Depression stands as a pervasive global mental health challenge, impacting approximately 5% of the world’s population as estimated by the World Health Organi-zation. Recent studies unveil an alarming surge, revealing a global prevalence rate of 27.6% for depression, an escalation poised to designate it as the primary cause of disease burden by 2030 [1-3]. The roots of depression are intricate, involving a multitude of factors such as genetics, environment, immunity, neurogenesis, biogenic amine depletion, and endocrine dysfunction in its pathogenesis [4,5]. The hypothesis of depression, initially introduced in the 1960s, laid a crucial foundation for depression medication [6]. With the advent of 5-hydroxytryptamine (5-HT) reuptake inhibitors (selective serotonin reuptake inhibitors, SSRIs) in the 1990s, this hypothesis gained broader acceptance among basic and clinical researchers [7-9]. This theory posits that depression arises from a decrease in 5-HT release within the central nervous system and a reduction in its synaptic gap concentration. 5-HT, a monoamine neurotransmitter, accumulates within nerve endings facilitated by the 5-HT transporter (SERT). This transporter shuttles 5-HT into the cytoplasm to replenish synaptic vesicles, thereby terminating its extracellular effects. The regulatory role of 5-HT spans human behaviors, mood, memory, and its involvement in treating psychiatric and neurological disorders. Consequently, it plays a pivotal role in both the development and treatment of depression. By impeding the SERT in the presynaptic membrane, the inhibition of presynaptic 5-HT reuptake toward the synaptic gap amplifies the impact of 5-HT on postsynaptic 5-HT receptors. This action facilitates the binding of 5-HT to the 5-HT₁ receptor, thereby exerting antidepressant and anxiolytic effects [10].

For a long time, however, depression diagnosis and response evaluation to antidepressants predominantly rely on clinical manifestations and scales, lacking objective laboratory diagnostic indicators [11,12]. Cerebrospinal fluid (CSF) 5-HT levels hold promise as deal biomarker for investigating brain-related illnesses due to its contact with interstitial fluid [13]. However, its invasive nature and associated risks, coupled with a scarcity of large-scale studies, limit its widespread use. Hence, the assessment of peripheral 5-HT levels emerges as the preferable clinical approach. In 1987, Sarrias et al. [14] proposed plasma 5-HT levels as a potential diagnostic tool for depression. Drawing from their physiological origin, structural and functional parallels, plasma 5-HT levels have been conjectured to mirror the 5-HT levels in neurosynaptic gap, while platelet 5-HT levels reflect 5-HT levels in central neurons [15]. Unfortunately, great difference in reference was noted between peripheral 5-HT levels and depression diagnosis and response assessment to antidepressants both in clinical evidence and animal experiments [16-19]. For instance, an extensive observational cohort study among postmenopausal woman revealed that depressed individuals displayed lower plasma 5-HT levels. However, this disparity did not attain statistical signifi-cance after adjusting for multiple comparisons. One study reported a significant decrease in serum 5-HT levels in Chronic unpredictable mild stress (CUMS) rats compared to the normal control group [20], the other found no significant difference in serum 5-HT levels between CUMS rats and the normal control group [21]. Moreover, a meta-analysis encompassing 19 studies on 5-HIAA in CSF failed to establish a correlation between depression and concentrations of 5-Hydroxyindoleacetic acid, the primary metabolite of 5-HT [22]. Therefore, systematic research is needed to obtain more evidence to validate and confirm the potential clinical value of specific peripheral 5-HT test. To address the inconclusive clinical issue, this article explores the feasibility of using 5-HT levels in serum, plasma, and platelets as peripheral biomarkers for depression diagnosis and response evaluation to antidepressants with animal depression models.

Animals

This research received approval from the Experimental Animal Ethics Committee of Sixth People’s Hospital South Campus, Shanghai Jiaotong University and was conducted under the oversight of the hospital’s Review Committee (No. 2022-0334). Thirty female C57BL/6 mice (18−20 g, aged 5−7 weeks) and 30 female Sprague-Dawley rats (160−200 g, aged 8−10 weeks) were procured from Jiangsu Huachuang Xinnuo Pharmaceutical Science and Technology Co., Ltd. (License No: SCXK 2020-0009). The animals were accommodated in a standard laboratory environment with a 12-hour light (06:00−18:00) and dark (18:00−06:00) cycle, maintaining temperatures between 22−25°C and relative humidity ranging from 40% to 70%. All animals were fed ordinary food by specific person and get their food and drink freely for 7 days acclimatization period before commencing the experiment.

