Abstract

Introduction:

Depression is a prevalent neuropsychiatric disorder, and traditional Chinese medicine formulations such as Yin Huo Decoction (YHD) have shown potential antidepressant effects, yet their underlying mechanisms remain incompletely elucidated. This study aimed to investigate the therapeutic effects and molecular mechanisms of YHD in a PCPA-induced depression model in mice.

Methods:

PCPA-induced depressive-like mice were treated with YHD, and changes in body weight, sucrose preference, and behavioral performance in the forced swim and tail suspension tests were assessed. Hippocampal neuron structure and Nissl body integrity were examined, and brain serotonin (5-HT) levels were quantified. Liquid Chromatograph Mass Spectrometer (LC–MS)-based metabolomic profiling was performed on serum, urine, and brain tissue to identify metabolic disturbances, while network pharmacology analysis was used to explore the intersection of YHD targets and depression-related pathways. Pathway enrichment analysis was conducted to clarify key regulatory pathways.

Results:

YHD treatment significantly improved body weight, sucrose preference, and depressive-like behaviors in PCPA-induced mice, and preserved hippocampal neuron structure and Nissl body integrity—effects comparable to fluoxetine. YHD also restored reduced brain 5-HT levels in PCPA model mice. Metabolomic analysis revealed distinct metabolic perturbations in the PCPA model (e.g., in tryptophan and riboflavin metabolism), which were largely reversed by YHD. Network pharmacology identified 156 intersecting targets between YHD and depression-related pathways, primarily involved in neuroactive ligand-receptor interactions, dopaminergic synapses, and inflammatory processes (e.g., TNF signaling and cytokine production). Key targets including AKT1, TNF, IL-6, and EGFR were identified as central to YHD’s action.

Discussion:

YHD alleviates PCPA-induced depression-like behaviors in mice by modulating 5-HT levels, correcting metabolic imbalances in tryptophan and riboflavin pathways, and regulating neuroinflammation, neurotransmitter systems, and cellular signaling via targets such as AKT1 and TNF. These findings provide a comprehensive mechanistic understanding of YHD’s antidepressant effects, supporting its potential as a therapeutic agent for depression.

1 Introduction

Depression is a common mental disorder characterized by a persistent low mood and/or anhedonia. The pathogenesis of depression is mainly linked to a depletion of monoamine neurotransmitters, hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis, and the overexpression of inflammatory cytokines (Miller and Campo, 2021; Jesulola et al., 2018; Zhou et al., 2022; Halaris, 2019). Clinical studies have shown that patients with depression display abnormal metabolism of the neurotransmitter 5-hydroxytryptamine (5-HT) and its related metabolites (Li et al., 2015). A reduction in 5-HT levels contributes to depressive symptoms by disrupting circadian rhythm homeostasis (Crouse et al., 2021; Bunney et al., 2015), highlighting the critical role of 5-HT in depression development (Marszalek-Grabska et al., 2021). Currently, the main pharmacological treatments for depression include selective serotonin reuptake inhibitors (SSRIs) and monoamine oxidase inhibitors (MAOIs). The commonly used first-line drugs include fluoxetine, citalopram and amitrityline, etc. However, such drugs can cause gastrointestinal reactions, weight gain, insomnia, anxiety, and even sexual dysfunction. Yin Huo Decoction (YHD), as an extract of natural drugs, has multi-component compound compatibility and multi-target synergy, with few side effects and no risk of drug resistance, and can enhance the body’s self-healing ability to a certain extent. It can often achieve the effect of “curing the root cause” (Li et al., 2020). As a result, there is growing interest in identifying alternative therapeutic options that offer improved safety and efficacy profiles. Traditional Chinese Medicine (TCM), known for its multi-component and multi-target properties, has shown promising potential in treating depression, offering both efficacy and low toxicity (Xu et al., 2022).

