{'fraction_non_alphanumeric': 0.040232379553270345, 'fraction_numerical': 0.02120129544923375, 'mean_word_length': 4.871789845128406, 'pattern_counts': {'":': 0, '<': 33, '<?xml version=': 0, '>': 0, 'https://': 2, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Antidepressant-Like Effect and Mechanism of Ginsenoside Rd on Rodent Models of Depression

Background There is growing evidence to suggest that ginsenoside Rd (GRd) has a therapeutic effect on depression, but the specific mechanisms behind its activity require further study. Objective This study is designed to investigate the antidepressant-like effect and underlying mechanisms of GRd. Methods In this study, the behavioral despair mouse model of depression and chronic unpredictable mild stress (CUMS) rat model of depression were established to explore the effects of GRd on depression-like behavior and its underlying mechanisms. Behavioral tests were used to evaluate the replication of animal models and depression-like behaviors. The hypoxia-inducible factor-1α (HIF-1α) blocker 2-methoxyestradiol (2-ME) was injected to determine the role of HIF-1α in the antidepressant-like effect of GRd. In addition, molecular biology techniques were used to determine the mRNA and protein expression of HIF-1ɑ signaling pathway and synaptic plasticity-related regulators, that is synapsin 1 (SYN 1) and postsynaptic density protein 95 (PSD 95). In silico binding interaction studies of GRd with focused target proteins were performed using molecular docking to predict the affinity and optimal binding mode between ligands and receptors. Results Our data show that GRd significantly reversed depression-like behavior and promoted mRNA and protein expression of HIF-1ɑ signaling pathway and synaptic plasticity-related regulators. However, the antidepressant-like effect of GRd disappeared upon inhibition of HIF-1α expression following administration of 2-ME. Furthermore, molecular docking results showed that GRd possessed significant binding affinity for HIF-1α, VEGF, and VEGFR-2. Conclusion Our results show that GRd exhibits significant antidepressant-like effect and that HIF-1α signaling pathway is a promising target for the treatment of depression.


Introduction
Depression is a mental affective disorder characterized by significant and long-time decreased interest, low mood, slow thinking, activity decline, and loss of appetite, which seriously affects the physical and mental health of patients. 1 According to a report from the World Health Organization, depression is predicted to become the main cause of the global burden of disease by 2030. 2 At present, the treatment strategy for depression still involves selective serotonin (5-HT) reuptake inhibitors (SSRIs) which have been traditionally used in the clinic. Unfortunately, SSRIs do not meet the criteria for an excellent therapy, including rapid onset of action, high curative rate, and fewer side effects. SSRIs are ineffective in up to 35% of patients and they cause many side effects, such as gastrointestinal adverse reactions (particularly nausea and vomiting) and neurological side effects (especially headaches and tremors). [3][4][5] Moreover, depression has become a serious worldwide public health problem and its complicated pathological mechanisms need animals were kept in the following standard conditions, including a 12/12 h light/dark cycle (light on 8:00 a.m. and light off 8:00 p.m.), at ambient temperature (21-23°C), humidity range of 30-50%, and adequate standard food and water. All steps involving animals were authorized by the Experimental Animal Ethics Committee of the Academic Committee of Beijing University of Chinese Medicine (project identification code: BUCM-2021082802-3002). The experimental procedures of our study were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Animal Study Design Behavioral Despair Mouse Model of Depression
Forty mice were randomly divided into four groups: control (Con), paroxetine (Par, 10 mg/kg, i.p.), low-dose GRd (L-GRd, 10 mg/kg, i.g.), and high-dose GRd (H-GRd, 20 mg/kg, i.g.). All drugs were administered once a day for 14 days, while mice in the Con group received an equal volume of saline. Behavioral tests were performed on day 14. All mice were intraperitoneally anesthetized with 1% pentobarbital. The hippocampus of each mouse was removed and stored on ice for analysis.

