Depressive disorder, which has a detrimental impact on health systems and psychological well-being, remains among the leading causes of burden, with prevalence estimates and disability weights that are comparatively higher than those of many other diseases(Zhao et al., 2021; Ferrari et al., 2022). The gut microbiome is the major microbial community that settles in the human body and affects the nutrition, metabolism and immune function of the host(Hou et al., 2022). Disturbances in the gut microbial ecosystem have been linked with psychological conditions, including depression(Ortega et al., 2022).

Depression is inextricably linked to changes in gut flora richness and diversity. In healthy people, Bacteroidetes and Firmicutes are the most dominant bacteria, while the abundance of other phyla is relatively small, and the dynamic balance between different bacterial groups is critical to maintain normal intestinal function (Yu et al., 2017; Cheng et al., 2020). Compared with normal people, the richness and diversity of intestinal microorganisms in depressed patients decreases significantly, usually manifested as a decrease in Firmicutes, an increased proportion of Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level, a decrease in Trichospiraceae and Ruminococcaceae at the family level, and a decrease in Faecium, Ruminococcus, Lactobacillus and Bifidobacterium at the genus level (Safadi et al., 2022). Although some studies may report contradictory flora changes due to differences in experimental conditions or subjects, the general consensus is that the flora composition of depressed patients is significantly different from that of healthy patients.

Similarly, changes in the gut microbiota have been reported in animal models of depression. The intestinal microbiota of depressed mice induced by chronic mild unforeseeable stimulation (CUMS) showed a reduced relative abundance of Corynebacterium, Colophilus, Lactobacillus and Faeccoccus (Sudo et al., 2004; Liang et al., 2015). Learned helplessness animal models of depression reduce the relative abundance of bacteria such as Lactobacillus and Clostridium spp. (Takajo et al., 2019). Li (Jianguo et al., 2019) investigated the potential correlation between depression-like symptoms and altered fecal metabolites in CUMS rats and found that altered intestinal microbiota may affect depression-like symptoms in CUMS rats through changes in intestinal metabolites. Transplantation of fecal microbiota from depressed patients into germ-free (GF) rodents resulted in altered depressive-like behavior compared with that in specific pathogen-free (SPF) rats (Yang et al., 2020). In addition, there were similar findings in the intestines of rodents subjected to the weaning isolation model, bilateral olfactory bulb resection model and social failure model. These mice showed typical depression-like behaviors, such as social avoidance and anhedonia, accompanied by intestinal flora changes [i.e., a decreased level of Firmicutes and an increased ratio of Bacteroidetes to Firmicutes (O’Mahony et al., 2009; Bharwani et al., 2017)]. These studies also provide evidence for the changes in the gut microbiota during the development of depression when they show the difference in the gut microbiota in depressed individuals and healthy individuals.

The gut microbiota can influence brain function and behavior through the microbiota-gut-brain axis (Zheng et al., 2021). However, depressive disorders are always long-lasting, and the gut microbial ecosystem is altered as the disease progresses. The specific role of microbiota metabolites in the long-term pathogenesis of depression has not been fully explored. Therefore, this study aims to explore the changes in intestinal microecology and microbial metabolites during the development of long-term depression.

In this study, we sought to investigate the composition and function of the gut microbiome in the long-term depression CUMS rat model. Animals were sacrificed after either 4 weeks or 8 weeks under CUMS to mimic long-term depression in humans. Based on 16S rRNA sequencing and metabolomics, we characterized the gut microbiota of depression and long-term depression, revealed the correlation between gut microbes and metabolites, and provided insights into the role of microbiota in the long-term development of depression.

