Correlation between depressive-like behavior and gut microbiota in mice with hypothyroidism

Abstract

BACKGROUND

The association between hypothyroidism and depression is well established, but the underlying mechanisms remain unclear.

AIM

To explore the potential role of the gut microbiota in depressive-like behaviors in a mouse model of hypothyroidism, with a focus on bacterial composition.

METHODS

Hypothyroidism was induced in mice using propylthiouracil. Depressive-like behaviors were assessed using the sucrose preference test (SPT), forced swimming test (FST), tail suspension test (TST), and open field test (OFT). Inflammatory cytokines, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α, and IL-10, were quantified, together with colon histopathology scores, brain-derived neurotrophic factor, nuclear factor κB, inhibitor of nuclear factor κB, and tight junction proteins (occludin, claudin-1, zonula occludens-1). Gut microbial composition was determined by 16S rRNA gene sequencing of fecal samples.

RESULTS

Propylthiouracil-treated mice exhibited pronounced depressive-like behaviors, intestinal barrier dysfunction, elevated peripheral and central inflammation, and gut microbiota dysbiosis. Pearson correlation analysis showed that Bilophila and Psychrobacter abundance positively correlated with sucrose preference in the SPT and locomotor activity in the OFT, and negatively correlated with immobility times in the FST and TST. Gordonibacter abundance was positively correlated with locomotion in the OFT and negatively correlated with immobility in the FST and TST. Prevotellaceae_UCG_001 was inversely correlated with immobility in the FST and TST. Streptococcus was positively associated with sucrose preference in the SPT.

CONCLUSION

The observed associations between specific bacterial taxa and behavioral indices support a potential connection between gut microbiota composition and depressive symptoms in mice with hypothyroidism.

Key Words: Hypothyroidism; Gut microbiota; Depression; Inflammation; Intestinal barrier

Core Tip: Our research has found that hypothyroidism-induced depression is associated with gut microbiota dysbiosis, and the intestinal barrier dysfunction and the activation of peripheral and central inflammation play important roles between the two. Furthermore, through Pearson correlation analysis, we found that specific bacterial genera including Bilophila and Gordonibacter, correlate with depressive-like behaviors, suggesting that microbiota-targeted interventions may offer therapeutic benefits for depression associated with hypothyroidism.

INTRODUCTION

Hypothyroidism is a prevalent endocrine disorder characterized by decreased levels of thyroid hormones, which can result in various symptoms, including depressed mood, reduced appetite, fatigue, and constipation. Recent research has established a strong correlation between hypothyroidism and depression onset. Patients with hypothyroidism are at a higher risk of experiencing depressive symptoms[1,2], with an incidence rate of approximately 60%[3]. Depression is a multifaceted psychological disorder influenced by biological, psychosocial, and lifestyle factors. Its primary clinical manifestations include persistent feelings of sadness, diminished interest in activities, and, in severe cases, suicidal tendencies, all of which significantly affect patients’ quality of life and overall physical health. Worldwide, depression is responsible for more disability-adjusted life years than any other condition[4].

Growing awareness of depression has prompted researchers to investigate its origins more thoroughly, revealing a nuanced connection between depressive disorders and the gut’s microbial community. The gut, often referred to as the “second brain”, engages in a bidirectional communication with the brain through the gut-brain axis, with gut bacteria playing a crucial role. An imbalance in gut bacteria can affect both the peripheral and central nervous systems via this axis, potentially contributing to the development and/or severity of depression[5]. Remarkably, animals without depression that receive gut bacteria from those with the condition exhibit behaviors indicative of depression[6]. Moreover, a substantial body of research suggests an interplay between depression and inflammation, where these two conditions can exacerbate one another in a cyclical manner[7,8]. Inflammatory markers can also traverse the gut-brain axis, highlighting their role in the development of depression[9]. A study by Simpson et al[10] identified diminished diversity in the gut bacteria of individuals with depression, characterized by a reduction in bacteria that combat inflammation and an increase in those that promote it. The decline in bacteria that counteract inflammation can compromise the gut’s mucosal barrier, elevating the risk of bacteria and their byproducts entering the bloodstream[11,12]. In depression, the regulation of inflammatory cytokines, such as interleukins (ILs), tumor necrosis factor, and interferons, is critical[13]. Variations in the levels of these cytokines may be associated with depression pathogenesis. Furthermore, research suggests that irregular cytokine production is associated with the modulation of the toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) inflammatory pathway[14]. Therefore, altered gut bacteria and the inflammatory signals they generate could penetrate the blood-brain barrier, affecting brain function and potentially contributing to depression[15]. Depression is also closely associated with brain-derived neurotrophic factor (BDNF), a protein that promotes neuronal growth and is central to depression pathogenesis[16,17]. Treatment with antidepressants can rescue reduced BDNF levels in individuals with depression[18]. Additionally, BDNF exerts an antidepressant effect in animals with depressive symptoms[19]. Clinical studies indicate a moderate association between hypothyroidism and depression (odds ratio = 1.30, 95% confidence interval: 1.08-1.57)[1]. However, the precise mechanisms linking hypothyroidism to depression remain insufficiently understood. The current study aims to explore the potential connection between gut bacteria and depression in mice with hypothyroidism, potentially paving the way for new therapeutic approaches that target the gut microbiome.

