Introduction

Major depressive disorder (MDD) is one of the most prevalent mental illnesses worldwide. It is characterized by persistent and significant mood depression, decreased interest, and, in severe cases, suicidal tendencies (Rehm & Shield 2019). Depression is predicted to become the world’s leading cause of disability by 2030, according to a report from the WHO (Nobis et al. 2020), with a global prevalence of 4.4% (Friedrich 2017; Huang et al. 2019). Several theories have been proposed regarding the pathogenesis of depression, which include monoamine neurotransmitter imbalance, neurogenetic disorders, oxidative stress disorder, immune regulation disorder, etc. (Otte et al. 2016; Wohleb et al. 2016). However, the pathogenesis of MDD remains unclear, and some patients do not receive timely and effective treatment due to the lack of objective indicators for diagnosis and treatment (Smith 2014). Therefore, it is crucial to investigate the pathophysiological mechanism of MDD thoroughly and identify reliable biomarkers that will enable the development of appropriate drug targets and accurate treatments as soon as possible.

Kynurenine (KYN) is an intermediate product of tryptophan (TRP) metabolism. Approximately 5% of tryptophan is converted into 5-hydroxytryptophan (5-HT) in enterochromaffin cells, whereas 95% of tryptophan is metabolized into KYN via the kynurenine pathway (KP) in various tissues, including the liver, kidney, and brain (Platten et al. 2019). Anomalies in KP metabolism are linked to the incidence and progression of neuropsychiatric ailments, chronic immune disorders, and cancer (Comai et al. 2020). Results of animal studies and clinical research have both documented an increase of serum KYN levels in a chronic restraint stress-induced depression rat model and depression patients with end-stage renal disease (Gibney et al. 2014; Koenig et al. 2010). These findings suggest that the tryptophan-kynurenine pathway might be closely associated with depression.

Indoleamine 2, 3-dioxygenase (IDO) is the first enzyme that limits the rate of tryptophan-kynurenine pathway. By breaking down the indole ring in tryptophan, it can be substituted by tryptophan into KYN (van Baren & Van den Eynde 2015). It has been reported that challenge of lipopolysaccharide (LPS) could induce not only depression-like behaviors in mice, but also an increased IDO activity, both in central and peripheral administration. Moreover, early administration of 1-MT, which is an IDO competitive inhibitor, has been demonstrated to exert an antidepressant-like effect by inhibiting IDO activity (O'Connor et al. 2009).

Tryptophan-2, 3-dioxygenase (TDO) is a heme-containing tetramer protein with specific L-tryptophanase activity, which is another metabolic enzyme of tryptophan-kynurenine pathway (Yu et al. 2016). It can transform tryptophan indole groups into canines by assisting oxygen entry (Papadopoulou et al. 2005). Increased TDO levels have been found in the liver and cerebral cortex of depressive rats, with a positive correlation with the degree of depression (Gibney et al. 2014). Moreover, depression symptoms could be relieved after treatment with allopurinol, a TDO2 inhibitor (Gibney et al. 2014).

Given the significant findings in animal studies, it is important to investigate the changes of IDO and TDO in clinical investigations and explore their potential as biomarkers for effective diagnosis of MDD. To achieve this, the baseline levels of serum IDO and TDO of MDD patients were measured and compared with healthy controls. Furthermore, the correlation between IDO and TDO levels and the degree of depression was analyzed, and their diagnostic values were evaluated. Finally, the abundance of IDO and TDO were measured again after two weeks or one month of treatment.

Materials and methods

Participants

This study was carried out at the Hefei Fourth People’s Hospital and Anhui Mental Health Center from December 2019 to October 2021. A total of 55 patients with major depressive disorder (MDD) experiencing their first episode of depression were selected. These patients were screened via psychiatric interviews by an experienced researcher in accordance with the guidelines of the structured clinical interview based on the Diagnostic and Statistical Manual for Psychiatric Disorders-Fifth Edition (DSM-V).

The inclusion criteria were as follows: (1) meeting the guidelines of the structured clinical interview based on the Diagnostic and Statistical Manual for Psychiatric Disorders-Fourth Edition (DSM-IV); (2) aged between 18 and 70; and (3) having not received any antidepressant treatment.