Depression-like Behavior Animal Models Establishment, Fluoxetine Treatment and Sample Collection Workflow

CUMS-induced depression animal model was established according to the protocol [23]. After 1 week of acclimation, thirty mice and rats were randomly assigned into two groups using a random number table respectively. No intervention was given to the control group animals (NC group, n = 10) while animals in the other group (CUMS group, n = 20) was subjected to CUMS for 4 weeks in mice and 15 weeks in rats. The stressors included: swimming at 4°C or at 45°C for 5 minutes; cage vibration for 20 minutes; restraint stress for 3 hours; damp bedding for 12 hours; noise interference (85 dB) for 1.5 hours; cycle disturbances; cage tilting (45°) for 12 hours; no sawdust for 12 hours. Two stressors were used per day, and each stressor was not used consecutively for two days. The schedule of the stressors was shown in Table 1. Animal subjected to CUMS was housed separately in a single cage, while others were housed in groups. After model establishment, the modeled animals (n = 20) were further stratified into two sub-groups: CUMS group (n = 10) and CUMS + fluoxetine (FLX) group (n = 10). Then animals in the NC group (n = 10) and CUMS group (n = 10) were gavaged an equal volume of drinking water daily while animals in CUMS + FLX (n = 10) group were gavaged with FLX of 10 mg/ml/d for 28-day. Blood samples were collected at the end of model establishment and drug administration, respectively. The animals’ weights were recorded weekly. After drug administration, animals were sacrificed and hippocampus, cerebral cortex, and colon tissues were collected. The 5-HT levels in blood and tissue were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, 5-HT-associated proteins in tissue and platelets such as SERT, tryptophan hydroxylase 1 (TPH₁), TPH₂, and 5-HT_1A were identified via Western blotting. The entire experimental procedure was illustrated in Figure 1.

Behaviors Tests

Behavioral tests including sucrose preference test (SPT), open field test (OFT), tail suspension test (TST), and forced swimming test (FST) were performed to validate the depression-behavior models. These tests were usually conducted between 9:00 and 14:00.

SPT: Animal was individually housed in separate cage, and water drinking training was implemented. Briefly, for mouse, one bottle containing a 1.5% sucrose solution alongside the other bottle of drinking water were supplied for 48 hours. For rat, two bottles of 1.5% sucrose water were supplied for the initial 24 hours, followed by one bottle of 1.5% sucrose water and the other bottle of drinking water for the subsequent 24 hours. Sucrose preference was calculated as follows: sucrose preference (%) = [amount of sucrose solution consumed/(amount of sucrose solution consumed + amount of drinking water consumed)] × 100%.

OFT: All animals were given 0.5 hour acclimatization period in the test room before the test. Once initiated, each animal was individually taken from cage and introduced into the central area of the field. Tracking Master V3.0 (ZSDC Science and Technology Development Co., Ltd.) was employed to monitor its activity within the field for a duration of 5 minutes. After the test, the field was wiped with alcohol to eliminate any residual odor and mitigate its potential influence on subsequent animal assessment.

TST: This test is only conducted in mice. All mice were given 0.5 hour acclimatization period in the test room before the test. Each mouse was caught from cage, and the tail was secured to hook and suspended in an inverted position with 30 cm distance between the head and the bottom of the enclosure. Tracking Master V3.0 was employed to record the immobilization time of mice in the last 4 minutes of the 6-minute test.

FST: The animal was placed in transparent cylindrical buckets with a water level of 30 cm for mouse and 60 cm for rat (the water temperature was 23−25°C, and the rat was trained to swim one day in advance). Tracking Master V3.0 was employed to record the immobility time of the animal in the last 4 minutes of the 5-minute test.