From a TCM perspective, depression is believed to result from an imbalance between Yin and Yang, a deficiency in Yang qi, and subsequent disruptions in mental activity. Specifically, kidney Yin deficiency and heart fire (inflammation) are considered the primary pathological factors, with kidney tonification being a key therapeutic approach in TCM (Kunlingzi and Lisheng, 2020). YHD is a classical TCM formula used to tonify the kidneys. It originates from the Qing Dynasty’s Syndrome Differentiation and Qiwen’ and comprises Rehmannia glutinosa (prepared root), Morinda officinalis, Ophiopogon japonicus, Schisandra chinensis, and Poria cocos (Ying et al., 2015). Previous studies have shown that YHD can alleviate perimenopausal depression in rats (Dongxue et al., 2023), as well as depression- and anxiety-like behaviors in both long-term and short-term ovariectomized mice (Shurong et al., 2023). Despite these promising results, existing research on YHD remains limited, and more comprehensive studies are needed to fully elucidate its therapeutic potential and underlying mechanisms.

P-chlorophenylalanine (PCPA) is a model of depression constructed by inhibiting the activity of tryptophan hydroxylase (TPH) and thereby reducing serotonin levels (Daut and Fonken, 2019; Zhikang and Fei, 2023). Therefore, we evaluated the antidepressant effect of YHD under this model and explored its molecular mechanism. A mouse model of depression was established by intraperitoneal injection of PCPA, a selective inhibitor of tryptophan hydroxylase, followed by YHD treatment via oral gavage. Behavioral assays, histological analyses, and enzyme-linked immunosorbent assays (ELISA) were perdormed to assess depression-like behaviors and 5-HT levels in brain tissue, thereby evaluating YHD’s therapeutic efficacy.

Furthermore, we also used high-throughput metabolomics was used to identify differential metabolites in serum, urine, and brain tissue, thereby elucidating the metabolic profile associated with YHD treatment. Finally, network pharmacology was employed to explore the molecular mechanisms underlying YHD’s antidepressant effects, and to provide a certain basis for the future research on the core targets and active ingredients of YHD in the treatment of depression.

2 Materials and methods2.1 Animals

Nine-week-old male KM mice (25 ± 2 g) were obtained from the Faculty of Laboratory Animal Science, Harbin Medical University. The mice were acclimated for 1 week prior to the start of the experiments. All animals were housed under specific-pathogen-free (SPF) conditions, maintained at a controlled temperature of 24 ± 0.5 °C and relative humidity of 55 ± 5%. The mice were provided ad libitum access to standard chow and water. In accordance with the ARRIVE 2.0 standard, we have supplemented the sample size of each experimental group in the original text and clarified the relevant matters regarding the blind method. All experimental procedures were conducted in accordance with institutional guidelines and relevant regulations governing the care and use of laboratory animals. Approval number: No.2023052601.

We selected 9-week-old male KM mice due to their well-established use in depression models and their stable genetic background, which allows for more consistent and reproducible results. These mice are mature, with stable hormonal levels that minimize potential confounding factors related to sex hormones. However, we acknowledge that the generalizability of our findings may be limited to male KM mice, and future studies should consider the potential differences in antidepressant efficacy between sexes and genetic strains.

2.2 Experimental groups and treatment

The PCPA model is a well-recognized and widely used model in rodents to induce depressive-like behaviors by reducing serotonin synthesis. As a selective and irreversible inhibitor of TPH, PCPA inhibits TPH activity, thereby reducing serotonin synthesis, which mimics the serotonin deficiency observed in depression.

The specific operation for constructing the depression model with PCPA is as follows: PCPA (D831376; Macklin, Shanghai, China) was dissolved in 0.9% physiological saline as the solvent and thoroughly mixed to prepare a suspension at a concentration of 45 mg/mL. The pH of this solution was approximately 5.5 as detected. In subsequent modeling experiments, the suspension was intraperitoneally injected into mice at a dose of 450 mg/kg once daily for 4 consecutive days. This protocol is consistent with the classic approach in previous studies and can significantly lower serotonin levels and induce depressive-like behaviors (Kukuia et al., 2022).