HIF-1α Inhibition
Forty mice were randomly divided into four groups: control (Con), 2-Methoxyestradiol (2-ME), high-dose GRd (H-GRd) and H-GRd+2-ME. Mice in the H-GRd group were intragastrically treated with GRd (20 mg/kg) once daily for 14 days. The GRd dose used was optimal dose identified in Behavioral Despair Mouse Model of Depression. Mice in the GRd +2-ME group were intraperitoneally injected with 2-ME (5 mg/kg) after intragastric administration of GRd. The Con group received equal volumes of saline and mice in the 2-ME group were intraperitoneally injected with 2-ME (5 mg/kg) after receiving saline. Behavioral tests were performed on day 14.

CUMS Rat Model of Depression
Sixty rats were randomly divided into six groups: control (Con), model (CUMS, CUMS+saline), CUMS+Par (CUMS +4.8 mg/kg paroxetine, i.p.), CUMS+L-GRd (CUMS+10 mg/kg GRd, i.g.), CUMS+H-GRd (CUMS+20 mg/kg GRd, i.g.), and H-GRd (20 mg/kg GRd, i.g.). All rats, except those in the Con and H-GRd groups, were exposed to unpredictable mild stressors for 28 days. Subsequently, each group of rats received corresponding drug for 21 days. The behavioral tests including open field tests (OFTs) and sucrose preference tests (SPTs) were performed on day 21 of dosing. All rats were anesthetized by intraperitoneal injection of 1% pentobarbital and the hippocampus of each rat was removed and stored on ice for analysis.

CUMS Protocol
With the exception of the Con and H-GRd groups, rats were exposed to one or two of the following stressors accompanied by single cage breeding for 28 days: deprivation of food and water, reversal of day and night, cage tilt, 4°C ice water swimming, noise, stroboscopic, wet bedding in cages, body restraint, horizontal shaking, and tail pinching. The same stressor was not repeated within a 7 day period to prevent the rats from predicting upcoming stressors.

Forced Swimming Tests
Mice were taken to the testing room to adapt for 1 h before forced swimming tests (FSTs). A single mouse was placed in a cylinder-shaped glass container (height: 25 cm, diameter: 10 cm) filled with tap water (water height: 10 cm, temperature: 24±1°C). When mice were swimming, their tails and limbs could not reach the bottom of container while the tip of their nose was above water to breathe. Containers were separated by an opaque plate to prevent two mice from observing each other. After 2 min of adaptive swimming, the cumulative immobility time of mouse within 4 min was automatically collected and analyzed by behavior analysis system (Etho-Vision XT9, Noldus, Netherlands). The standard criterion for determining the immobility of mice was as follows: mouse stopped struggling and floated on the water, or made only minor movements necessary to keep its head above the water.

OFTs
Animals were taken to the testing room to adapt for 1 h before OFTs. The tests used an opaque square autonomous activity box (mice: 45 cm × 45 cm × 30 cm; rats: 100 cm × 100 cm × 40 cm). The bottom of the box was divided into twenty-five identical squares with nine squares in the middle set as the central area. A single mouse or rat was placed in the center of the autonomous activity box and the behavior analysis system described in Forced Swimming Tests was used to record the movement distance, velocity, frequency of entering the center, and the duration of the stay in the center within 5 min. The environment was kept quiet with stable light during the tests. Before each experiment, the area was cleaned with alcohol to remove all feces and to remove any odor left by previous mice.

SPTs
The SPT is a classic method to assess anhedonia in animals. Briefly, rats were individually placed in cages equipped with two bottles of 1% (w/v) sucrose solution for adaptation training for 24 h. Subsequently, one of the bottles of sucrose solution was replaced with tap water for 24 h. During the third 24 h period, rats were deprived of food and water. Immediately after the third phase, rats had free access to two bottles of solution in the cage for 2 h, one bottle filled with 100 mL of 1% sucrose solution and the other bottle containing 100 mL of tap water. The positions of the two bottles were alternated every hour to prevent positional preference. The consumption of both solutions were recorded and the sucrose preference was calculated as the sucrose preference rate (%): sucrose consumption/(sucrose consumption + tap water consumption) × 100.