The exclusion criteria were as follows: (1) combination with other mental illness or nervous system disease history; (2) other body diseases (chronic inflammatory disease, diabetes, cardiovascular disease, thyroid disease or Cancer, etc.); (3) substance abuse (drug, nicotine, alcohol, etc.); (4) pregnancy and or breastfeeding.

Parallelly, 55 healthy controls were enrolled from the Anhui Medical University Health Checkup Center. The sex, age and years of education of HC matched those of MDD patients. The study was conducted according to the Declaration of Helsinki and approved by the ethics committee of the Anhui Mental Health Center, and the Informed consents were obtained from all the participants or their guardians.

Clinical assessment

Fifty-five patients with first-episode depression were selected as the MDD group. 55 healthy individuals were selected as the HC group. HAMD-24 was used to assess depression symptoms and their severity in the MDD group at baseline (MDD-baseline), at two weeks after treatment (MDD-2 weeks), and at the end of a month of treatment (MDD-1 month).

Measurement of serum IDO and TDO concentrations

Serum samples were collected from the HC group and the MDD group before and after treatment. Blood samples were collected from the participants’ vein between 7:00 A.M. and 8:00 A.M. after fasting for at least 8 h, and immediately centrifuged at 1, 200 g for 10 min at 4 °C. The serum was extracted and stored at − 80 °C, and all the samples were analyzed within 6 months. Double antibody sandwich enzyme-linked immunosorbent assays (ELISA) were employed to quantify the serum concentrations of IDO and TDO (Jianglai Bio, Shanghai, China) according to the manufacturer’s instructions. In brief, diluted serum (1:5) with sample buffer was used to conduct ELISA, in duplicate. All measurements were performed in duplicate and expressed as ng/ml. Samples were measured using ELISA reader (Rayto RT-6100, Shanghai, China), and data were analyzed with ELISACalc software version 0.2 (ELISACalc software, Bethesda, USA).

Statistical analysis

Statistical analyses were carried out using the SPSS version 19.0 (SPSS, Chicago, Illinois, USA) and GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Data in figures are presented as means ± standard deviation (SD). Student’s t-test and χ2 analysis were used to compare demography and clinical characteristics of HC and patients with MDD. Analysis of variance (ANOVA) was used to determine significant differences between HC and patients with MDD (before and after treatment). A paired-sample t-test was performed to compare serum IDO and TDO concentrations of MDD before and after treatment. The relation between HAMD-24 scores and serum IDO and TDO levels were analyzed with Pearson’s correlation test. The ROC curve analysis was used to determine the area under the curve (AUC) and cut-off values of serum IDO and TDO levels. The criterion for significance was P < 0.05.

Results

Demographic and clinical characteristics of the participants

As shown in Table 1, there was no statistically significant difference in age (t = 0.176, P = 0.865), sex (χ2 = 0.000, P = 1.000), or BMI (t = 1.315, P = 0.263) between HC and patients with MDD at baseline. The HAMD-24 scores of the MDD group were significantly higher than HC group (t =  − 19.14, P < 0.001).

Table 1 Demographic and clinical characteristics of HC group and MDD group
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Comparison of serum IDO and TDO levels between HC and patients with MDD (before and after treatment)

The serum concentrations of IDO and TDO were measured using ELISA, and the intra-assay variation of standards was less than 5%. Analytical range of the serum IDO assay was 0.31 to 20 ng/ml, with a 5.5% intra-assay coefficient of variation. Analytical range of the TDO assay was 0.156 to 10 ng/ml, with a 4.7% intra-assay coefficient of variation.

Serum IDO and TDO levels of HC and patients with MDD (before and after treatment) are shown in Table 2. The serum IDO and TDO levels in the MDD groups were higher than those in the HC group, whether treatment is performed or not, and for how long.

Table 2 Serum concentrations of IDO and TDO in HC and MDD patients (ng/ml)
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Comparison of serum IDO and TDO levels before and after treatment

Serum IDO and TDO levels of patients with MDD (before and after treatment) are shown in Table 3. Notably, both the serum IDO and TDO levels were decreased after 2 weeks or 1 month of treatment. Moreover, the serum TDO level of MDD (1 month) group was remarkably lower than that of the MDD (2 weeks) group. In terms of the HAMD-24 score, the mean HAMD-24 score in the group after treatment (whether treatment after 2 weeks or 1 month after treatment) was significantly lower than that in the MDD (Baseline) group. And the mean HAMD-24 score of MDD (1 month) group was remarkably lower than that of the MDD (2 weeks) group.