In this study, we identified the different gut microbiome and fecal metabonomics among control, 4-week CUMS and 8-week CUMS rats. We found distinguishable microbial phenotypes in the depression rat groups. Compared with the control group, the CUMS rats showed decreased species richness indices in microbial composition ( Figure 2A ). The diversity of the human gut microbiota has been considered as one of the forms of evidence of health for decades (Eckburg et al., 2005). Normal intestinal flora diversity and flora metabolites are closely related to human health (Maier et al., 2018), and decreased species richness may indicate poor health conditions and disordered physiological processes. Bacteroides fragilis is a gram-negative brevibacterium with capsules in the intestine. It can activate and induce IL-10 to produce Tregs through its cell component capsular polysaccharide A and play an anti-inflammatory role. A decrease in the abundance of such bacteria reduces the immunity of mammals (Maynard et al., 2012). Normal coliform bacteria take Bacteroides, Bifidobacterium, Clostridium and Escherichia coli as the dominant bacteria, synthesize various complex digestive enzymes and metabolize sugars through glycolysis, pentose phosphate and anaerobic decomposition. When the abundance of these dominant bacteria decreases, it weakens the host’s absorption of nutrients (Backhed et al., 2004). (Knuesel and Mohajeri, 2021) found that the diversity of intestinal flora in patients with depression decreased compared with that of healthy people. Other studies showed that the large reduction in Bacteroides in the intestine of patients with depression was closely related to depression symptoms and loss of pleasures (Mason et al., 2020; Wenzel et al., 2020). Therefore, the decreased species richness may indicate poor health conditions and disordered physiological processes.

Furthermore, the gut microbial composition of 8-week CUMS rats was significantly different from that of 4-week CUMS rats. More co-occurrence relationships were detected in the 4-week CUMS group than in the 8-week CUMS group. The top 20 differential microbes between rats under 4 weeks of CUMS and 8 weeks of CUMS included Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus reuteri, Bifidobacterium animalis, Helicobacter, Bacteroides, Roseburia, Blautia Marseille, Psychrobacter, Lachnospiraceae bacterium, Lactobacillus intestinalis, Clostridiales bacterium, Clostridium, Allobaculum, and Rodentibacter ratti. Among them, five species belong to Lactobacillus, while species of the same family have been recently reported to alleviate depression-related symptoms in a CUMS mouse model by regulating brain xanthine oxidase activity (Xu et al., 2022). To identify distinguishable microbial characteristics between 4-week CUMS and 8-week CUMS treatments, LEfSe analysis revealed the differential OTUs between the two groups ( Figures 4A, B ). The long-term depression group was characterized by disturbance of Alistipes indistinctus, Bacteroides ovatus, and Alistipes senegalensis at the species level ( Figure 4B ). Alistipes, primarily isolated from medical clinical samples, is a relatively new genus that is highly relevant in dysbiosis and disease, including mental signs of depression (Parker et al., 2020). A recent study revealed gut microbiota characteristics of ADHD and found that low levels of Bacteroides ovatus were associated with host cognitive impairment (Li et al., 2022). Bacteroides ovatus colonization has also been reported to influence the abundance of intestinal short-chain fatty acids and neurotransmitters (Horvath et al., 2022). Our study further detected its role in long-term depression development.

We also found that the development of depression was associated with disturbances in fecal metabolomics, which is always considered the functional readout of the gut microbiome since the gut microbiota is involved in the regulation of the metabolic pathways of the host. Compared with the control group, there were 508 differential fecal metabolites in the depression rats, of which 247 metabolites increased and 261 metabolites decreased. In addition, increased D-pinitol and decreased indoxyl sulfate, trimethylamine n-oxide, and 3alpha,7alpha-dihydroxy-12-oxocholanoic acid were observed. The active natural inositol D-pinitol reduces pancreas insulin secretion and increases circulating ghrelin levels in rats (Navarro et al., 2020); herein, the increase in D-pinitol in the long-term depression group may provide insight into the mechanism of depression-related weight gain. These differential metabolites were further used for KEGG pathway enrichment analysis. Among the top 20 pathways revealed, pantothenate and CoA biosynthesis was most significantly enriched ( Figure 5E ). Specifically, these differentially expressed metabolites were related to valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; beta−alanine metabolism; phenylalanine metabolism; phenylalanine, tyrosine and tryptophan biosynthesis; riboflavin metabolism; thiamine metabolism; pyrimidine metabolism; the sulfur relay system; lysine degradation; biosynthesis of amino acids; glycerophospholipid metabolism; alanine, aspartate and glutamate metabolism; ubiquinone and other terpenoid−quinone biosynthesis; arginine and proline metabolism; alpha−linolenic acid metabolism; tyrosine metabolism; sulfur metabolism; and fatty acid degradation. Among them, the pathway with the highest enrichment factor was valine, leucine and isoleucine biosynthesis. A recent study showed that deficiency in the essential amino acids l-isoleucine, l-leucine and l-histidine can be used as predictors of moderate depression in elderly women (Solís-Ortiz et al., 2021). The enrichment of this KEGG pathway also reflected the important role of leucine and isoleucine in long-term depression development.