Real-Time Fluorescence Quantitative Polymerase Chain Reaction Analysis
To detect mRNA expression of relevant genes, total RNA was first extracted from hippocampal tissue using the Hipure Total RNA Mini Kit (MAGEN) according to the manufacturers instructions. An ultraviolet spectrophotometer (UV-2000, Unico) was used to measure RNA concentration of each sample and RNA samples were maintained on ice to prevent degradation. Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and the Power SYBR Green PCR Master Mix (Invitrogen) according to the manufacturer's instructions. The following conditions were used on a T100 Thermal Cycler PCR machine (Bio-Rad, USA): 42°C for 1 h, 70°C for 5 min, storage at 4°C. Amplification and quantitative detection were performed in a Real-Time PCR machine (Bio-Rad, USA) using the following protocol: initial denaturation at 95°C for 10 min, denaturation at 95°C for 10s, annealing at 60°C and extension for 30s with a total of 50 amplification cycles. We used the 2 −ΔΔCt method to calculate the relative quantitative analysis of the results. All primer sequences are shown in Table 1.

Western Blotting
Hippocampal tissue was homogenized in RIPA lysis buffer with protease-and phosphatase inhibitors using an electric homogenizer then lysed on ice for 20 min before centrifugation at 13,000 rpm for 20 min at 4°C. The supernatant was collected and the pellet was discarded to obtain the whole cell lysates. The total protein concentration was determined according to the instructions of a BCA protein quantification kit. A total of 40 µg of protein was separated by electrophoresis on a 5-8% sodium dodecyl sulfate-polyacrylamide gel and then transferred to a 0.45 µm PVDF membrane by wet transfer at 100 V constant voltage for 1.5 h. Subsequently, the membrane was blocked with 5% (w/v) non-fat powdered milk at room temperature for 2 h. After 3×10 min washes in TBST buffer, the membrane was incubated with primary antibody at 4°C overnight and then further washed 3 times before incubation with secondary antibodies at room temperature for 1 h. Blots were developed using an ECL chemiluminescence detection kit. The labeled proteins were visualized using an Azure multifunctional molecular imaging system (C600, Azure, USA).

Molecular Docking
Three-dimensional (3D) structures of focused target proteins including HIF-1α, VEGF, and VEGFR-2 implicated in depression were obtained from the online RCSB Protein Data Bank (PDB; https://www.rcsb.org/). PyMOL 2.4 (Delano Scientific LLC, Italy) was used to remove water molecules and small molecule ligands from protein structure. AutoDock Tools was then used to add hydrogen and atom types, to calculate charge, and finally for conversion into the PDBQ format. A two-dimensional (2D) structure of the small molecule ligand (ie GRd) was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Chem3D 19.0 software was then used to convert this into a 3D structure and to optimize the mechanical structure to minimize energy. AutoDock Tools was used to detect the root of the ligand and select the rotatable bond. Finally, this was saved in a PDBQT format. Molecular docking experiments were performed in Autodock Vina (PyRx 0.8).

Statistical Analysis
Data are expressed as mean ± standard error mean (SEM), and data with normal distribution and homogeneity of variance were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The Kruskal-Wallis test was used for data with non-normal distribution. Statistical analysis was performed using SPSS 26 (SPSS, Chicago, Illinois, USA) and GraphPad Prism 8.0 (GraphPad Software Inc, San Diego, CA, USA). A p value < 0.05 was considered to be statistically significant.

Effects of GRd on Immobility Time in FSTs
The FST is still one of the most commonly used methods for screening antidepressants in all animal models. Compared to the Con group, the forced swimming immobility time of H-GRd, D-GRd, and Par groups were significantly shorter (*p < 0.05; **p < 0.01) ( Figure 1).

Effects of GRd on OFTs
OFTs were designed to eliminate the interference of immobile time in the FST. There were no significant differences in movement distance, velocity, frequency of entering the center, and the duration of stay in the center between all groups ( Figure 2).