Table 3 Serum concentrations of IDO and TDO of MDD before and after treatment (ng/ml)
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Correlation between serum IDO and TDO levels and HAMD-24 score serum concentrations

As can be seen from the Fig. 1, IDO level was significantly positively correlated with HAMD-24 score: the richer the IDO concentration, the higher the severity (r = 0.531, P < 0.001) (Fig. 1A). Similarly, a positive correlation was also found between serum TDO level and HAMD-24 score (r = 0.328, P < 0.001) (Fig. 1B).

Fig. 1
figure 1

Correlation of the serum concentrations of IDO (A) or TDO(B) with HAMD-24 scores in patients with MDD

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Diagnostic values of serum IDO and TDO in discriminating MDD Patients from HC

The diagnostic performance of IDO and TDO levels in discriminating patients with MDD from healthy controls were performed by ROC curve analysis (Fig. 2). The results showed that the AUC values of TDO and IDO were 0.999 (P = 0.000; 95% CI: 0.996–1.000) and 0.966(P = 0.000; 95% CI: 0.939–0.992). At a cut-off point of 6.20 ng/ml for TDO, the sensitivity and specificity were 98.2% and 96.4%, respectively; at a cut-off point of 51.72 ng/mL for IDO, the sensitivity and specificity were 98.2% and 80.0%, respectively. These results indicated a predictive value of serum concentrations of TDO and IDO for discriminating MDD patients from the controls, with a higher diagnostic value of TDO than IDO.

Fig. 2
figure 2

ROC curve for serum IDO and TDO in the identification of patients with MDD

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Discussion

The pathogenesis of depression is highly complex, and depletion of monoamine neurotransmitters, particularly 5-HT, is considered a crucial factor for the treatment of depression. Tryptophan is a precursor of 5-HT, but under conditions of micro-inflammatory environment induction, the rate-limiting enzymes IDO and TDO activate the tryptophan metabolism bypass pathway, resulting in the production of KYN, which reduces the production of 5-HT and leads to depression. Moreover, KYN metabolites themselves also contribute to the pathophysiological process of depression (Höglund et al. 2019). Therefore, it is rational to hypothesize that IDO and TDO have a significant role in the development of depression (Badawy 2017a).

TDO is mainly found in the liver but also in the brain (Kanai et al. 2009); whereas IDO mainly exists in extrahepatic tissues such as the brain, blood, spleen, kidneys, and lungs (Kim & Jeon 2018). In the present study, although the serum levels of IDO (80.39 ± 14.73) and TDO (8.93 ± 1.45) differed significantly in patients with MDD at baseline, they were both remarkably higher than that in the HC. Previous research has also shown that the levels of IDO were significantly higher in major depressed patients at baseline than in normal controls (Al-Hakeim et al. 2020). It might be partly ascribed to the hyper-inflammation status MDD patients, which was demonstrated in our previous studies (Xu et al. 2022, 2023) and others (Anisman & Hayley 2012; Wu et al. 2022). When the body experiences inflammation or stress, it triggers an increase in pro-inflammatory cytokines like IFN-α, IFN-γ (Fatokun et al. 2013; Hughes et al. 2016), and TNF-α (Lai et al. 2023), leading to the over-activation of IDO in extrahepatic tissues of depressed patients (Ogyu et al. 2018).

On the one hand, TRP synthesizes KYN, which causes an increase in quinolinic acid (QUIN), over-activates N-Methyl-D-Aspartate (NMDA) receptors, damages the hippocampus, and worsens oxidative stress response. On the other hand, TRP depletion induces a decrease in brain 5-HT, which has neuroprotective effects, ultimately leading to depressive-like behavior (Maes et al. 2011). Consistently, it has been reported that serum IDO activity was increased while 5-HT activity was reduced in depressed mice, together with a negative correlation between serum IDO activity and 5-HT level (Carabelli et al. 2020; Gao et al. 2021).

