In the present study, we used UPLC-MS to detect different metabolites in the plasma of controls and MDD patients. And we followed MDD patients’ hospitalization for two months. Samples of this metabolomics study were collected under the non-interventional medical condition, the self-control of patients avoid the influence of confounding factors, which could reflect the metabolic changes of patients under the real treatment condition.
Finally, we constructed a new network consisting of 4 SMs and 8 enrichment pathways. The relative contents of these SMs showed a regular trend of change in the plasma samples of each group, and significantly correlated with the scores of HDRS-17 scale. These results suggest that they are correlated with the severity of MDD and may have the potential to be biomarkers for the diagnosis and treatment of MDD. 8 enrichment pathways associated with the 4 SMs are involved in the possible mechanisms of MDD changes in metabolic processes, which are explained in detail as follows.
Lipid is important for maintaining normal brain function and activity, abnormal lipid metabolism is thought to be linked to many cerebral disorders, including depression(9). Among the 4 SMs selected, AA, TXB2 and 9-HPODE are important participants in lipid metabolism. In present study, it was found that the lipid metabolism processes of MDD patients, including arachidonic acid metabolism, linoleic acid metabolism, primary bile acid biosynthesis, cholesterol metabolism, etc. were significantly altered compared with healthy controls.
Linoleic acid (LA) is one of the essential fatty acids in human body, which is important for regulating homeostasis(10, 11). 9-HPODE is one of the products of linoleic acid peroxidation, which has been shown to be involved in the oxidative stress of brain tissue caused by Alzheimer’s disease(12). The results of our study showed that the plasma content of 9-HPODE in MDD patients was significantly decreased compared with the healthy controls, and its content was increased after treatment. Moreover, the relative content of 9-HPODE was also negatively correlated with the subjects' HDRS-17 scores. However, the relationship between linoleate peroxide and depression has not been clarified in previous studies. Therefore, the role of linoleate metabolism in the process of depression still needs further studies to confirm.
Arachidonic acid (AA) is an important product of linoleic acid metabolism. Cyclooxygenase (COX), lipoxygenase (LOX), Cytochrome P450 (CYP450) and other enzymes catalyze the formation of eicosanoid family of mediators, including thromboxanes (TXs), prostaglandins (PGs), leukotrienes (LTs), hydroxy eicosatetraenoic acid (HETE), which are closely related to the regulation of the inflammatory process(13, 14). Once an imbalance of pro-inflammatory and anti-inflammatory shows in the central nervous system, diseases including depression may appear(15, 16). In our study, plasma contents of AA and its downstream stable pro-inflammatory product, TXB2 in MDD patients increased significantly compared with healthy controls, and then showed a downward trend after treatment, and their relative levels were positively correlated with the subjects' HDRS-17 score. In addition, although the relative contents of Leukotriene B4 (LTB4, the pro-inflammatory metabolite of AA) and the unstable metabolite (±) 15-HETE did not change regularly among the groups, their contents in 0m group were significantly increased compared with controls. The results indicated that the metabolic process of AA was significantly activated in MDD patients, suggesting that inflammatory events may occur in MDD patients.
AA is closely related to retrograde endocannabinoid signaling. Endocannabinoid system (ECS) consists type 1 and type 2 cannabinoid receptors (CBR1 and CBR2), endogenous ligands such as N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol(2-AG), and a series of related enzymes and transporters. ECS plays an important role in the regulation of central nervous system functions such as energy metabolism, inflammation, behavioral selection and synaptic plasticity(17). AEA and 2-AG which are derivatives of AA generally play a role in the signal transmission of different brain regions and synapses through a retrograde mechanism, namely(18). The metabolic glutamate receptors (mGRAs), serotonergic receptors 5-HT2a and 5-HT2c, can regulate synaptic transmission by triggering the endocannabinoid retrograde signaling(19). ECS is involved in both short-term and long-term depression, and the loss of retrograde signaling may be one of the causes of depression(20). In our study， the contents of AA and L-glutamate were significantly increased in plasma of 0m group compared with control group. Arachidonic acid is one of the hydrolytic product of two endocannabinoids(21), and glutamate is thought to be one of the neurotransmitters that activate the ECS(22). However, since the changes in key intermediate links such as receptors and enzymes related to the endocannabinoid retroactive signaling pathway are still unknown, further studies are needed to confirm the potential connection between the ECS and MDD.