Blood and Tissues Sample Preparation and Measurement

Animals were kept unconscious with isoflurane for gas anesthesia. The orbital venous plexus blood was collected by glass capillary at 10:00 am. One part of the blood was kept in a regular EP tube and the other part was kept in an eppendorf (EP) tube containing anticoagulant citrate dextrose (ACD) (1 ml/6 ml whole blood). Blood in regular EP tube was allowed to clot for 2 hours at room temperature, centrifuged at 3,000 rpm for 5 minutes, and the upper supernatant (serum) was stored at −80°C. Blood in EP tube (with ACD) was centrifuged at 800 rpm for 20 minutes, the supernatant platelet-rich plasma (PRP) was collected and centrifuged at 4,000 rpm for 20 minutes, yielding platelet poor plasma (PPP, later for short, plasma). The plasma was collected and stored at −80°C. The remaining white precipitate at the bottom was hypotonically ruptured by adding 50 μl of double-distilled water and stored at −80°C. The remaining PRP was used for platelet counting, with the PRP volume and platelet number recorded for analysis.

Blood sample (50 μl of serum, plasma) was mixed with 100 μl of acetonitrile, while platelets were repeatedly frozen and thawed three times and mixed with 100 μl of acetonitrile, then samples were vortex-mixed for 30 seconds to precipitate the proteins and were centrifuged at 14,800 rpm for 5 minutes. The supernatant was filtered through a needle filter, then 80 μl was extracted into the injection bottle with 1.6 μl of 3,4-dihydroxybutyric acid (DHBA, internal standard, IS, 10 μg/ml), and then 2 μl of the supernatant was injected into the LC-MS/MS system.

After the animals were sacrificed, cerebral cortex, hippocampus and colon were collected and stored at −80°C. The cerebral cortex and hippocampus samples were homogenized with 0.1 ml water for 10 seconds using a Tissue homogenizer (Next Advance, Inc.). To prevent possible protein degradation, the homogenization was performed in ice bath. The total volume of each homogenate was equal to the sum of the brain sample weight (g)/(1 g/ml) and 0.1 ml (the volume of solvent added for homogenization), as the density of brain tissue is approximately 1 g/ml. An aliquot of 50 μl brain homogenate was mixed with 100 μl acetonitrile to precipitate protein. Then the mixture was vortexed and centrifuged at 4°C, 14,800 rpm for 5 minutes. The supernatant was collected and 80 μl was extracted into the injection bottle with 1.6 μl of DHBA (internal standard, IS, 10 μg/ml), and then 2 μl of the supernatant was injected into the LC-MS/MS system. Colon samples were prepared as the same method for brain homogenates.

5-HT levels were measured with LC-MS/MS system (1290 Infinity LC, 6530 UHD and Accurate-Mass Q-TOF/MS; Agilent Technologies) according to reference [24]. The chromatographic separation conditions were as follows: chromatographic column: C₁₈ column (Agilent, 4.6 × 50 mm, 1.8 μm). Sample was eluted in the mobile phase at a flow rate of 0.6 ml/min at 25°C: H₂O with 0.1% methane acid (A) and acetonitrile with 0.1% methane acid (B). Gradient elution values as follows: The column was ramped from 45 to 70% B from 0−2 minutes, maintained at 100% B from 2.1−3.0 minutes, kept at 45% B from 3.1−5.0 minutes. For MS/MS detection transitions m/z 177.2 → 160.1 and m/z 153.0 → 109.0 for 5-HT and the IS were followed, respectively. The ion source was kept at 4,000 V and at 350°C in positive ion mode. The heater gases were set at 11 L/min, while the collision gas was set to 9. The scanning mode employed was multiple reaction monitoring. The method was validated in terms of selectivity, linearity, sensitivity, and precision and was fit for the intended purpose.

Cerebral cortex, hippocampus, and colon tissues were ground in RIPA buffer at 4°C with pre-added 1% protease and phosphatase inhibitors. The mixture was allowed to incubate for 30 minutes before undergoing centrifugation at 12,000 rpm for 10 minutes at 4°C. Proteins within the platelets were extracted using a specialized platelet protein extraction kit. The supernatant above was quantified using the BCA protein assay kit (Solarbio Science & Technology Co., Ltd.), analyzed by 10% sodium dodecyl sulfate polyacylamide gel electrophoresis (SDS-PAGE; NCM Biotech), and transferred onto a nitrocellulose filter membrane (Pall Corporation). After 1 hour of blocking, the membranes were incubated with the primary antibody (5-HT_1A, TPH₁, TPH₂ antibody were purchased from Immunoway, GAPDH and SERT antibody were purchased from ABclonal Inc.; dilution rate: 5-HT_1A, TPH₁, TPH₂ and SERT were 1:1,000, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was 1:10,000) overnight at 4°C, followed by incubation with secondary antibodies (goat anti-rabbit antibody and goat anti-mouse antibody were purchased from ImmunoWay Biotechnology Company; dilution rate: goat anti-rabbit and goat anti-mouse were 1:10,000), and detected with an enhanced chemilumin-escence (ECL) buffer (NCM Biotech). The intensity of each band was determined by ImageJ software.