In early studies, it was demonstrated that TDO affects the level of serum tryptophan availability in tissues (Badawy 2017b). As TDO activity is linked to the synthesis of brain TRP and 5-HT, TDO is inversely proportional to central nervous system function (Kanai et al. 2009). Serum cortisol levels are elevated in depressed patients (Strawbridge et al. 2017). Elevated cortisol levels may increase TDO activity, which could explain the 5-HT deficiency observed in major depressive disorder (Badawy 2013). The hypothesis is that increased cortisol levels lead to glucocorticoid resistance, downregulation of glucocorticoid receptors, and reduced signaling through cortisol to increase TDO (Cohen et al. 2012; Sorgdrager et al. 2017). Results of our present study showed that after antidepressant treatment, no matter 2 week or 1 month, the serum IDO and TDO levels of MDD patients were remarkably decreased. Moreover, after one month of treatment, the serum TDO levels were significantly lower than that of the treatment for two weeks. However, there was no difference in IDO levels between 2 weeks and 1 month of treatment. These findings indicate a more sensitive value of TDO than that of IDO to the antidepressant treatment. Giving the findings that inhibiting the activity of TDO can have neuroprotective effects (Lanz et al. 2017), together with the known inhibitive effect of antidepressant against TDO (Bano et al. 2010), our results should provide new therapeutic targets for treating depression.

HAMD-24 is the most widely used interview scale for selecting patients with depression (Wang et al. 2017). Here, it was used to assess the occurrence and severity of depression. This study shows that the mean HAMD-24 score in the group after treatment (whether treatment after 2 weeks or 1 month after treatment) was significantly lower than that in the MDD (Baseline) group. After treatment in 2 weeks or 1 month, a certain degree of symptom relief in patients with depression. Moreover,

this study revealed a significant positive relationship between the levels of TDO and IDO and HAMD-24 scores in the MDD group. The higher the TDO and IDO levels, the higher the HAMD-24 score, and the more severe the patient's symptoms. Given the heterogenous nature of MDD, there is an opinion that single biomarker might not be specific enough for use as a diagnostic tool. In other word, it might be more suitable to develop multiple biomarkers covering separate hypotheses/pathways (Bilello 2016; Papakostas et al. 2013; van Buel et al. 2019). However, based on the findings in the present study, not only the increased abundance of TDO and IDO in MDD patients with first episode of depression, but also their positive correlation with HAMD-24 score, together with their sensitivity to antidepressant, it is reasonable to take TDO and IDO as candidates to develop as potential tools for diagnosing depression or evaluating the antidepressant effect.

To further investigate the potential value of TDO and IDO levels as diagnostic biomarkers for MDD, we conducted ROC analysis. The results showed that IDO and TDO levels could distinguish MDD patients with AUCs of 0.999 and 0.966, respectively. As the AUC value in ROC analysis should be at least 0.7 (Gao et al. 2022), these findings suggest that IDO and TDO levels have clinical screening value as biomarkers for identifying patients with MDD. Our results provide a strong foundation for uncovering the role of the KP pathway in the pathogenesis of MDD and for developing clinically objective diagnostic methods for this disorder.

However, several limitations of this study need to be addressed. Firstly, this was a single-center study with a small sample size, which requires a conservative interpretation of the current data. Moreover, gender and age effects were ignored in the present study, due to the fact that most of the subjects were males and similarly aged 30–50. However, gender and age are contributors affecting the onset of depression and IDO metabolism (Marttila et al. 2011), which should be taken full consideration in our further studies. Secondly, we did not collect and analyze data on treatment methods for MDD patients, such as physiotherapy, pharmacotherapy, and psychotherapy (Emmanuel 2019; Jones & Husain 2021; Pandarakalam 2018), to explore the effects of different treatments on IDO and TDO levels. Notably, IDO and TDO mediated KP metabolism abnormalities are involved in the occurrence and development of many other diseases including tumors, inflammatory immune-related diseases, and neuropsychiatric diseases and/or neurodegenerative disorders such as AD (Platten et al. 2019; Savonije & Weaver 2023). It is precisely because KP is a finger in every pie (Savitz 2020) and depression is a multifactorial disease with a considerable symptomatic overlap with other psychiatric and somatic disorders, it is necessary to weight and combine with other candidates including inflammation (TNFα, IL-1β) (van Buel et al. 2019), HPA axis (steroids) (Almeida et al. 2021; Bilello 2016) and neurotrophism (BDNF, VEGF) (Phillips 2017) in subsequent studies to further improve the specificity of depression detection.

In conclusion, the data presented here demonstrate that serum levels of IDO and TDO are closely associated with MDD. In the future, IDO and TDO levels may be developed as potential clinical serological indicators for the diagnosis of depression. Furthermore, developing new antidepressant drugs targeting IDO and TDO could bring hope to patients with depression.