Moreover, AEA can also produce activity through the activation of transient receptor potential V1(TRPV1) channel in addition to the retrograde signaling pathway. The TRP channel family is increasingly considered as a sensor of nociceptive stimulation and can also be indirectly regulated by a variety of inflammatory mediators(23). Arachidonic acid metabolism can activate the TRP channels, especially the TRPV4, TRPV1 and TRPM8 channels (24, 25). We found that compared with healthy controls, plasma contents of arachidonic acid and its metabolites LTB4 and (±)15-HETE were increased in MDD patients, both of which are activated molecules of the TRP channels. Although the link between depression and the TRP channels has not been clearly understood (26), the possibility of the regulation of TRP channels as the potential mechanism of MDD cannot be ruled out, but the confirmation of the pathway needs more exploration.
What is known to all that the decrease of 5-hydroxytryptamine (5-HT) level in the brain is one of the main causes of depression. 5-HT can stimulate the release of AA by inducing phospholipaseA2 (PLA2) signaling through the 5-HT2 receptor. Then AA and its metabolites can in turn inhibit the release of neurotransmitters including 5-HT(27-29). Moreover, the KEGG pathway network constructed in present study showed that long-term depression was also associated with the above processes. Our study found that arachidonic acid metabolism was activated in MDD patients, suggesting that there may be crossover between arachidonic acid metabolism and 5-HT system, but there are still many complex processes involved between them, further studies on the receptors and enzymes involved in the intermediate processes are needed.
Bile acid not only has the characteristics of local cleaning agent, but also is an important signal molecule in the human body. They may lead to MDD by altering intestinal epithelial cell permeability, and may also regulate neuroinflammation through the signaling pathway mediated by takeda G protein-coupled receptor5 (TGR5), thus influencing the depressive phenotype of animals(30). In addition, the changes of expression of farnesoid X receptor (FXR) which can be activated by bile acid is one of the reasons induced depression animal behavior and neuroinflammation(31). And the activation of FXR can alleviate liver inflammation by inhibiting arachidonic acid metabolism and reducing the level of the pro-inflammatory metabolite LTB4, which in turn inhibits the nuclear factor κ light-chain enhancer of activated B cells (NF-κB)(32). We found that compared with control group, the plasma contents of cholesterol, TXB2 and LTB4 in 0m group were significantly increased, while glycochenodeoxycholic acid and glycocholic acid were significantly decreased, indicating abnormal bile acid metabolism process in MDD patients. However, the change trend of related indicators is not completely consistent with previous studies(33). Whether the expression of FXR changes in MDD patients, and whether the disturbance of bile acid system causes the activation of neuroinflammation, these questions require further exploration before they can be answered.
In our study, we also found changes in histidine metabolism in MDD patients. Compared with control group, the contents of L-glutamic acid and N-acetylhistamine in 0m group were increased, and the contents of Imidazoleacetic acid and 3-N-methyl-L-histidine were decreased. Moreover, the relative content of Imidazoleacetic acid showed an upward trend after treatment. L-histidine can produce L-glutamic acid through a series of complex metabolic pathways. And glutamate system plays an important role in the pathophysiological processes of depression(34). Unfortunately, the relationship between histidine metabolism and depression has not been directly reported and the relevant mechanisms need to be explored in the future.
Changes in plasma metabolic profile
The result shows that HDRS - 17 scores of 0 m, 1 m and 2 m group were significantly increased compared with controls, and the HDRS-17 score of MDD patients gradually decreased with the prolongation of treatment time, indicated that patient's depressive symptoms were gradually relieved during the treatment. Moreover, there were significant differences in plasma metabolic profiles between MDD patients and controls, but metabolic transformations before and after treatment were not significant. These results suggest that the effect of two months of conventional treatment on the change of metabolic abnormalities in MDD patients is limited. Depression is a heterogeneous disease with a complex mechanism, and the classification of the subtypes of depression is not perfect at present, which leads to great differences in the internal metabolic changes of patients with similar depressive symptoms. Our study suggests that short-term conventional drug therapy can effectively improve patients' mental symptoms, while it cannot accurately act on the abnormal related metabolic pathways in patients, resulting in the inconsistency between the external behavioral symptoms and the changes in the internal biochemical indicators. Fortunately, we also found some changes of plasma metabolic profiles and pathway disturbances in MDD patients during treatment, which may be a potential target for antidepressants and provide a new idea for individualized and precise treatment of MDD.
There are limitations to our study. As a pilot study at the exploratory stage, this study only included 12 control group and 12 MDD patients, and did not strictly control the subjects' gender and age. Although we followed up the patient for two months during his hospitalization, we failed to to obtain the patient's prognosis information after discharge. MDD is a multi-target chronic disease with complex mechanism, which not only involves changes in the metabolic process, but is also closely related to hormone levels，protein and gene levels related to each pathway. The network of possible mechanisms for MDD that we got from the metabonomic alone is deficient in some intermediate details.