Background
As the latest World Health Organization (WHO) report showed, depression is a high-risk psychiatric disease that affects people of all races around the world, and ranks among the world’s largest contributors of years lived with disability. It is also one of the major causes of the global economic burden of disease [
1]. However, up to 60% of patients show no respond to the initial antidepressant treatment, which is called treatment resistant depression (TRD), which may be a subtype of depression patients with unique pathophysiological features [
2,
3]. Due to lack of sufficient relief from current antidepressant therapy, TRD patients have an increasing risk of suffering from chronic psychosocial disorders, relapses and suicide [
4].
The abnormality of hypothalamic–pituitary–adrenal (HPA) axis is one of the most concerned research areas in depression [
5‐
7]. Stress can activate the HPA axis, including increased release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) [
8,
9]. In clinical studies, the dysregulation of HPA axis is also a common feature found in depressive patients [
10]. Numerous CRH receptors are resident in extra-hypothalamic area, and most cells of the 5-hydroxytryptamine (5-HT) and noradrenaline (NA) systems are also included herein [
11]. CRH can activate 5-HT neurons in the dorsal rape nucleus, and regulate NA activity. Glucocorticoids can stimulate 5-HT and NA systems via mineralocorticoid and glucocorticoid receptors. Therefore, the disorder of HPA axis and its interaction with central monoaminergic system might be one of the main causes of TRD [
12,
13].
As an interesting animal model of TRD, chronic administration of ACTH to rodent, mimicking the stress induced the HPA axis activation, results in resistance to the treatment of imipramine in the forced swimming test (FST) [
14], as well as resistance to other antidepressants [
15,
16]. Though, ACTH-induced depression rat model has been widely accepted as an animal model for TRD, the underlying mechanism of this model is not completely understood.
Deregulation of the gut microbial community has been extensively associated with the diseases of the central nervous system (CNS). The crosstalk between the gut microbiota and the CNS, so-called gut microbiota–brain axis, occurs through neuroendocrine, enteric, autonomic, and immune system pathways, and may impact the host physiological and pathological mechanisms, including the HPA axis activation, and the communications between neurotransmitters and immune system [
17]. Therefore, the perturbed gut microbiota could be associated with neurological diseases via the gut microbiota–brain axis [
18]. The alterations of specific species of gut microbiota may contribute to the occurrence and the development of depression, while depressive states may cause the changes of specific species of gut microbiota, which eventually result in more severe depression. These specific species of gut microbiota are mainly included in the bacteria phyla of
Firmicutes,
Actinobacteria,
Bacteroidetes and
Proteobacteria [
19]. Meanwhile, a number of studies have shown that probiotics, such as
Lactobacillus and
Bifidobacterium, have beneficial psychoneurological effects [
20,
21]. It is reported that CBM588, a specific phenotype of the strain
Clostridium butyricum, in combination with antidepressants is effective and well tolerated in the treatment of TRD [
4]. Recently, a 16S ribosomal RNA gene sequencing of feces based study showed that the antidepressant effects of ketamine, an
N-methyl-
d-aspartate receptor (NMDAR) antagonist showed rapid and sustained antidepressant effects in TRD patients, might be partly mediated by the restoration of altered gut microbiota [
22].
The crosstalk between gut microbiota and brain might be indirect. The metabolites of gut microbiota, which circulate in the systematic circulation and metabolism, may play a vital role in the gut microbiota–brain axis. Metabolomics is a systematic biological method for measuring the changes of endogenous small molecule metabolites. It has been increasingly applied to the diagnosis of diseases, the discovery of disease biomarkers, and the pathogenesis study of diseases [
23]. In metabolomics studies, urinary sample is non-invasive, and more convenient for collection compared to other samples (blood or specific organ). Furthermore, the greatest advantage of urinary sample for metabolomics studies is that it contains a combination of metabolites in a relatively longer window period (12 or 24 h), while the metabolites in blood can only represent the transient state of the research object. At present, urinary samples of animal or human have been widely used for metabolomics research to reveal the pathophysiological mechanism of diseases systemically [
24]. Therefore, to characterize the global characteristics of ACTH-induced TRD, it is necessary to combine the gut microbiota study with the metabolomic signatures.
In the present study, an integrative gas chromatography-time-of-flight mass spectrometer (GC-TOFMS)-based urinary metabolomics and 16S rRNA gene sequencing approach was performed on chronic ACTH-treated rat model with depression phenotype to reveal the differentiated gut microbiota and urinary metabolites. Further, the relevancies of above two were discovered to reveal the interactions between the gut microbiota and the host metabolism in ACTH-induced TRD rats. The results will help to enhance comprehensive understanding of the pathogenesis of ACTH-induced TRD.