Increasing research evidence shows that the intestinal microbiota helps to maintain the metabolic homeostasis of the host, and in contrast, an imbalance in the microbiota affects the level of metabolites (such as branched-chain amino acids, hormones, vitamins, and short-chain fatty acids), leading to the development of obesity, insulin resistance, depression and other diseases (Caspani et al., 2019). It can be seen that the metabolic group and intestinal microbiota are closely related. At present, 16S sequencing technology is mostly used for the analysis of the intestinal microbiome, but this technology has certain limitations and cannot indicate the transcriptional activity of genes in each bacterial genome or distinguish between live bacteria and dead microbiota. Fecal metabolomics can better explain the metabolic interaction between the host, diet and intestinal microbiota and can provide functional data of microbiota to compensate for the lack of sequencing. Zierer J et al. found that the fecal metabolome reflected the composition of the intestinal flora to a large extent. The composition of the intestinal flora explained 67.7% of the changes in the level of 710 metabolites on average. There was a high correlation between the intestinal microbiome and fecal metabolome (Zierer et al., 2018). It has been reported in the literature that depression is related to main microbial metabolites (short-chain fatty acids - SCFAs -, bill acids, amino acids, tryptophan - trp - derivatives, and more) (Ortega et al., 2022). We conducted a study on the correlation between gut microbes and fecal metabolites and found that at least nine gut microbes were significantly correlated with at least twenty fecal metabolites, such as Bacteroides uniformis with trimethoprim (ID1376) (Silva et al., 2000), Bacteroides vulgatus with N-acetyl-d-glucosamine (ID953) (Nihira et al., 2013; You et al., 2023), Helicobacter with trigonelline (ID1194) (Kyung et al., 2019), and Helicobacter with N-acetyl-d-glucosamine (ID953) (Dai et al., 2021). Some correlations between intestinal microorganisms and metabolites found in our study are consistent with those reported in the literature, but the names of metabolites are more specific, and the correlation between them has also been reported in other diseases, such as metabolic disorders and tumors. In long-term depression, the experimental results can provide ideas for further study.

In summary, we outlined the landscapes of gut microbiota and fecal metabolites in rats under 4-week and 8-week CUMS using multiomics data analysis. We found that the gut microbiome was distinguishable between depression and long-term depression rat models, and the altered microbiota may participate in the long-term development of depression through metabolism regulation. Our findings provide a new perspective for understanding the pathogenesis of long-term depression.

The data are divided into two parts, which have been uploaded. SubmissionID: SUB12502472, BioProject ID: PRJNA917078, https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB12502472/overview. SubmissionID: SUB12518854, BioProject ID: PRJNA919495, https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB12518854/overview.

The animal study was reviewed and approved by the Ethics Committee of Institute of Basic Theory for Traditional Chinese Medicine, China Academy of Chinese Medical Sciences.

Conception and design: YXS and ZZhan. Performed the experiments: YBL, JLL, HXL, YKZ, YJL, ZBS, ZZhai and YPY. Performed the microbiome and metabolomic analysis: YXS, JLL, RC, MW and YJC. Drafted the manuscript: YXS and YBL. Final approval of the completed manuscript: YBL and ZZhan. All authors contributed to the article and approved the submitted version.