To evaluate the antidepressant effects of YHD, mice were treated with the decoction following model induction. YHD is composed of Rehmanniahg nbAQ glutinosa (Shu Dihuang), Morinda officinalis (Ba Jitian), Ophiopogon japonicus (Mai Dong), Poria cocos (Fu Ling), and Schisandra chinensis (Wu Weizi), all sourced from Beijing Tongrentang Pharmaceutical Co. (Harbin, China). Mice were administered YHD at a dose of 4.05 g/kg via intragastric gavage (i.g.) once daily for 7 days.

Sixty-four KM mice were housed under SPF conditions (temperature: 24 ± 2 °C, humidity: 55 ± 10%) with a 12:12 light/dark cycle. The mice had ad libitum access to commercial SPF chow and autoclaved water. Following a one-week acclimatization period, they were randomly assigned to four groups: Control group (C): healthy mice receiving purified water (i.g.) for 7 days. Model group (M): PCPA-treated mice receiving purified water (i.g.) for 7 days. YHD group (Y): PCPA-treated mice receiving YHD (4.05 g/kg, i.g.) once daily for 7 days. Fluoxetine group (F): PCPA-treated mice receiving fluoxetine (2.6 mg/kg; 5558A, Eli Lilly and Company) via i.g. administration once daily for 7 days.

Twenty-four hours after the final PCPA injection, YHD and fluoxetine treatments were initiated in the respective groups. Control and model groups received an equivalent volume of purified water. Following the final dose, behavioral tests were conducted over 2 days. Then, under deep anesthesia with isoflurane (2 ~ 3%; S190815, Yuyuan, Shanghai, China), mice were transcardially perfused with ice-cold sterilized saline, and then were collected for futher analysis. The experimental design is illustrated in Figure 1.

Schematic diagram showing the detail of PCPA stimulation and drug treatment in PCPA-induced depression mice model.

2.3 Behavioral tests2.3.1 Sucrose preference test (SPT)

The Sucrose Preference Test (SPT) was used to assess anhedonia, a core symptom of depression. Prior to testing, mice were habituated to a 1% sucrose solution. On the first day of habituation, mice were presented with two identical bottles containing 1% sucrose solution. After 24 h, one bottle was replaced with purified water. To avoid side preference, the positions of the bottles were alternated every 6 h. Mice were deprived of water for 24 h before the test. On the testing day, each mouse was provided with one bottle of 1% sucrose solution and one bottle of purified water. The positions of the bottles were swapped every hour to minimize positional bias. Finally, the consumption of sucrose after 2 h was recorded and calculated by the following formula: SPT (%) = [Sucrose consumption / (Sucrose consumption + Water consumption)] × 100.

2.3.2 Forced swimming test (FST)

The Forced Swimming Test (FST) was employed to evaluate depression-like behavior, specifically behavioral despair. Each mouse was placed individually in a transparent Plexiglas cylinder (40 cm height × 20 cm diameter) filled with water to a depth of 25 ± 3 cm, maintained at 24 ± 1 °C. Mice were observed for 6 min, and the duration of immobility during the final 4 min was recorded. Immobility was defined as the absence of active movements, with the mouse floating passively or making only minimal movements to keep its head above water.

2.3.3 Tail suspension test (TST)

The Tail Suspension Test (TST) was conducted to assess behavioral despair in mice. Following the final intragastric administration, mice were individually suspended by the tail using adhesive tape, positioned 1 cm from the tail tip. The mice were suspended at a height of 25 cm above the surface for 6 min. The duration of immobility during the last 4 min was recorded. Mice were considered immobile when they remained completely motionless and passive.

2.3.4 Open field test (OFT)

The Open Field Test (OFT) was utilized to assess anxiety-like behavior and general locomotor activity. The open field apparatus consisted of a square arena (100 cm × 100 cm × 40 cm) with a black floor divided into nine equal squares. Following the last administration, each mouse was placed in the center of the arena and allowed to explore freely for 5 min under dim lighting and a quiet environment. Behavioral parameters recorded included the number of grid crossings (locomotor activity), entries into the central area (anxiety-related behavior), instances of rearing (standing on hind legs), and grooming behaviors. The apparatus was thoroughly cleaned with 75% ethanol between trials to eliminate odor cues.