Discussion
Depression is a multifactorial disease which is regulated by genetic and environmental factors. As a systematic disease, it is a complex phenotype associated abnormalities ranging from central nervous system to many peripheral systems [
30‐
32]. As an important component of the neuroendocrine system, HPA axis is a complex collection of direct influences and feedback interactions, and controls the response to stress and regulates many body processes including digestion, the immune system, mood and emotion, sexuality, energy storage and expenditure [
33,
34]. In humans, gut microbiota contains the largest number of bacteria and the greatest number of species compared to other areas of the body. It not only plays a barrier to pathogenic organisms, but also serves as an endocrine organ by providing short-chain fatty acids (SCFAs), vitamin, and xenobiotics to the host [
35]. Although the vital role of HPA axis dysfunction or gut microbiota dysregulation in pathogenesis of TRD has been widely accept, the interactions between the structural features of gut microbiota and host metabolic phenotype in depression accompanied by HPA axis dysfunction have rarely been reported until now. In the present study, an integrative metabolomic signatures and microbial community profiling was employed on ACTH-induced depression rats compared with normal control rats.
For FST and TST (Additional file
1: Fig. S1), striking helpless and depression-like behaviors observed in ACTH group were consistent with other reports [
25,
36]. Elevated levels of ACTH and CORT in serum and negative feedback of CRH level (Additional file
1: Fig. S2) confirmed that ACTH injection induced hyperactivity of HPA axis, which was reported to be closely related to depression [
37,
38].
The metabonomic results indicated that the differential metabolites were mainly related to disorders of energy metabolism, amino acid metabolism, ascorbate and aldarate metabolism, and inositol phosphate metabolism (Fig.
3). The disorder of energy metabolism was mainly related to pyruvate metabolism and glycolysis or gluconeogenesis. Pyruvic acid is an important intermediate in the glucose metabolism and its carboxylate anion is called pyruvate. Pyruvate is the end product of glycolysis and can be converted to acetyl coenzyme A (acetyl-CoA) by oxidative decarboxylation, which further participates in tricarboxylic acid (TCA) cycle to generate ATP [
39]. It was reported that perturbed energy metabolism is ubiquitous in animal models of depression [
40,
41]. In ACTH group, pyruvic acid concentration was significantly reduced (Additional file
2: Table S1), interfering with glycolysis or gluconeogenesis pathway, which may further lead to TCA circulatory dysfunction and depressive symptoms. Threonine, the essential dietary amino acid, is the precursor to isoleucine. Since Threonine is mainly found in the central nervous system, it can be of great help in the treatment of different types of depression. Threonine contributes to the synthesis of glycine and serine, and plays a key role in maintaining the integrity and barrier function of the intestinal mucosal [
42]. Previous studies showed that abnormal amino acid metabolism might usually happen in depression and disturbed amino acid level might be served as clinical trait-biomarker for depression [
43,
44]. Our research found that
l-threonine level was significantly reduced in ACTH group and played a key role in the glycine, serine and threonine metabolic pathways, which may have great impact in the occurrence of depression (Additional file
2: Table S1). As the major metabolite of human, hippurate is formed by the reaction of many aromatic compounds such as benzoic acid and toluene with glycine and is involved in phenylalanine, tyrosine and tryptophan biosynthesis [
45] (Fig.
3). Glycine is one of the excitatory amino acids closely related to major depression [
46]. The increase of hippurate concentration is closely related to the change of glycine concentration. It has been reported that gut microbiota can signal to the brain through the regulation of tryptophan metabolism, thus affecting the development, function and behavior of the brain, leading to the occurrence of neurodevelopmental diseases derived from gut microbiota [
47]. In our study, the hippurate concentration in ACTH group was significantly higher than that in Control group (Additional file
2: Table S1), and a critical signal molecule in the brain-gut axis, 5-HT, was significantly decreased (Additional file
1: Fig. S2), possibly leading to the occurrence of brain-gut axis disorders and depression.
In addition, ascorbate and aldarate metabolism was also disrupted in ACTH-induced depression rats compared to Control group. Ascorbic acid, an important antioxidant in the CNS, promotes the synthesis of neurotransmitters (5-HT and NE) [
48]. Compared with Control group, the level of ascorbic acid in ACTH group was significantly reduced (Additional file
2: Table S1), resulting in lower levels of 5-HT and NE in serum (Additional file
1: Fig. S2). The abnormality of these monoamines is widely accepted as the main biological mechanism of depression, and targeted by most of antidepressants.