MATERIALS AND METHODS

Animal treatments

Eight-week-old C57BL/6J mice weighing between 20 g and 23 g, were acquired from Chongqing Lepet Bio-technology Co., Ltd. and maintained under controlled environmental conditions, including a 12-hour light/dark cycle and a temperature of 23 ± 2 °C. The mice were randomly allocated to two groups, each consisting of six animals. The first group received regular tap water, whereas the second group was given tap water supplemented with 0.05% propylthiouracil (PTU). The study duration was 30 days. Prior to the commencement of the experiment, the mice were allowed a one-week adaptation period during which they had unrestricted access to both water and food.

Sucrose preference test

The sucrose preference test (SPT) evaluates anhedonia in rodents[20]. Prior to the SPT, mice were acclimatized for 2 days to drinking from two bottles. On the first day, both bottles contained regular drinking water. On the second day, the water in both bottles was substituted with a 1% sucrose solution. On the third day, the mice underwent a 24-hour fasting period without access to food or water. On the fourth day, the mice were presented with one bottle of regular drinking water and one bottle of 1% sucrose solution for a 2-hour assessment. The positions of the bottles were alternated every 30 minutes to mitigate positional bias. Bottle weights were recorded before and after the assessment to quantify the consumption of regular drinking water and the sucrose solution. The sucrose preference rate was calculated as the sucrose consumption divided by the total consumption of sucrose and regular drinking water, multiplied by 100%.

Tail suspension test

The tail suspension test (TST), originally described by Steru et al[21], measures the duration of immobility in response to tail suspension following their established methodology. In this procedure, mice were suspended using adhesive medical tape placed 2 cm from the end of the tail, elevated to a height of 25 cm above the ground. A team of three observers blinded to the treatment groups recorded the duration of immobility during the last 4 minutes of a 6-minute trial, following an initial 2-minute acclimatization period. Immobility was defined as the absence of struggle or movement, indicating a state of despair. Prolonged immobility in this test suggests a greater level of depressive behavior.

Forced swimming test

The forced swimming test (FST) serves as a standard procedure for assessing depressive-like behavior in rodents. Mice were placed in a transparent cylindrical container (20 cm diameter, 30 cm height) filled with water to a depth of 15 cm, maintaining the water temperature at 24 ± 1 °C. A group of three observers blinded to the treatment conditions recorded the duration of immobility during the final segment of a 6-minute trial, following a 2-minute acclimatization period. Immobility was defined as the cessation of escape attempts, with the mouse floating passively, which is interpreted as a manifestation of despair. An increased duration of immobility in the FST indicates a more pronounced depressive phenotype.

Open field test

Mice were placed in a box measuring 50 cm × 50 cm × 50 cm, with the floor divided into 16 squares; the central four squares were labeled as the central area, whereas the remaining 12 squares comprised the peripheral area. At the start of the experiment, mice were introduced into the center of the open field, and their movements were recorded over a 5-minute period. Tracker software was employed for further analysis and to generate trajectory maps of their movement within the open field. Indicators for depression included the total distance traveled by the mice, the frequency of entries into the central area, and the duration of stay in the central area.