2.4 Sample collection and tissue preparation

After the completion of behavioral experiments, blood samples were collected via orbital bleeding. Following this, a thoracotomy was performed to expose the heart, and a small incision was made in the right atrium. Three mice from each group were selected for perfusion: an intragastric administration needle was inserted into the apex of the heart, and perfusion was conducted until both the liver and the perfusate appeared pale, indicating complete circulation clearance.

Immediately post-decapitation, the brains were carefully dissected on an ice-cooled dish. The olfactory bulbs and cerebellum were removed, and the brains were bisected into left and right hemispheres using a sterile blade. The hemispheres were fixed in 4% paraformaldehyde for 48 h. Following fixation, the brain tissues were embedded in paraffin for subsequent Hematoxylin–Eosin (HE) and Nissl staining.

The remaining 13 mice were decapitated without perfusion, and their brains were similarly dissected into hemispheres. These samples were rapidly frozen in liquid nitrogen and stored at −80 °C for enzyme-linked immunosorbent assay (ELISA) and metabolomics analyses.

2.5 Hematoxylin–eosin (HE) staining

HE staining was performed following standard protocols. Briefly, fixed brain tissues were dehydrated through a graded ethanol concentrations and then embedded in paraffin at 60 °C. Paraffin-embedded tissues were sectioned, deparaffinized, and rehydrated. Sections were stained with hematoxylin and eosin solutions (DH0006, Beyotime Biotechnology) for 5 min. Following staining, the sections were dehydrated, cleared, and mounted with coverslips. Histopathological changes in the brain tissue were observed under a light microscope.

2.6 Nissl staining

Nissl bodies, which serve as markers of neuronal health by indicating the synthesis of structural proteins necessary for cellular function, diminish or disappear in response to neuronal damage. After undergoing the same dehydration, clearing, infiltration, embedding, and sectioning procedures as in HE staining, brain sections were stained with 0.1% toluidine blue solution (C00117, Beyotime Biotechnology). Following staining, sections were dehydrated, cleared, and sealed. Neuronal morphology and the presence of Nissl bodies were examined under a light microscope.

2.7 Enzyme-linked immunosorbent assay (ELISA)

The 5-HT quantification via ELISA, total protein was extracted from homogenized tissue of the left hemisphere of the mouse brain. The tissue was homogenized in 200 μL RIPA buffer (P0013C, Beyotime Biotechnology, Shanghai, China) supplemented with 2 μL phenylmethanesulfonyl fluoride (PMSF; ST506, Beyotime, Shanghai, China) and 4 μL phosphatase inhibitors (P1081, Beyotime, Shanghai, China). The homogenates were centrifuged at 17,949 × g for 5 min at 4 °C, and the supernatants were collected. Protein concentrations were quantified using a BCA protein assay kit (BL521A, Biosharp, Hefei, China). The 5-hydroxytryptamine (5-HT) levels in brain tissue were measured according to the manufacturer’s instructions using commercial ELISA kits (H104-1-1, Nanjing Jiancheng Bioengineering Institute).

2.8 Untargeted metabolomics analysis using HPLC–QTOF-MS

An ExionLC™ AD system coupled with a TripleTOF™ 5,600+ mass spectrometer (SCIEX) was employed to perform untargeted metabolomics analysis.

2.8.1 Chromatography detection conditions

Utilizing a column, ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm), the experiment conducted at 40 °C, with an injection volume of 5 μL and a detection time of 23 min. The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and 0.1% formic acid-acetonitrile (phase B). The elution program is presented in Table 1.

Time (min)Flow (mL/min)A (%)B (%)00.395520.395560.3703070.37030100.3406011.50.34060130.3208013.50.32080160.31090170.3109017.50.30100200.3010020.50.3955230.3955

Gradient elution procedure.