Data Analysis

All data were presented as mean ± standard error of mean. Comparisons between two groups were performed using the two independent samples ttest (Wilcoxon rank test was performed when the parameter test was not satisfied). Comparisons between multiple groups were performed using one-way analyses of variance (ANOVA) and Tukey test. pvalue < 0.05 was considered statistically significant.

Chronic Stress-induced Depression-like Behavior Models Validation and Effects of FLX in Mice and Rats

In general, the behavioral tests (OFT, SPT, FST, and TST) results confirmed the successful establishment of depression-like behavior models. After CUMS, the animals exhibited a significant decrease in sucrose preference (mice and rats: p < 0.05), decreased distance ventured into the central area (mice and rats: p < 0.01), and notably elevated immobility duration in the TST (mice: p < 0.05) and FST (mice: p < 0.01; rats: p < 0.05) compared to the animals in NC group (Fig. 2). Meanwhile there was significant reduction in weight gain at the end of model establishment in CUMS group compared to the animals NC group (mice and rats: p < 0.01) (Fig. 3).

After 28 days of FLX treatment, the depression-like behaviors (OFT, SPT, and FST) were remarkably improved in CUMS + FLX group compared to CUMS group both in mice and in rats’ models (Fig. 2). However, there was no significant increase in immobility time (TST test) in mice model.

Effect of Chronic Stress on Peripheral and Tissue 5-HT Levels in Mice and Rats

In mice model, there was a significant increase in serum 5-HT levels and a significant reduction in plasma 5-HT levels in CUMS-model group (p < 0.05) compared with the NC group, while platelet 5-HT levels had not much change (Fig. 4A-4C). In rats’ model, there was a significant increase in serum 5-HT levels while plasma and platelet 5-HT levels showed little change in the CUMS group compared with the NC group (Fig. 4J-4L).

In mice model, there was a significant decrease of 5-HT levels in cerebral cortex (p < 0.05; Fig. 4G) and a significant decrease of 5-HT levels in hippocampus (p < 0.01; Fig. 4H) in CUMS group compared to NC group, whereas there was a decreased tendency of 5-HT levels in colon (Fig. 4I), although this decrease was not significant. In rats’ model, there was a decreased tendency of 5-HT levels in cerebral cortex, hippocampus, and colon in CUMS group compared to NC group (Fig. 4P-4R). although there were no significant changes.

Effect of FLX on Peripheral and Tissue 5-HT Levels in Mice and Rats

After 28 days of FLX treatment, peripheral and tissue 5-HT levels were showed in Figure 4. In mice model, serum 5-HT levels and platelet 5-HT levels were significantly decreased in CUMS + FLX group (p < 0.01; Fig. 4D, 4F), meanwhile plasma 5-HT levels had not much change compared with CUMS group (Fig. 4E). There was an increasing tendency of 5-HT levels in colon, cerebral cortex, and hippocampus in CUMS + FLX group compared with CUMS group (Fig. 4G-4I). In rats’ model, serum, plasma and platelet 5-HT levels significantly decreased in CUMS + FLX group compared with the CUMS group (p < 0.01; Fig. 4M-4O). There was little change of the tissues 5-HT level in CUMS + FLX group, compared with CUMS group (Fig. 4P-4R).