Fecal collection and tissue dissection

Following behavioral testing, fecal samples were collected from each mouse. The mice were subsequently anesthetized using isoflurane, after which blood was drawn via exsanguination through ocular removal. The blood was processed by centrifugation at 3000 g at 4 °C for 10 minutes to isolate the serum. Euthanasia was performed via cervical dislocation, after which the hippocampal region was extracted from the decapitated mice. Furthermore, colonic tissue, specifically 1 cm proximal to the cecum, was harvested, divided into two segments, with one preserved in a 4% paraformaldehyde solution and the other frozen at -80 °C.

Histopathological analysis

The colon tissue was initially fixed in 4% paraformaldehyde and subsequently dehydrated in a series of ethanol solutions (70%, 80%, 90%, 95%, and 100%), with each treatment lasting 1 to 2 hours, including two treatments with 100% ethanol. Following dehydration, the tissue was clarified in xylene, with each treatment lasting 30 minutes and repeated twice. The clarified tissue was immersed in paraffin at 60 °C for 30 minutes and subsequently embedded in a paraffin block. The embedded tissue was sectioned into 5 μm slices using a microtome, and sections were examined under a microscope.

Immunofluorescence

The paraffin-embedded sections were incubated in citrate buffer in a microwave at temperatures ranging from 95-100 °C for 10-20 minutes, after which they were allowed to cool to ambient temperature. Permeabilization was achieved with 0.1%-0.3% Triton X-100 in phosphate buffered saline (PBS) for 10-15 minutes, followed by rinsing with PBS. To minimize nonspecific binding, sections were blocked with either 5% bovine serum albumin or 10% normal goat serum for 60 minutes. The sections were then incubated with appropriately diluted primary antibodies, either overnight at 4 °C or for 1-2 hours at 37 °C. After primary antibody incubation, the sections underwent three rounds of PBS washing for 5 minutes each. They were then treated with fluorescent secondary antibodies for 1 hour at room temperature in a light-protected environment. Following secondary antibody incubation, another set of three PBS washes for 5 minutes each was performed. DAPI staining was conducted by immersing the sections in a DAPI-PBS solution for 5 minutes in the dark, followed by a PBS rinse. The sections were subsequently mounted and stored in darkness until examined under a fluorescence microscope.

16S rRNA analysis of fecal samples

Polymerase chain reaction (PCR) products were purified using magnetic bead technology, equally pooled based on concentration, and thoroughly mixed. PCR products were evaluated for target bands, which were extracted and prepared for library construction. Library concentrations were quantified using Qubit and quantitative PCR assays. Data extraction for each sample was conducted offline in accordance with the Barcode sequence and PCR primer sequence. Barcode and primer sequences were trimmed, and FLASH (version 1.2.11) was employed to merge the reads for each sample, generating the initial tags data (raw tags). Cutadapt software was subsequently utilized to align with the reverse primer sequence and remove residual sequences to reduce interference in subsequent analyses. Fastp software (version 0.23.1) was applied to rigorously filter the merged raw tags, yielding high-quality tags data (clean tags). These clean tags underwent chimera sequence removal through comparison with species annotation databases - Silva for 16S/18S and Unite for internal transcribed spacer - to identify and eliminate chimera sequences, resulting in the final effective data (effective tags). The effective tags were further refined using the DADA2 module or deblur within QIIME2 software (version QIIME2-202202), with DADA2 set as the default, to derive the final amplicon sequence variants and feature table. Species annotation was performed with QIIME2 software against the Silva 138.1 database for 16S. QIIME2 software was also utilized for rapid multiple sequence alignment to establish phylogenetic associations among all amplicon sequence variant sequences. Finally, sample data were normalized, using the sample with the lowest data volume as the benchmark for normalization. Regarding the links provided for FLASH, Silva, and Unite databases, there may have been an issue with accessing these websites due to network constraints or the validity of the uniform resource locators. Should you require information from these databases, it is advisable to verify the uniform resource locators for accuracy and attempt to access them again later. If the issue persists, seeking alternative sources or contacting the database administrators for assistance may be necessary.