Effects of GRd on HIF-1α-VEGF Signaling Pathway
Changes in the HIF-1α-VEGF signaling pathway were examined in the behavioral despair mouse model of depression to explore potential mechanisms underlying the antidepressant-like effect of GRd. We found that mRNA and protein expression of HIF-1α and VEGF in low and high dose groups of GRd were significantly higher than those in the control group ( Figure 3; *p < 0.05; **p < 0.01; ***p < 0.001).

Effects of HIF-1α Inhibitor on the Behavioral Despair Mouse Model of Depression
Effects of 2-ME on Immobility Time in the FST We used 2-ME, a widely used and effective inhibitor of HIF-1α, to block HIF-1α expression to explore its role in the antidepressant-like effect of GRd. Our data show that the forced swimming immobility time of the GRd group was significantly shorter than that of the control group ( Figure 4; **p < 0.01). This was not the case for the GRd+2-ME group mice. These data suggests that (1) the antidepressant-like effect of GRd can be eliminated by 2-ME, and (2) HIF-1α is essential for this effect. In addition, the forced swimming immobility time of mice treated with 2-ME alone was no different from that of the control group, which ruled out the potential of 2-ME-mediated interference.

Effects of 2-ME on OFTs
There were no significant differences in movement distance, velocity, frequency of entering the center, and the duration of stay in the center between all groups ( Figure 5).

Effects of GRd on CUMS Depressive Model in Rats
Effects of GRd on SPT The SPT is a classic test for anhedonia in animals. The sucrose preference of rats in the CUMS group was significantly lower than that of control group ( Figure 6; ### p < 0.001). In comparison, the sucrose preference of rats following treatment with low and high doses of GRd was significantly higher than the CUMS group ( Figure 6; *p < 0.05, **p < 0.01). There were no differences in sucrose preference between the GRd and paroxetine groups. These results indicate that GRd can reverse CUMS-induced anhedonia in rats.

Effects of GRd on OFTs
The OFT is also a classic behavioral test. The movement distance, velocity, frequency of entering the center, and the duration of stay in the center of CUMS group were all lower than the control group (Figure 7; ## p < 0.01; ### p < 0.001). However, the observation indexes of OFTs in GRd treatment group were better than in the CUMS group (Figure 7; *p < 0.05; **p < 0.01; ***p < 0.001). SPT and OFT data showed that the CUMS model was successfully replicated and that GRd reverses CUMS-induced depression-like behavior to exhibit a significant antidepressant-like effect.

Effects of GRd on the HIF-1α-VEGF Signaling Pathway
RT-PCR and Western blotting were used to measure mRNA and protein expression of HIF-1α and VEGF, respectively. The data show that mRNA and protein expression of HIF-1α and VEGF in the CUMS group were significantly downregulated compared to the control group (Figure 8; # p < 0.05; ### p < 0.001). Both low-and high-dose GRd significantly increased mRNA expression of HIF-1α and VEGF, and HIF-1α protein expression in CUMS rats (Figure 8; **p < 0.01; ***p < 0.001). In addition, high-dose GRd significantly increased VEGF protein expression in the CUMS rats ( Figure 8; **p < 0.01).

Effects of GRd on mRNA and Protein Expression of VEGFR-2
VEGFR-2, a high-affinity receptor of VEGF, is implicated in depression. We measured mRNA and protein expression of VEGFR-2 to understand whether GRd can act on VEGFR-2 to exert its antidepressant-like effects. The data showed that hippocampal mRNA and protein expression of VEGFR-2 in the CUMS group were severely downregulated compared to the control group (Figure 9; ## p < 0.01). Following treatment with low-dose GRd, VEGFR-2 protein expression increased significantly (Figure 9; ***p < 0.001). Further, treatment with high-dose GRd, significantly increased both mRNA and