2.8.2 Mass spectrometry detection conditions

Positive and negative ion detection modes were used, with the first and second mass spectrometry scan ranges set at 100–1200 m/z and 50–1,200 m/z, respectively. Nitrogen gas was used for all gas paths. For specific parameters, please refer to Table 2.

ParameterLC–MSLC–MS/ MSScan TypeTOFTOFIon Spray Voltage Floating (ISVF)±5,500 V±5,500 VSource Gas 1 (GS1)60 psi60 psiSource Gas 2 (GS2)60 psi60 psiCurtain Gas (CUR)35 psi35 psiTemperature (TEM)550 °C550 °CDeclustering Potential (DP)100 V100 VCollision Energy (CE)±10 V±40 VCollision Energy Spread (CES)—20 V

Mass spectrometry conditions.

2.8.3 Serum sample collection and processing

Allow the blood to stand for 30 min. Centrifuge at 4000 rpm for 15 min at 4 °C to collect the supernatant. Precipitate proteins with methanol. Centrifuge at 13000 rpm for 15 min at 4 °C, then evaporate the supernatant using nitroge. Resuspend in methanol and vortex to mix thoroughly. Centrifuge the blood homogenate at 13000 rpm for 10 min at 4 °C. Filter the supernatant through a 0.22 μm membrane filter, then inject into the machine. Quality control (QC) samples were prepared by mixing 10 μL of each sample.

2.8.4 Urine sample collection and preparation

Collect mouse urine, mix with distilled water at a 1:2 ratio (total volume 300 μL), vortex for 10 s to obtain a uniform urine suspension. Subsequent methods are equivalent to blood homogenization. Prepare QC samples as described previously.

2.8.5 Brain tissue sample collection and preparation

Brain tissue homogenates were prepared by homogenizing the right hemisphere of the mouse brain in a 1:1 methanol–water solution. Subsequent methods are equivalent to blood homogenization. Prepare QC samples as described above.

2.9 Data processing

An untargeted metabolomics approach was utilized to characterize differential metabolites in serum, urine, and brain tissue. Data processing and metabolite identification were conducted using MS-DIAL software to integrate comprehensive metabolic profiles. For targeted metabolomics, data were processed using SCIEX OS software. Quantitative results were derived by applying the peak area ratios (analyte/internal standard) to corresponding standard curves to calculate the concentration of each metabolite.

Metabolomics data were processed using the following methods: The raw data were converted to the ABF format using the Analysis Base File Converter, followed by preprocessing with MSDIAL software. The metabolite identification library was based on LC–MS/MS data in both Positive and Negative Ion Modes1. Multivariate statistical analysis was conducted using SIMCA software, which included unsupervised Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal PLS-DA (OPLS-DA). Differential metabolites were analyzed using MetaboAnalyst2, allowing for the identification of potential metabolic pathways. The trends of differential metabolites across various metabolic pathways were compiled to construct a metabolic disturbance map.

2.10 Network pharmacology analysis2.10.1 Identification of YHD targets and depression-associated genes

TCMSP, SwissTargetPrediction and PharmMapper databases were used for prediction and analysis to obtain the potential targets of YHD. In addition, targets associated with depression were collected from DisGeNET, OMIM, and GeneCards databases. The target data of YHD and depression were imported into the Venn graph making website for intersection target analysis to provide further research.

2.10.2 Protein–protein interaction (PPI) network and core target analysis

The common targets of YHD and depression obtained from network pharmacology analysis were imported into the STRING database for PPI network construction, and disconnected nodes were excluded to enhance the robustness of the network. The generated interaction files were imported into Cytoscape software to visualize the PPI network.

2.10.3 GO enrichment and KEGG pathway analysis

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were conducted using the Metascape platform. The top five terms from biological processes (BP), cellular components (CC), and molecular functions (MF) were selected based on p-values for visualization.

2.10.4 Construction of the “YHD components–depression–target–pathway” network

Active YHD components capable of crossing the blood–brain barrier, together with the intersecting targets and KEGG-enriched pathways, were integrated into Cytoscape to construct a comprehensive network illustrating the relationship between YHD components, depression-related targets, and pathways.