Statistical analysis

Experimental data were analyzed using SPSS software (version 20.0) and visualized with GraphPad Prism (version 8.0). Results are presented as mean ± SEM. Tests for normality and homogeneity of variance were conducted prior to analysis. If the data met these criteria, one-way analysis of variance was employed to analyze inter-group differences, with least significant difference for post-hoc pairwise comparisons. For normally distributed data with unequal variances, the Welch test was applied. The Kruskal-Wallis test was used for data not adhering to a normal distribution. A P value less than 0.05 was considered statistically significant.

RESULTS

PTU-induced hypothyroidism in mice showed depression-like behavior

The PTU-induced hypothyroidism mouse model is a well-established experimental approach[22]. As shown in Figure 1A, the study includes a control group (control, n = 6) and a hypothyroid group (HO, n = 6). The control cohort received standard drinking water, whereas the HO cohort was administered water containing 0.05% PTU to induce hypothyroidism over a 30-day period. Following this duration, we conducted behavioral assessments [SPT, FST, TST, open field test (OFT)] on the mice, subsequently performing euthanasia and sample collection. The HO group demonstrated significant weight loss compared to the control group after 30 days of consuming 0.05% PTU water (P < 0.001, Figure 1B). Baseline examinations indicated a significant reduction in free thyroxine, thyroxine, and thyroid-stimulating hormone levels (free thyroxine: P < 0.05; thyroxine: P < 0.05; thyroid-stimulating hormone: P < 0.05, Figure 1C-E), confirming the successful establishment of a hypothyroid state. We then proceeded with behavioral testing. One commonly utilized method to examine anhedonia, a prominent depressive symptom in rodents, is the SPT[20]. The SPT results revealed that the HO group exhibited a significantly lower preference for sugar water compared to the control group (P < 0.001, Figure 1F).

Figure 1 Mice with hypothyroidism exhibit depression-like behaviors. A: Experimental setup: Mice were divided into control and hypothyroid groups (HO), with the HO group receiving propylthiouracil in their drinking water for 30 days; B: Body weight significantly decreased in the HO group over time; C-E: Hormonal analysis reveals reduced levels of free thyroxine and total thyroxine, and increased thyroid-stimulating hormone in the HO group, confirming hypothyroidism; F: The sucrose preference test shows a reduced preference in the HO group, suggesting anhedonia; G and H: The tail suspension and forced swimming tests indicate increased immobility times in the HO group, reflecting depressive-like behavior; I: Movement paths in the open field test demonstrate reduced activity in the HO group; J-L: The open field test results show decreased total distance traveled, fewer entries into the center, and less time spent in the center for the HO group, indicating anxiety-like behavior. Results are depicted as mean ± SEM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 indicate statistical significance. HO: Hypothyroid group; PTU: Propylthiouracil; T4: Thyroxine; FT4: Free thyroxine; TSH: Thyroid-stimulating hormone.

Immobility time in the TST and FST is a well-established measure of depressive-like behavior[23]. The HO group showed significantly increased immobility time in both TST and FST compared to the control group (TST: P < 0.001; FST: P < 0.001; Figure 1G and H). The OFT assesses depressive behavior by evaluating total distance traveled, number of entries into the central area, and time spent in the center[24]. Movement patterns of the control and HO groups during the OFT are depicted in Figure 1I. When compared to the control group, the HO group traveled a significantly shorter distance (P < 0.001, Figure 1J), made fewer entries into the central area (P < 0.001, Figure 1K), and spent less time in the central area (P < 0.001, Figure 1L). These findings indicate that PTU-treated mice exhibit behaviors characteristic of depression.

Peripheral and central inflammation is activated in hypothyroidism mice

A growing body of research indicates that inflammation plays a crucial role in depression onset[8,25,26]. Patients with depression frequently exhibit higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines in both peripheral and central nervous systems[13]. We assessed cytokine levels in the peripheral and central nervous systems of mice (Figure 2A-D), specifically inflammatory cytokines in the colon and hippocampus. The HO group shows significantly higher levels of IL-6, IL-1β, and tumor necrosis factor-α, and lower IL-10 levels compared to the control group. Furthermore, we utilized enzyme linked immunosorbent assay to analyze cytokine expression in serum, colon, and hippocampus (Figure 2E-H). These results suggest that hypothyroidism leads to elevated pro-inflammatory and reduced anti-inflammatory cytokines in both peripheral and central nervous systems. Figure 2I displays hematoxylin-and-eosin-stained colon sections from both groups. The control group exhibited minimal inflammatory cell infiltration in the mucosal lamina propria, with a slightly loose submucosa and no significant inflammatory cell infiltration. In contrast, the HO group showed increased mucosal lamina propria infiltration, with inflammatory cells aggregating in clusters in some areas, and a loose, edematous submucosa with scattered inflammatory cell infiltration.