Effects of GRd on PI3K/AKT/mTOR Pathway
We used RT-PCR and Western blotting to explore VEGF-and VEGFR-2-mediated changes in mRNA and protein expression in the PI3K/AKT/mTOR signaling pathway, respectively. Our data showed that the mRNA expression of PI3K, AKT, and mTOR, and the protein expression of p-PI3K, p-AKT, and p-mTOR in the hippocampus of CUMS model rats were significantly lower compared to the control group ( Figure 10; # p < 0.05; ## p < 0.01; ### p < 0.001). Moreover, low-dose GRd significantly increased mRNA expression of AKT and p-PI3K, p-AKT, and p-mTOR protein expression in the hippocampus of CUMS rats (Figure 10; *p < 0.05; **p < 0.01; ***p < 0.001). High-dose GRd significantly increased

Effects of GRd on Synaptic Plasticity-Related Regulators
We also sought to understand whether GRd exerts an antidepressant-like effect by regulating synaptic plasticity-related regulators (ie SYN 1, PSD 95). We found that compared to the control group, SYN 1 and PSD 95 protein expression were significantly reduced ( Figure 11; ## p < 0.01; ### p < 0.01). This could be reversed by low-and high-dose GRd ( Figure 11; *p < 0.05; **p < 0.01; ***p < 0.001). These data suggest that GRd may exert its antidepressant-like effects by regulating the expression of synapse-related regulatory factors.

Molecular Docking of GRd with HIF-1α, VEGF, and VEGFR-2
A negative binding energy between a ligand and receptor is generally suggestive of spontaneous binding. The smaller the binding energy, the greater the docking activity and the greater the possibility of action. A binding energy below −5 kcal/ mol is indicative of strong protein-ligand binding. Our molecular docking data suggest that the binding energy between GRd and HIF-1α, VEGF, VEGFR-2 were all less than −7 kcal/mol (−7.2, −8.3, −7.8 kcal/mol, respectively). This is indicative of a high likelihood of spontaneous binding. PyMOL was used to visualize these modelled binding data. The number of hydrogen bonds formed between GRd and HIF-1α, VEGF, and VEGFR-2 are 7, 7, and 10, respectively. The amino acid residues involved in these interactions are shown in Table 2. Further, molecular docking patterns of GRd with target proteins are shown in Figure 12.