Effect of Chronic Stress on TPH, 5-HT_1A Receptor and SERT Expression in Tissue and FXL Treatment in Mice Model

As observed in Figure 5A, TPH₂ and 5-HT_1A receptor expression in cerebral cortex were decreased significantly in CUMS model compared to the NC group (p < 0.05). But not much change in TPH₂, 5-HT_1A receptor and SERT expression in hippocampus were observed in CUMS group compared to the NC group (Fig. 5B). TPH₁ expression in colon was significantly decreased in CUMS group compared to NC group (p < 0.01), while 5-HT_1A receptor expression had not much changed (Fig. 5C). As for platelets, there was a significant increase of SERT expression (p < 0.01) in CUMS group compared to NC group, 5-HT_1A receptor expression had not much changed (Fig. 5D).

After FLX treatment of fluoxetine, TPH₂ and 5-HT_1A receptor expression in the cerebral cortex were enhanced in CUMS + FLX group compared to CUMS group (p < 0.05; Fig. 5A). SERT expression in the hippocampus was decreased significantly (p < 0.05) in CUMS + FLX group compared with CUMS group, while TPH₂ expression level had not much changed, and 5-HT_1A receptor expression increased significantly (p < 0.05; Fig. 5B). In the colon, there was a significant increase in the TPH₁ expression in CUMS + FLX group than CUMS group (p < 0.05), meanwhile 5-HT_1A expression had not much changed (Fig. 5C). In the platelets, FLX inhibited the elevated SERT expression significantly in CUMS + FLX group compared with CUMS group (p < 0.01) but didn’t change the 5-HT_1A receptor expression (Fig. 5D).

Effect of Chronic Stress on TPH, 5-HT_1A Receptor and SERT Expression in Tissue and FXL Treatment in Rats Model

As observed in Figure 5, similar to the result in mice model above, 5-HT_1A receptor expression in cerebral cortex was decreased in CUMS group compared with NC group, but not much change in TPH₂ expression. SERT expression in cerebral cortex was increased in CUMS group compared with NC group (Fig. 5E). In rat’s hippocampus, TPH₂ and 5-HT_1A displayed a decreased expression while SERT expression displayed an increased expression in CUMS group compared with NC group (Fig. 5F). In rat’s colon, there was a significant reduction in TPH₁ expression (p < 0.01) with a substantial increase in SERT expression (p < 0.05) in CUMS group (Fig. 5G). In rat’s platelets, SERT expression exhibited a significant increase (p < 0.01; Fig. 5H) in CUMS group compared with NC group.

After FLX treatment of fluoxetine, SERT expression in the cerebral cortex and hippocampus were slightly inhibited in CUMS + FLX group compared with CUMS group (Fig. 5E, 5F), though the changes were not significant. In the colon (Fig. 5G), TPH₁ expression was increased significantly (p < 0.01) while SERT expression was decreased slightly, and 5-HT_1A receptor expression increased slightly in the CUMS + FLX group compared with the CUMS group. Lastly, compared with CUMS group, SERT expression in platelets showed a significant decrease (p < 0.01) and 5-HT_1A receptor expression in platelets showed an increasing trend in the CUMS + FLX group (Fig. 5H).

The unpredictable chronic mild stress model of depression was first proposed by Katz in 1982 and further developed by Papp and Willner [25]. Considered by many to be the animal model of depression with the greatest validity and translational potential, CUMS model maximises the simulation of patients suffering from chronic psychological stress in the clinic and is widely used to simulate depression-related behaviors in humans. Com-pared with some other stress models such as the chronic restraint stress, social defeat stress and early life stress, CUMS overcomes the stress habituation. In this model, rats or mice are chronically exposed to a sustained bombardment of unpredictable micro-stressors, leading to the development of a large number of behavioural changes, including a decreased response to rewards, a behaviour that is associated with the clinical core symptom of depression, anhedonia. However, the drawback of the model is that it is difficult to perform CUMS experiments, which requires a lot of work, space and time [26]. In this paper, the use of two animal breeds with highly stable genetic backgrounds, C57BL/6 mice and SD rats, excludes the influence of genetic factors of depression and ensures the consistency of experimental data. Epidemiological statistics show that the global prevalence of 12-month major depression is 5.8% in woman and 3.5% in man [27]. The gender difference in depression was generally believed that twice as many women suffer from major depression than men, whereas there was no significant difference in serum serotonin levels by sex [28]. In addition, most of the current research focuses on male animals and ignores a role in the female gender, with many potential compounds being discarded in drug development [29]. Therefore, this study was conducted on female animals.

