oxidized state by oxygen (Mohamad Ishak and Ikemoto 2023). A study suggests that in an osteoporosis model induced by estrogen deficiency, dietary supplementation with PQQ can inhibiting oxidative stress and osteocyte senescence to alleviate bone loss effectively (Geng et al. 2019). Consequently, the supplementation of PQQ was acknowledged for its significant role in promoting longevity, as well as its antioxidant and anti-inflammatory effects (Ouchi et al. 2013; Sasakura et al. 2017; Yin et al. 2019). However, limited attention was given to the effects of PQQ treatment on GD mice and gut microbiota. Therefore, the objective of this study is to explore the potential therapy effects of PQQ in regulating GD and reconstructing intestine homeostasis.
Eight-week-old female BALB/c mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). They were maintained in a specific pathogen-free facility (Shanghai Tenth People’s Hospital Animal Center, China) under standard conditions with 22 ± 2°C, 60 ± 5% humidity and 12-hour light/dark cycles. Mice were fed with a standard diet and water. All animal experiments were approved by the Animal Care Institution and Ethics Committee of Shanghai Tenth People’s Hospital (20KT127).
The construction of recombinant adenovirus Ad-TSHR289 encoding TSHR: the subunit and identification of virus transfection ability were carried out by Shanghai Jikai Gene Technology Co., Ltd. (China). All the Ad-TSHR289 were mixed with phosphate-buffered saline (PBS, pH = 7.3) in a total volume of 100 μl for immunization. Then, the prepared Ad-TSHR289 solution was injected into mice for three times at 3-week intervals.
PQQ (purity > 98%) was provided by Shanghai Maclin Biochemical Technology Co., Ltd. (China). The mice were randomly divided into six groups (5 mice per group): 1) control group (treated with PBS only); 2) GD group (treated with Ad-TSHR289 only); 3) GD + PQQ low group (treated a low concentration of PQQ, i.e., 20 mg PQQ/kg BW/day); 4) GD + PQQ mid group (treated with a middle concentration of PQQ, i.e., 40 mg PQQ/kg BW/day); 5) GD + PQQ high group (treated with a high concentration of PQQ, i.e., 60 mg PQQ/kg BW/day) PQQ was dissolved in PBS in 0.2 ml per mouse and administered orally from 4 weeks to 8 weeks after the last injection (Qu et al. 2022); 6) GD + MMI (methimazole) (Meilun Biotech Co., Ltd., China) group (treated with MMI, i.e., 2.5 mg MMI/kg BW/day). MMI was dissolved in PBS in 0.2 ml per mouse and administered orally from 4 weeks to 8 weeks after the last injection. After the final oral gavage, mice were sacrificed for further tests.
After the mice were euthanized, the separated thyroid and small intestine tissue were fixed with 10% buffered formalin, paraffin-embedded sections were stained with the hematoxylin and eosin (H&E) (Sigma-Aldrich®, USA), and the histopathological changes, including the severity of inflammatory cell infiltration and the extent of crypt damage were observed under light microscopy to access the intestinal barrier integrity. In this study, the board-certified pathologist blinded to the experimental condition samples scored the samples.
Total thyroxine (T4) and TSH receptor autoantibodies (TRAb) in mouse serum were measured using Mouse T4 Elisa Kit (YCQZ-10219; shycbio, China), Mouse TRAB Elisa Kit (YCMZ-35270; shycbio, China) according to the manufacturers’ instructions.
Total RNA was extracted from the small intestine by using the Total RNA Isolation Kit V2 RC112 (Vazyme Biotech Co., Ltd., China). Reverse transcription was carried out using HiScript® III All-in-one RT SuperMix Perfect for qPCR R333 (Vazyme Biotech Co., Ltd., China). RT-qPCR was performed on the LightCycler® 96 System (Roche Diagnostics (Shanghai) Ltd., China) using Taq Pro Universal SYBR qPCR Master Mix Q712 (Vazyme Biotech Co., Ltd., China). The relative expression of mRNAs was performed by the 2ΔΔCt method using GAPDH mRNA for normalization. The primer information is shown in Table SI.