Discussion
Many ginsenosides are not easily absorbed from the intestine into the blood. Therefore, these components require metabolism by the microbiota in the gastrointestinal tract to be broken down into more easily absorbed compounds. Studies have shown that the antidepressant effect of red ginseng depends on the metabolite GRd being absorbed into the blood. Moreover, GRd has the highest absorption rate in the blood following oral administration of red ginseng in human and mice. 22 Our previous studies have shown that Ginsenoside Rb1 (GRb1) induces antidepressant activity. GRd, a metabolite of GRb1, which is produced by removal of a β-D-glucose, was also identified in the blood, brain, feces, and urine of normal and depressed mice after administration of GRb1. In addition, our network pharmacological analysis results showed that GRd is implicated in antidepressant activity. 30 The behavioral despair mouse model of depression is a proven depression model which places animals in irresistible adversity to breed despair. The immobility of animals after forced swimming stress is sensitive to antidepressants, making it an ideal behavioral experiment for preliminarily testing of the pharmacodynamics of antidepressants. 31,32 Compared to the control group, both high and low doses of GRd can significantly shorten the forced swimming immobility time. This suggests that GRd improves the behavioral despair of mice and exerts a significant antidepressantlike effect.
The CUMS depression model simulates unpredictable stress events and induces various long-term physical, behavioral, neurochemical, and neuroendocrine changes, similar to those observed in depressed patients. 33,34 Exposure to CUMS can reduce the movement distance, velocity, frequency of entering the center, the duration of stay in the center in OFTs, and the sucrose preference rate in SPTs, which shows that CUMS depression model is successfully reproduced. Both high and low doses of GRd reversed the depression-like behavior of CUMS rats and showed a significant antidepressant effect.
HIF-1, a base-helix-loop-helix PAS heterodimer composed of active subunit HIF-1α and constitutive subunit HIF-1β, mediates the adaptive response of cells to hypoxia. The transcriptional activity and protein stability of HIF-1α are extremely sensitive to changes in oxygen concentration. Moreover, HIF-1α is crucial for maintaining the integrity and function of HIF-1. 35 Under conditions of hypoxia, HIF-1α binds to hypoxia response elements (HREs) on a variety of target genes to induce transcription of target genes. Many studies have shown that HIF-1α, not only in response to hypoxic stress but also in response to severe psychoemotional stress, is a promising target for the treatment of depression. HIF-1α prevents oxygen homeostasis disorders, promotes neuroplasticity, and exerts neuroprotective effects by activating a series of target genes involved in regulation of angiogenesis, vasomotor control, energy and ion metabolism, erythropoiesis, and apoptosis. Triggering these protective mechanisms may have a beneficial impact on the depression, thereby preventing the development of a depressive state caused by severe psychological and emotional stress. [36][37][38] Recently, many studies have shown that VEGF, a secreted angiogenic mitogen regulated by HIF-1α, plays a beneficial role in the treatment of depression. 39,40 Long-term stress decreases cell proliferation near the vascular system and expression of VEGF. Decreased expression of VEGF can be seen in the prefrontal cortex and hippocampus of patients with chronic stress-induced major depressive disorder. Further, VEGF protein expression in the cerebrospinal fluid of individuals who have attempted suicide is significantly lower compared to healthy people. [41][42][43] In comparison, intracerebroventricular infusion of VEGF provides an exceedingly effective antidepressant-like effect. 44 This antidepressant effect is associated with the ability of VEGF to improve cognitive function, promote neurogenesis, and facilitate hippocampal synaptic plasticity. [45][46][47][48] Comprehensively, activation of the HIF-1α-VEGF signaling pathway may be implicated in the treatment of depression, which is highly consistent with our experimental results. Compared to the CUMS group, high and low doses of GRd can significantly increase mRNA and protein expression of hippocampal HIF-1α and VEGF in both the behavioral despair mouse model of depression and the rat CUMS model.
Intriguingly, 2-ME, a natural metabolite of estradiol and an effective HIF-1α inhibitor, inhibits the transcriptional activity and protein expression of HIF-1α by inducing microtubule depolymerization and the production of reactive oxygen species. 49,50 In our HIF-1α inhibition experiment, the forced swimming immobility time of mice treated with 2-ME following oral administration of GRd was not shorter than the control group. This indicates that the antidepressant effect of GRd can be blocked by 2-ME, further confirming that HIF-1α is essential for the antidepressant effect of GRd.
The antidepressant effect of GRd benefits from the increased expression of hippocampal the synaptic plasticityrelated regulators, SYN 1 and PSD 95. There is increasing evidence to suggest that synaptic plasticity disorder may be a pathological mechanism involved depression. Moreover, an improvement in synaptic plasticity plays a critical role in the treatment and prognosis of depression. [51][52][53] Synaptic plasticity is generally coordinated with the synapse-associated proteins, mainly including presynaptic SYN 1 and postsynaptic PSD 95. SYN 1, a neuron-specific phosphoprotein associated with synapses, is distributed in almost all nerve terminals and located on the surface of the presynaptic vesicle membrane. SYN 1 regulates the transportation and circulation of synaptic vesicles to promote neurodevelopment, neuronal information transmission, and synaptic plasticity. 54-56 PSD 95, a protein located on the cytoplasmic side of the postsynaptic membrane and related to synaptic signal transmission, plays a significant role in regulating neuron survival and synaptic function. Further, PSD 95 is widely involved in the onset and treatment of cognitive and emotional related mental diseases. 57,58 Many studies have shown that protein expression of SYN 1 and PSD 95 are decreased in the prefrontal cortex of patients with major depression. A decrease of presynaptic SYN 1 and postsynaptic PSD 95 in the hippocampus and prefrontal cortex due to chronic stress may lead to depressive behavior and a severe decline in cognitive function in rodents. 59,60 Conversely, increased SYN 1 and PSD 95 expression can improve depression. 61,62 Consistent with our findings, GRd greatly reversed the significant decrease in SYN 1 and PSD 95 protein levels in the hippocampus of CUMS rats. This shows that the antidepressant-like effect of GRd is linked to the expression of hippocampal synaptic plasticity-related regulators.
There is a close correlation between the activation of the HIF-1α-VEGF signaling pathway and increased expression of synaptic plasticity-related regulatory factors. Both are beneficial for improving depression. [63][64][65] Our results show that the activation of the HIF-1α-VEGF signaling pathway may promote the expression of synaptic plasticity-related regulatory factors through regulation of the PI3K-AKT-mTOR signaling transduction pathway mediated by vascular  856 endothelial growth factor receptor 2 (VEGFR-2, also known as Flk-1). The downstream signal transduction molecular mechanism of VEGF is complex. VEGFR-2, a high-affinity receptor tyrosine kinase of VEGF, which is highly expressed in the rodent hippocampus, can activate the PI3K signaling pathway to stimulate cell proliferation and survival. 66,67 VEGF-Flk-1 signaling brings into play a considerable role for synaptic plasticity in hippocampal dependent processes, such as learning and memory, and regulating synaptic transmission. These effects are important in the behavioral responses of animal models receiving multiple antidepressants and can, therefore, be used as valuable predictors of antidepressant activity. [68][69][70][71] Mammalian target of rapamycin (mTOR), one of the downstream signaling molecules of PI3K, is activated and regulated by the PI3K-AKT pathway, which is a pivotal target for glutamatergic and cholinergic drugs which exert rapid antidepressant effects. [72][73][74] Studies have shown that the mTOR signal is closely related to synaptic plasticity. When the signal is stimulated, it will rapidly increase the expression of synaptic proteins (eg PSD 95 and SYN 1) and the number