Myo-inositol, a stereoisomer of inositol, is a carbocyclic sugar, which is widely present in brain and other mammalian tissues. It mediates cell signal transduction through various hormones, neurotransmitters and growth factors and is involved in osmoregulation. Myo-inositol has the highest concentration in the brain and plays an important role here, combining other neurotransmitters and some steroid hormones with their receptors [
49]. It’s also an essential participant and regulator of inositol phosphate metabolism [
50]. Chiappelli et al. [
51] found that the occurrence of depression in schizophrenic patients was closely related to the reduction of myo-inositol levels by proton magnetic resonance spectroscopy analysis. This finding is consistent with the decrease in myo-inositol levels in ACTH group (Additional file
2: Table S1).
In the microbial community profiling, the PLS-DA analysis and the compositional structure both presented the significant differentiation between the groups (Fig.
4a, b). According to the reports, the increase of the ratio of
Firmicutes to
Bacteroidetes of phylum level was commonly observed in both depression patients [
52] and patients with irritable bowel syndrome [
53]. Therefore, the increased ratio of
Firmicutes to
Bacteroidetes is considered to be a marker of the gut–brain axis disorders, which also occurred in ACTH group (Additional file
1: Fig. S4).
The LEfSe difference analysis showed that the differential microbiota was mainly concentrated on the cumulative number of
Oscillospira and
Ruminococcus (Fig.
4c).
Oscillospira is a mysterious bacterial genus that has never been cultured but is continuously detected by sequencing of 16S rRNA gene in the human microbiome [
54]. In terms of the metabolites excreted by
Oscillospira, it is inversely correlated with inflammatory diseases and BMI [
55]. BMI represented the best predictive value for gut dysbiosis and metabolic alterations. It is speculated that
Oscillospira may produce short-chain fatty acid butyrate, similar to trends of other genera (e.g.,
Roseburia and
Faecalibacterium) [
56]. Butyrate concentrations were negatively correlated with anxiety levels. In addition,
Ruminococcus, a genus of class
Clostridia which pointing to an energy metabolism dysfunction, was identified as one of characteristic genus of individuals with inflammatory bowel disease (IBD) [
57].
By integrating gut microbiota data with differential metabolites, our results highlighted the tight crosstalk between the gut microbiota and host metabolism in ACTH-induced depression rats. As shown in Fig.
5, the relevant genus of gut microbiota predicted from each of differential urinary metabolites were
Ruminococcus,
Akkermansia,
Lactobacillus and
Klebsiella.
Akkermansia muciniphila, the only currently known species within genus
Akkermansia, has the ability to continuity regeneration of the intestinal epithelium and degrade mucin, and also producing acetic acid, propionic acid and oligosaccharides. These products become the substrate for
Faecalibacterium prausnitzii, one of the main producers of butyrate in the intestine. Anaerobic bacteria that produce butyrate help to inhibit inflammation in the gastrointestinal tract, and prevent an increase in intestinal permeability [
58]. In our study, 6 metabolites were statistically relevant to
Akkermansia, including pyruvic acid, 4-hydroxybenzoic acid, myo-inositol, hippurate,
d-arabitol and ascorbic acid, many of which are closely related to depression, as we discussed above.
Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, is the main component the lactic acid bacteria group (i.e. they convert sugars to lactic acid). It was reported ingestion of
Lactobacillus strain could regulate emotional behavior and central GABA receptor expression in mouse via the vagus nerve [
59], as well as improve behavioral, cognitive, and biochemical aberrations caused by chronic restraint stress [
60]. As shown in Fig.
5, there are 3 metabolites relevant to
Lactobacillus, namely pyruvic acid, hippurate and
d-arabitol. Arabitol is a sugar alcohol, which can be formed by reducing arabinose or lyxose. Some organic acid tests examined the presence of
d-arabitol, which may indicate overgrowth of intestinal microbes. Arabitol showed very similar inhibition effects to its isomer xylitol on oral
Lactobacilli [
61]. As we discussed above, the correlation analysis between differential metabolites and bacteria revealed that active metabolites of
Akkermansia and
Lactobacillus were closely related to the host inositol phosphate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, which indicated there was a significant crosstalk between the gut microbiota and the host metabolism in ACTH-induced TRD rats.
In addition, it has been reported that the abundance of
Klebsiella and
Ruminococcus was changed in patients with MDD compared with healthy controls [
62,
63]. In our study, these two intestinal microbes were observed to have varying degrees of correlation with
l-threonine, pyruvic acid, mannitol and hippurate, which are closely related to the development of depression as we discussed earlier, but there was no statistical significance (
P > 0.05). These results suggest that the changes in gut microbiota and urinary metabolic phenotype are associated with the development of depression. The relevance between the gut microbiota of genus level and the differential urinary metabolites further confirmed the correlation between disturbances of gut microbiota and the disturbed microorganism-host metabolic balance in ACTH-treated rats.
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