Fecal samples of mice were collected after the final oral gavage, snap-frozen in liquid nitrogen, and stored at –80°C until use. The DNA of total bacteria in mice feces was extracted with the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Inc., USA) according to the manufacturer’s protocols. The purity and concentration of DNA were detected by NanoDrop™ 2000 (Thermo Fisher Scientific Inc., USA). The thermocycler polymerase chain reaction system GeneAmp™ 9700 (Applied Biosystems, USA) was utilized to amplify the bacterial 16S ribosomal RNA (rRNA) genes hypervariable region 3 (V3) and 4 (V4) with the primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). To ensure the accuracy of the subsequent data analysis, each sample was amplified three times. The products were further purified using the Axygen® AxyPrep DNA Gel Extraction Kit (Corning, USA), and quantitative analysis was carried out using QuantiFluor™-ST Fluorometer (Promega Corporation, USA), according to the manufacturer’s protocol. Sequencing was performed on an Illumina® MiSeq™ platform (Illumina, Inc., USA) by combining the purified amplicons at equimolar concentrations and performing end pair sequencing analysis.
The analysis of 16S rRNA gene sequencing data was performed in the Majorbio Cloud Platform (
This study presents the data as means ± SEM. To assess significance, the mean values of independent groups were compared using Student’s
The GD model in mice was initially established using Ad-TSHR289, and its validity was confirmed through the measurement of serum TSHR and T4 levels using Elisa, as well as the examination of the thyroid gland using HE staining (Fig. 1). Our findings revealed a significant increase in serum TSHR and T4 levels (Fig. 1A and 1B), along with a noticeable enlargement of the thyroid gland (Fig. 1C) in the GD group compared to the controls. Subsequently, we evaluated the therapeutic effects of PQQ at varying concentrations, namely low, middle, and high. The results showed that serum TSHR and T4 were remarkably decreased (Fig. 1A and 1B). The size of the thyroid gland was typically reduced (Fig. 1C) in three cohorts of PQQ replenishment compared with the GD group, suggesting the significant effect of PQQ treatment on GD, among which middle concentration group demonstrated the best efficacy (Fig. 1).
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, *
Alterations in the thyroidgut axis are considered one of the modern views of GD pathogenesis (Knezevic et al. 2020). Consequently, our study aimed to investigate the impact of PQQ treatment on the small intestine system. In the GD model,
Fig. 2.
PQQ supplement decreases inflammation and oxidative stress response and alleviates small intestine epithelial injury. Relative expression of mRNA in colons of A)
Data are represented as mean ± SEM, *
In our previous studies, we have investigated the close relationship between GD and gut microbiota (Jiang et al. 2021). Given the significant efficacy of PQQ at moderate concentrations, we were particularly interested in examining the impact of PQQ with a moderate concentration on the alteration of gut microbiota in GD. The analysis of rank-abundance curves and rarefaction curves of the Sobs index at the OTU level revealed that the species in the three groups exhibited a uniform and abundant distribution, with the sequencing data being sufficient in quantity (Fig. 3A and 3B). Specifically, there were a total of 15 phyla, 25 classes, 57 orders, 86 families, 162 genera, and 236 species, and 739 OTU in the controls; 9 phyla, 15 classes, 36 orders, 55 families, 116 genera, 183 species and 667 OTU in the GD group; and 10 phyla, 14 classes, 44 orders, 78 families, 163 genera, 266 species, and 1045 OTU were in the PQQ group. By examining a diversity using Sobs, Ace, and Chao indices at the OTU level, it was observed that the diversity and abundance were significantly reduced in the GD group than controls and greatly improved in the PQQ group (Fig. 3C, 3D and 3E). These findings suggest that the administration of PQQ could effectively restore the diversity of intestinal flora that GD has disrupted. In the analysis of β diversity, including principal component analysis (PCA), principal coordinate analysis (PCoA) based on bray_curtis, binary_jaccard, and unweighted_ unifrac distance, as well as non-metric multidimensional scaling (NMDS) at the genus level, it was evident that the bacterial composition in the GD group differed significantly from that of the control and PQQ groups (Fig. 3F–3J). In contrast, the microbial structure exhibited a high degree of similarity between the control and PQQ groups (Fig. 3F–3J). The above results demonstrated that the PQQ supplement could reconstruct the microbiota composition influenced by GD.
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. **
The taxon compositions of the three groups were determined based on the genus level using a Venn diagram (Fig. 4A). The results indicated that
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.
Differential distributed genera validated by the Kruskal-Wallis
Fig. 5.
Spearman correlation analysis and PICRUSt prediction under PQQ supplement in the GD group.