2.11 Statistical analysis

Statistical analyses were performed using SPSS 21.0 software. Data are presented as mean ± SD for normally distributed variables. Comparisons across multiple groups were analyzed using one-way ANOVA, followed by Tukey’s Honest Significant Difference (HSD) test for pairwise comparisons. Statistical significance was defined as p < 0.05 (significant), p < 0.01 (highly significant), and p < 0.001 (extremely significant).

3 Results3.1 Effects of YHD on body weight and depressive-like behaviors in PCPA model mice

To evaluate the effects of YHD on PCPA-induced depression model mice, we first monitored body weight changes for 6 days post-modeling. The body weight of the M group was significantly lower than that of the C group (p < 0.05). Post-treatment with YHD or fluoxetine significantly increased the body weight of PCPA model mice (p < 0.05) (Figure 2A).

Effects of YHD on the body weight and behavioral test in each group of mice. (A) Changes in body weight of mice within 6 days after modeling (n = 16 per group). (B) Effect of YHD on sugar water preference rate in PCPA model mice (n = 16 per group). (C) Effect of YHD on the immobility time of swimming in PCPA model mice (n = 16 per group). (D) The effect of YHD on the immobility time of tail suspension in PCPA model mice (n = 16 per group). (E–H) The effect of YHD on the open field experiment of PCPA model mice (n = 16 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01 vs. model group.

In the sucrose preference test, the M group showed a significant reduction in sucrose preference compared to the C group (p < 0.01), while YHD (Y) and fluoxetine (F) treatments significantly improved preference (p < 0.01) (Figure 2B). In the forced swim test, the M group exhibited increased immobility time compared to the C group (p < 0.01), which was reduced by both YHD and fluoxetine treatment (p < 0.01) (Figure 2C). Similarly, in the tail suspension test, the M group had significantly increased immobility time (p < 0.001), which was alleviated by YHD and fluoxetine (p < 0.001) (Figure 2D). In the open field test, the M group displayed reduced movement and exploratory behaviors, with fewer grid crossings, central area entries, rearing, and grooming events (p < 0.01, p < 0.001). YHD and fluoxetine treatments significantly improved these behaviors (p < 0.05, p < 0.01) (Figures 2E–H).

These results confirm the successful establishment of the PCPA-induced depression model and demonstrate that YHD effectively alleviates PCPA-induced weight loss and motor dysfunction. Furthermore, YHD was consistent with fluoxetine results in improving anhedonia, hopelessness, and anxiety.

3.2 Histological effects of YHD on hippocampal neurons, Nissl bodies, and brain 5-HT content in PCPA model mice

HE and Nissl staining were used to assess hippocampal neuron morphology in PCPA mice, evaluating the effects of YHD treatment. HE results showed that hippocampal neurons in the control (C) group exhibited a well-preserved cell structure, compact arrangement, and distinct nucleoli. In contrast, the M group showed significant neuronal degeneration, characterized by scattered and disorganized neurons with unclear structures, pyknosis of the nuclei, and a reduced number of cells. However, in the F and Y groups, the structure of hippocampal neurons was relatively intact, with a more uniform distribution and an increased number of cells (Figure 3A). Disruption of Nissl substance integrity reflects impaired neuronal function and is one of the key pathological features of depression (Duman and Monteggia, 2006; Peng et al., 2015).

Results of histomorphological observation. (A) Effect of YHD on hippocampal neurons in PCPA model mice at 10 × magnification (n = 3 per group). (B) The effect of YHD on the Nissl body in the hippocampus of PCPA model mice at 10 × magnification (n = 3 per group). (C) Effects of YHD on 5-HT content in PCPA mice (n = 3 per group). *p < 0.05, **p < 0.01 ***p < 0.001 vs. control group; #p < 0.05, ##p < 0.01 vs. model group.

Similarly, Nissl bodies were abundant in the hippocampal neurons of the C group, whereas in the M group, the Nissl bodies were lighter and fewer in number. In contrast, the F and Y groups displayed darker, more abundant Nissl bodies, along with a significant increase in the number of cells compared to the M group (Figure 3B). These findings suggest that YHD improves the morphology and number of hippocampal neurons and Nissl bodies in PCPA model mice.