Figure 2 Inflammation is present in both peripheral and central regions of hypothyroid mice. A-D: Quantitative polymerase chain reaction analysis reveals elevated levels of inflammatory cytokines [interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α, IL-10] in both the colon and hippocampus of hypothyroid group (HO) mice; E-H: Enzyme linked immunosorbent assay results indicate higher concentrations of these cytokines in the serum, colon, and hippocampus of HO mice; I: Hematoxylin and eosin staining of colon tissue shows increased inflammatory cell infiltration in the HO group; J-L: Quantitative polymerase chain reaction demonstrates increased mRNA levels of nuclear factor κB (NF-κB) and decreased levels of inhibitor of NF-κB α and brain-derived neurotrophic factor in the hippocampus of HO mice; M: Western blot analysis confirms higher protein levels of NF-κB and lower levels of inhibitor of NF-κB α and brain-derived neurotrophic factor in the hippocampus. Results are presented as mean ± SEM, with ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 indicating statistical significance. HO: Hypothyroid group; IL: Interleukin; TNF: Tumor necrosis factor; NF-κB: Nuclear factor κB; IκB: Inhibitor of nuclear factor κB; BDNF: Brain-derived neurotrophic factor.

Depression is associated with activation of the hippocampal TLR4/NF-κB inflammatory pathway[15]. Chronic inflammation in depression can activate this pathway through various mechanisms[27], leading to a cycle of increased peripheral and central inflammation that may result in depression. Additionally, heightened inflammation can reduce BDNF expression[28], a recognized biomarker for depression[19]. NF-κB and inhibitor of NF-κB (IκB-α) play critical roles in the TLR4/NF-κB pathway[29]. When activated, the transcription factor NF-κB can drive the overexpression of pro-inflammatory cytokines, whereas IκB-α is a negative regulator and binds to NF-κB to prevent nuclear translocation and inhibit inflammation. We quantified the mRNA levels of NF-κB, IκB-α, and BDNF in the hippocampus of both groups. As illustrated in Figure 2J-L, the HO group exhibited significantly higher NF-κB levels (P < 0.05) and significantly lower BDNF and IκB-α levels (P < 0.001) compared to the control group. We also confirmed protein levels of these factors (Figure 2M). These findings suggest that hypothyroid mice experience activated hippocampal inflammation and reduced BDNF expression.

Intestinal barrier function is impaired in PTU-induced hypothyroidism mice

Disruption of intestinal barrier function significantly contributes to the pathogenesis of neurodegenerative conditions, including depression[30]. When the intestinal barrier is compromised, it can initiate intestinal inflammation that extends to the central nervous system via systemic circulation, resulting in neuroinflammation and potentially leading to depression[31]. To evaluate the status of the intestinal barrier in hypothyroid mice, we employed quantitative real-time PCR, Western blotting, and immunofluorescence to examine the levels of colonic zonula occludens-1, occludin, and claudin-1. As illustrated in Figure 3, the levels of these tight junction proteins in the colon were markedly lower in the HO group compared to the control group. These findings indicate that hypothyroidism impairs intestinal barrier function in mice.

Figure 3 The intestinal barrier is compromised in hypothyroid mice. A-C: The mRNA expression levels of genes encoding tight junction proteins zonula occludens-1 (ZO-1), claudin-1, and occludin in the colon are significantly reduced in the hypothyroid group (HO) compared to the control group; D: Western blot analysis reveals decreased protein levels of ZO-1, claudin-1, and occludin in the HO group; E: Immunofluorescence imaging demonstrates weaker staining for ZO-1, claudin-1, and occludin in the colon tissues of HO mice, further confirming reduced expression. Results are presented as mean ± SEM, with ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 indicating statistical significance. HO: Hypothyroid group; PTU: Propylthiouracil; ZO-1: Zonula occludens-1.