In this study, we successfully established a chronic stress-induced depressive-like behavior animal model in mice and rats. And we simultaneously measured 5-HT levels in serum, plasma, and platelets for the first time. In general, the results showed that chronic stress significantly increased serum 5-HT levels, decreased plasma 5-HT levels, but did not change much platelet 5-HT levels in mice model. FLX treatment significantly decreased serum and platelet 5-HT levels but did not change much plasma 5-HT levels. Like those results in mice model, chronic stress significantly increased serum 5-HT levels, but did not change much plasma 5-HT and platelet 5-HT levels in rats’ model. FLX treatment significantly decreased serum, plasma, and platelet 5-HT levels.

Since 5-HT cannot pass through the blood-brain barrier, peripheral and central 5-HT systems function independently. However, the correlation between peripheral 5-HT levels and central 5-HT levels has always been research interest [30]. Many studies focused on serum 5-HT levels or platelet 5-HT levels or plasma 5-HT levels, the results were disparate results [17,18,31]. In this work, there was no evidence to suggest a consistent correlation between 5-HT levels in brain tissue and peripheral blood. However, unlike the diverse variations in various peripheral blood 5-HT levels, chronic stress led to a reduction in hippocampal and cortical 5-HT levels in mice and rats, while fluoxetine might reverse this decrease in this work.

5-HT, also known as serotonin, is synthesized from tryptophan by TPH to 5-hydroxytryptophan, which is then synthesized by 5-hydroxytryptophan decarboxylase in central nervous system (CNS) neurons and intestinal chromaffin cells in the digestive tract of animals [32]. TPH is divided into two isoforms, TPH₁ (which is responsible for the majority of the peripheral 5-HT) and TPH₂ (which is responsible for the pool of the central neurotransmitters in the CNS) [33]. 5-HT is usually stored in presynaptic vesicles and released into the synaptic gap as required for neurotransmission. Serotonergic signaling is terminated primarily by the uptake of 5-HT from the synaptic gap back into the presynaptic neuron, which is achieved primarily through SERT on the presynaptic membrane. The initial FLX intake usually inhibits SERT, leading to elevated extracellular 5-HT levels within synaptic clefts. However, prolonged use of FLX causes an excess of extracellular 5-HT, triggering activation of presynaptic 5-HT_1A auto-receptors. This activation subsequently reduces 5-HT synthesis, and lowers intracellular 5-HT levels [34]. The reduced uptake by SERT leads to decreased intraneuronal 5-HT levels. After 2 weeks of FXL treatment, desensitization of 5-HT auto-receptors occurs, and serotonin synthesis is restored [35,36]. In this work, plasma 5-HT levels did not change significantly after FLX treatment in mice model but showed decrease after FLX treatment in rats’ model. The variation might stem from species-specific differences and different rates of drug response. In this work, rats were exposed with a longer modeling process compared to mice (15 weeks vs. 4 weeks), suggesting that rats exhibited a slightly delayed response to chronic stress. In addition, it is difficult to ascertain that PPP is truly devoid of platelets and extra-platelet 5-HT might be bound to albumin or other serotonin-binding proteins [37], which would pose an unknown impact on determination of physiological-active 5-HT outside of platelets. And during the preparation of PRP or PPP, centrifugation process might have an uncertain effect on the release of 5-HT, which would further influence to the 5-HT concentration of plasma components.