A) Kruskal-Wallis
B) LEfSe analysis showed that the relative abundance of 14 genera was significantly different among all groups (LDA score > 3.0 or < –3.0,
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,
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
The etiology of GD might be intricately linked to autoimmunity and oxidative stress (Hoang et al. 2022). However, the specific role of PQQ, a natural antioxidant compound, in GD mice remains unclear. In our study, we sought to ascertain the impact of PQQ supplementation on GD mice and their gut microbiota, and the key findings were as follows: i) The serum TSHR and T4 were decreased and the thyroid gland size was typically reduced under PQQ supplement in GD. ii) PQQ reduced inflammation and oxidative stress response and attenuated small intestine epithelial injury. iii) PQQ played a significant role in restoring the diversity and composition of microbiota. iv) Differential distributed genera validated by the Kruskal-Wallis
In the initial phase of our study, we assessed the influence of different concentrations of PQQ on GD. It was observed that low, middle, and high concentrations of PQQ all demonstrated a beneficial therapeutic impact on thyroid function, with the middle concentrations yielding the most favorable outcomes. Furthermore, Kumar et al. (2014) documented the alleviation of detrimental effects, such as increased serum T3 and T4 levels, in hyperthyroidism rats after administering PQQ. Our findings further verified the PQQ’s repair results on the thyroid gland. Following this, we assessed the potential impacts of PQQ on the intestinal system. Surprisingly, PQQ demonstrated the capacity to not only regulate thyroid autoimmune conditions but also alleviate inflammation and oxidative stress within the barrier of the intestinal tract. It is noteworthy that the maintenance of an intact intestinal barrier is currently acknowledged as a pivotal factor in overall health (Assimakopoulos et al. 2018). Recent research showed that impaired barrier function and microbial dysbiosis are connective with gastrointestinal disease, autoimmune disease, and a broken metabolic status in the host represented (Aldars-García et al. 2021; Fuhri Snethlage et al. 2021; Jonscher et al. 2021). In some animal models, the exposure to PQQ improved tight junction protein expression and increased jejunal barrier function, demonstrating that PQQ might act through the intestine to affect peripheral tissues (Friedman et al. 2018; Yin et al. 2019; Huang et al. 2020). Similarly, we also defined the resistance function of PQQ on the impaired gut barrier. More experiments are needed to associate thyroid autoimmune with a destructed intestinal mucosal barrier.
We next performed the 16S rDNA sequencings and conducted the microbial analyses for GD under PQQ replenishment. In our previous study, we reported that a diversity on the OTU level in Ace, Sobs, Chao, Simpson, Shannon, and Coverage indices were decreased in the GD group compared to controls (Jiang et al. 2021). In this study, we further validated this conclusion and found the potential of PQQ in improving the a diversity affected by GD. In the piglet model, the Shannon index of the PQQ group was higher than that of the controls (Huang et al. 2020). The findings of this study indicated that the administration of PQQ could potentially restore the abundance of specific microbial genera. The β diversity analysis revealed significant alterations in bacterial distribution due to the GD. However, PQQ could preserve the original composition of the gut microbiota. Similar results were observed in studies conducted on piglet and obese mice models (Friedman et al. 2018; Huang et al. 2020), suggesting that the protective effects of PQQ might be attributed to its ability to maintain the stability of the gut microbiota.
On the phylum level, the abundance of Firmicutes and
Therefore, it is hypothesized that besides the heightened basal metabolic rate, the thyroid hormones may also influence the composition and functionality of the microbiota, thereby contributing to alterations in weight. On the genus level, the abundance of
In our study, the KEGG database was utilized to assess the functions of distinct genera exclusive to the control, GD, and PQQ groups. We found that the “Metabolic pathway” and “Biosynthesis of secondary metabolites” exhibited in the predicted results, suggesting that these pathways were highly associated with the occurrence of controls, GD, and PQQ groups. In addition, the COG database was also involved in predicting the function in three groups. We found that the top function prediction in the three groups was characterized as “S: Function unknown”. The findings imply that there might still be an unexplored metabolic pathway in the progression of GD, and it should be the target of future research. Meanwhile, Collinearity Analysis and Spearman Correlation Analysis showed that
Our research has several limitations that should be acknowledged. Firstly, the evaluation of the effects of PQQ treatment on the thyroid and intestine lacks comprehensiveness. Additionally, the sample size and animal model employed in our study are small and single. Finally, the underlying mechanisms of PQQ supplement between thyroid and gut microbiota are unclear. Therefore, further investigations incorporating larger sample sizes, clinical research, and comprehensive analyses are imperative for future studies.
In summary, the supplementation of PQQ regulates thyroid function and mitigates thyroid injury, as well as reduces inflammation and oxidative stress response, thereby attenuating small intestine epithelial injury. Moreover, in microbial analyses, PQQ restores the diversity of gut microbiota and maintains the bacteria composition, initially impaired by GD. Notably, PQQ changes the primary composition and abundance of GD’s intestine microbiota by moderating