Alterations in gut microbiota diversity and composition in hypothyroid mice

Hypothyroid mice have significant alterations in gut microbiota diversity and composition. The Venn diagram (Figure 4A) illustrates shared and unique operational taxonomic units between the HO and control groups. The alpha diversity, measured by the Chao1 index (Figure 4B), is significantly higher in the HO group compared to the control group, suggesting increased richness. However, the Simpson index (Figure 4C) indicates reduced evenness in the HO group, reflecting a less balanced microbial community. The Shannon index (Figure 4D) further corroborates these changes in diversity. Principal component analysis plots (Figure 4E and F) demonstrate clear separation between the HO and control groups, highlighting notable differences in microbial composition. These findings suggest that hypothyroidism leads to significant shifts in gut microbiota diversity and structure.

Figure 4 Gut microbiota biodiversity and composition in hypothyroidism mice. A: Operational taxonomic units Venn diagram; B: Chao1; C: Shannon; D: Simpson: E: Principal component analysis; F: Principal coordinates analysis. HO: Hypothyroid group; PCA: Principal component analysis.

Relative abundance of gut microbiota in hypothyroid mice and correlation analysis of differential bacteria

As illustrated in Figure 5A and B, we compared the ten microbial taxa exhibiting the most pronounced changes in abundance between the control and HO groups at both the phylum and genus levels. At the phylum level, the HO group demonstrated increased relative abundances of Patescibacteria, Desulfobacterota, Spirochaetota, Campylobacterota, and Deferribacterota, in contrast to the control group, which showed decreases in Actinobacteriota and cyanobacteria. At the genus level, the HO group revealed increased relative abundances of Ligilactobacillus, Escherichia-Shigella, and the Prevotellaceae_NK3B31_group, whereas the Lachnospiraceae_NK4A136_group, Odoribacter, Alloprevotella, Allobaculum, Lactobacillus, and Prevotellaceae_UCG-001 exhibited decreased relative abundances. Cladogram diagrams are frequently employed to illustrate the phylogenetic associations and hierarchical structures among various microbial taxa. Linear discriminant analysis is typically used to analyze intestinal microbiota composition data, with the aim of identifying biomarkers that effectively differentiate between microbial communities. Generally, a linear discriminant analysis score exceeding 2 is regarded as statistically significant. As depicted in Figure 5C and D, we conducted LEfSe analysis, revealing a total of 76 species with significant differences, of which 43 were enriched in the control group and 33 in the HO group. Based on the results presented in Figure 5E, the correlation heatmap illustrates the associations between different bacterial taxa and various behavioral and physiological parameters in hypothyroid mice. Red and blue colors indicate positive and negative correlations, respectively. Notably, certain bacteria, such as Bilophila and Psychrobacter, exhibit significant correlations with specific behavioral tests, highlighting their potential role in the altered gut microbiota associated with hypothyroidism. The heatmap visually represents these associations, with statistical significance denoted by asterisks.

Figure 5 Relative abundance of gut microbiota in hypothyroidism mice. A: At the phylum level, microbiota composition differs between hypothyroid group (HO) and control groups; B: At the genus level, notable changes in bacterial abundance were observed; C: LEfSe analysis identified specific differential bacteria associated with the HO group in the cladogram; D: Linear discriminant analysis scores confirm significant differences in taxa within the HO group; E: The correlation heatmap illustrates associations between differential bacteria and behavioral/physiological parameters, with certain bacteria (e.g., Bilophila and Psychrobacter) associated with depression-like behaviors. ^aP < 0.05, ^bP < 0.01. HO: Hypothyroid group.