Among the peripheral biomarkers of depression diagnosis of interest in this study, mice and rat models of chronic stress-induced depressive-like behaviors showed a same result of significantly increased serum 5-HT levels. This result was consistent with some studies. For example, the serum 5-HT concentrations in a cohort of depressed patients were notably higher than those in a healthy control group before drug treatment [31]. Serum 5-HT levels usually consist of the bioactive 5-HT in plasma and 5-HT released during platelet aggregation and coagulation. And according to our experimental results, it was found that the vast majority of serum 5-HT originated from platelets 5-HT. Platelet 5-HT levels largely depend on the functioning of uptake-related mediators, especially SERT and 5-HT_1A receptors. Plasma 5-HT has a rapid turnover rate and its concentration results from the balance between synthesis and release by enterochromaffin cells and deamination by monoamine oxidase (MAO) in the liver and renal [38]. Due to rapid metabolization and degradation through MAO, free serotonin plasma concentrations are negligible [39]. Plasma MAO activity levels may be indicative of the rate of 5-HT catabolism. However, MAO activity was not measured in this study, which is a limitation of our work. Patients with complex neurological and neuropsychiatric disorders have already been described with altered platelet function several decades ago [40-42]. Recent studies also found platelet abnormalities in patients with psychiatric-related disorders [43,44]. Some studies reported increased platelet activation in individuals with depression, compared to healthy controls [45], and platelet release of 5-HT relies on its activation. We speculated that CUMS led to increased platelet activation in mice and rats, with an increased amount of 5-HT released from platelets in the serum, and therefore increased serum 5-HT concentrations. The platelet 5-HT levels in CUMS group did not show a significant increase both in mice and rats’ model seemed to be in line with the findings of several studies. For example, one study found no significant difference in platelet 5-HT levels between depressed patients and controls [46], and researchers in the other study observed that platelet 5-HT levels in untreated depressed patients were similar to those of healthy controls, and platelet 5-HT net uptake rate was found to be significantly increased in severely depressed patients compared to the healthy group [47], which was consistent with our western blotting results of a significant increase in SERT protein expression in the CUMS group of mice and rats compared to the NC group.

In the present study of peripheral biomarkers of response to antidepressant drug treatment, our findings indicated that 5-HT levels in peripheral platelets and serum were significantly reduced in both model mice and model rats treated with FLX. Similar to our results, it has been suggested in the study that peripheral serotonin decreased after FLX treatment [48]. One study also reported that citalopram-treated CUMS rats demonstrated a significant reduction in platelet 5-HT levels [49], and another study found a significant decrease in serum 5-HT after two weeks of fluoxetine treatment in depressed patients [50]. FLX blocks 5-HT transporters in the presynaptic membrane and maintains 5-HT in the synaptic gap. Platelets contain the same 5-HT transporter as neurons, suggesting that FLX blocks the transporter in the platelet membrane and prevents 5-HT from entering the platelet. In our study, the increase in SERT expression levels in platelets of model mice and model rats was reversed by FLX treatment, which may have resulted in a significant decrease in platelet 5-HT levels after a substantial reduction in platelet reuptake. In addition, FLX treatment may have reduced up-regulated platelet activity, resulting in a decrease in the amount of 5-HT released by platelets in the serum, and therefore a decrease in serum 5-HT.

About 50% of patients exhibit a positive response to SSRIs among the most prevalent antidepressant treatments. There is still work to be done to develop depression medications for the remaining patients who fail to remit effectively [28]. Consequently, more efforts should be done to develop potential biomarkers to respond antidepressants efficacy [51,52]. Based on the depression pathogenesis and drug development efficacy, 5-HT might potentially serve as biomarker for depression diagnosis and response to antidepressants. Clinical practice advocates for the development of single or combined biomarkers to ensure precise diagnosis and prognosis prediction for depression [53,54]. Our preliminary work might conclude that although these peripheral indexes are not direct reflectors of central 5-HT levels, increased serum 5-HT levels might be feasible to serve as a potential biomarker for diagnosis of depression, meanwhile decreased serum 5-HT and platelet 5-HT levels might respond to antidepressant treatment.

There are some limitations for this work due to the small sample size. Nevertheless, our results suggested the reliability of increased serum 5-HT level as biomarker for the diagnosis, and the reduced serum and platelet 5-HT levels as biomarkers for efficacy evaluation of antidepressants in depression. In addition, a clinically viable method of analyzing 5-HT, characterized by ease of handling, high sensitivity and specificity, as well as good reproducibility, may pave the way for its inclusion in a comprehensive diagnostic portfolio for clinical utilization.

No potential conflict of interest relevant to this article was reported.

Conceptualization: Feng Xu. Data acquisition: Zuanjun Su, Zhicong Chen, Canye Li, Jinming Cao, Jingjing Duan, Ting Zhou, Zhen Yang, Yuanchi Cheng, Zhijun Xiao. Formal analysis: Zuanjun Su, Zhicong Chen. Writing−original draft: Zuanjun Su. Writing−review & editing, Funding acquisition: Feng Xu. All authors read and approved the final manuscript.