5-HT, a key neurotransmitter involved in depression, has been shown to be significantly reduced in PCPA-induced depression models (Gumuslu et al., 2013). The brain tissue content of 5-HT was measured using ELISA. As reported previously, the 5-HT content in the M group was markedly decreased compared to the C group (p < 0.001). Both YHD and fluoxetine treatments reversed the reduction in 5-HT levels observed in the M group (p < 0.01, p < 0.05) (Figure 3C). This indicates that YHD can increase the 5-HT content in the brain of PCPA model mice.

3.3 Effects of YHD on serum metabolic profile and differential metabolites in PCPA model mice

In order to further explore the effect and mechanism of YHD on anxiety and depression-like behaviors in PCPA mice, we used LC–MS combined with MSDIAL software to characterize differential metabolites in serum, urine, and brain tissue, and assess the therapeutic potential of YHD. The total ion current diagrams of serum samples in positive and negative ion modes are shown below. (Figure 4A). PCA analysis showed strong clustering of QC samples, with retention time and relative peak area RSD values below 7%, indicating good reproducibility and stability of the method (Figure 4B). Supervised pattern recognition techniques, such as OPLS-DA and PLS-DA, were used to exhance sample separation. OPLS-DA analysis indicated clear separation between the C and M groups in both positive and negative ion modes (Figure 4C), suggesting the metabolomics of the model group was disordered and a metabolic profile shift in the treatment groups. Both the Y and F groups clustered more closely with the C group, indicating that YHD and fluoxetine treatments can improve the metabolomic disorder induced by PCPA model (Figure 4D).

Effects of YHD on serum metabolic profile and differential metabolites of PCPA mice. (A) Total ion flow diagram of serum sample in positive group or in negative group. (B) PCA diagram of serum samples. (C) OPLS-DA diagram of serum sample. (D) PLS-DA diagram of serum sample (n = 10 per group). (E) OPLS-DA replacement test chart of serum sample. (F) Clustering heat map of biomarkers in serum samples (n = 10 per group). (G) Biomarker content map of serum samples (n = 10 per group). (H) Analysis of potential biomarker pathways in the serum of PCPA model mice. *p < 0.01, ***p < 0.001 vs. C group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. M group (H) Analysis of potential differential metabolite pathways in serum of PCPA model ice.

In the OPLS-DA model permutation test (n = 200), the R2 and Q2 values from random permutations were smaller than the original values, and the Q2 intercept value was less than 0 (Figure 4E), validating the model without overfitting. The heatmap illustrates the effect of YHD on the serum metabolic profile of PCPA-induced mice and reveals the changes in differential metabolites. We identified 26 metabolites that were significantly altered between the C and M groups. Of these, 25 metabolites showed a tendency to revert toward normal levels following fluoxetine or YHD treatment (Table 3). Suggesting that YHD effectively improved metabolic disorders in PCPA model mice (Figure 4F). The relative contents of these 26 differential metabolites are shown in Figure 4G. Pathway enrichment analysis using MetaboAnalyst found 9 pathways related to differential metabolites, predominantly involving tryptophan metabolism and riboflavin metabolism (Figure 4H). These results indicate that YHD has a therapeutic effect on PCPA model mice, primarily through modulation of tryptophan and riboflavin metabolism.

NO.Formularm/zTRAdductsIdentificationM group VS C groupY group VS M groupF group VS M group1C6H14N2O2147.11280.56[M + H]+L-Lysine↓***↑##↑##2C4H9NO2104.07070.68[M + H]+2-Aminoisobutyric acid↓**↓##↓##3C5H5NO2112.04910.69[M + H]+Pyrrole-2-carboxylic acid↓**↑##↑##4C5H11NO2118.08430.7[M + H]+L-Valine↓**↑#↑##5C5H15NO4P184.07470.72[M + H]+Phosphorylcholine↑***↑##