857
of spinal synapses, [75][76][77] which is consistent with our research results. After CUMS rats were subjected to chronic stress, we found that mRNA and protein expression of VEGFR-2, PI3K, AKT, and mTOR were significantly reduced. These can all be reversed by GRd.
In recent years, molecular docking has become an important technology in the field of computer-aided drug design. This method is a theoretical simulation method for studying the interactions between molecules (eg ligands and receptors) and predicting their binding mode(s) and affinity. 78 Our molecular docking studies revealed additional insights into the effects of GRd on HIF-1α signaling pathway related target proteins for therapeutic intervention in depression. In the present study, a molecular docking study of GRd (ligand) was performed against focused target proteins including HIF-1α, VEGF, and VEGFR-2. Our data reveal that GRd docks well into HIF-1α, VEGF, and VEGFR-2 with docking scores of −7.2, −7.8, and −8.3 kcal/mol, respectively. This suggests that all three proteins may have a high binding affinity for GRd. We also analyzed the hydrogen bonds between GRd and the target protein. The number of hydrogen bonds formed by GRd with HIF-1α, VEGF, and VEGFR-2 were 7, 7 and 10, respectively. The details of the binding amino acid residues are shown in Table 2. In addition, the molecular docking patterns of GRd with the target protein are shown in Figure 12. The modelled antidepressant mechanism of GRd is shown in Figure 13.

Conclusion
Our data indicate that GRd exhibits a significant antidepressant-like effect. The observed beneficial effects may be attributed to the activation of the HIF-1α-VEGF signaling pathway, VEGFR-2-mediated PI3K-AKT-mTOR signaling pathway, and increased expression of the synaptic plasticity-related proteins, SYN 1 and PSD 95.

Institutional Review Board Statement
The study was approved by The Experimental Animal Ethics Committee of the Academic Committee of Beijing University of Chinese Medicine (project identification code: BUCM-2021082802-3002).