DISCUSSION

There is a strong association between hypothyroidism and depression; however, the precise mechanism underlying this association remains unclear. This study aims to investigate the potential correlation between gut microbiota and depression in hypothyroid mice, focusing on intestinal microorganisms. We utilized PTU to establish a hypothyroidism model for our research. The results indicated that mice with induced hypothyroidism displayed significant depressive-like behaviors, which were accompanied by intestinal barrier disruption, activation of both peripheral and central inflammatory responses, and alterations in gut microbiota. An increasing body of research indicates that disturbances in the gut microbiota contribute significantly to the development of depression. These imbalances can result in a compromised intestinal barrier and heightened intestinal permeability, often referred to as “leaky gut”[32]. This condition allows Gram-negative bacteria to enter the bloodstream, where their surface lipopolysaccharide is recognized by TLR4, initiating the TLR4/NF-κB inflammatory pathway. This activation leads to the release of pro-inflammatory cytokines, thereby exacerbating both local and systemic inflammation. This inflammatory state not only damages the intestinal barrier but also affects the central nervous system via the blood-brain barrier, potentially resulting in neuroinflammation and neurotransmitter imbalances that may contribute to depression[15]. Studies have revealed variations in gut microbial composition among individuals with depression[33], characterized by a consistent pattern of increased pro-inflammatory bacteria and decreased anti-inflammatory bacteria[34].

We analyzed the gut microbiota of mice in both control and HO groups using 16S rRNA sequencing. Although no significant differences in α diversity were observed, β diversity revealed a clear separation, indicating an imbalance in the gut flora of the HO group. At the phylum level, the HO group exhibited a reduction in cyanobacteria, which are known for their antidepressant effects due to the production of mood-enhancing metabolites[35]. Additionally, there was an increase in Campylobacterota, which is associated with the exacerbation of depression-like behaviors in chronic stress models[36]. At the genus level, pro-inflammatory bacteria such as Escherichia-Shigella and Prevotellaceae_NK3B31_group increased[37,38], whereas anti-inflammatory genera including Lachnospiraceae_NK4A136_group, Odoribacter, Alloprevotella, Allobaculum, Lactobacillus, and Prevotellaceae_UCG-001 decreased[39-43]. This aligns with findings that individuals with depression tend to have fewer butyrate-producing bacteria and a higher abundance of pro-inflammatory bacteria[44,45]. Butyrate, a microbiota-derived metabolite, enhances epithelial barrier integrity by upregulating tight junction proteins[46]. Decreased butyrate levels may compromise this integrity, facilitating the translocation of microbes and their products into the bloodstream, thereby propagating inflammation. Furthermore, butyrate exhibits neuroprotective and anti-inflammatory properties by inhibiting NF-κB signaling[47,48]. Restoring the balance of gut microbiota and increasing the population of butyrate-producing bacteria could represent a novel therapeutic strategy for addressing depression. Using LEfSe, we analyzed the correlation between differential species and behavioral indicators in mice. Streptococcus, a Gram-positive bacterium, was negatively correlated with sucrose preference, suggesting that its increased abundance may contribute to depressive symptoms. Its prevalence rises in patients with intestinal barrier damage, leading to inflammation and further barrier impairment[49]. Increased abundance of Streptococcus has also been observed in individuals with depression[50], implying its role in the disorder through inflammatory responses and barrier dysfunction.

Gordonibacter exhibited positive correlations with distance traveled in the OFT and negative correlations with immobility in the FST and TST, indicating its potential role in alleviating depressive symptoms. As an Actinobacteria, Gordonibacter can metabolize polyphenols to produce anti-inflammatory metabolites, thereby reducing intestinal and systemic inflammation. It also supports barrier integrity by producing butyrate, which prevents leakage and inflammation[51]. Psychrobacter was positively correlated with the sucrose preference rate in the SPT and total distance in the OFT, whereas it was negatively correlated with immobility time in the FST and TST, suggesting that Psychrobacter is associated with reduced depressive-like behavior. Psychrobacter is a Gram-negative bacterium within the Gammaproteobacteria class. Currently, there are no studies demonstrating that Psychrobacter produces butyrate or other anti-inflammatory metabolites. Based on our findings, we propose that Psychrobacter may have the potential to generate anti-inflammatory metabolites, but this requires further investigation. Such studies will clarify the potential role of Psychrobacter in depression and establish a foundation for developing new treatment strategies for the disorder.

CONCLUSION

In conclusion, our study demonstrated that hypothyroid mice exhibited an imbalance in intestinal flora, accompanied by inflammatory activation and intestinal barrier damage. This condition led to systemic inflammation, ultimately affecting brain function and inducing depressive-like behavior. By analyzing the correlation between intestinal microorganisms and indicators of depressive-like behavior, we identified a potential association between the two. This finding provides new targets and directions for therapeutic strategies focused on intestinal flora and for exploring the pathogenesis of depression.