Tao-Hong-Si-Wu Decoction improves depressive symptoms in model rats via restorations of neural transmitters and ameliorations of BDNF-CREB-arginase I axis disorders

Depressive disorder (DD) has become a global health problem. Applications of Chinese medicine have been demonstrated to be potential in the treatment of DD. Tao-Hong-Si-Wu decoction (TSD), a traditional Chinese medicine formula is widely used to treat ameliorate anemia, liver and heart dysfunctions, and inammation. Such symptoms are also associated with DD. Hence, our initial hypothesis was to observe and explore the targets of the effect of TSD on DD. The DD model was established by chronic unpredictable mild stress (CUMS) in rats. The measurements of body weight and behavioral tests were performed to conrm the success of modeling and observe the effect of TSD on the model animals. A gas-chromatography coupled with mass spectrometry (GC-MS)-based metabolomic analysis was conducted to reveal the metabolic characteristics related to the curative effect of TSD. Serum serotonin and arginase I (Arg I), which are associated with the key feature metabolites responsible for the effect of TSD on depression, were assessed by an ELISA assay. Proteins in the Brain-derived neurotrophic factor (BDNF)/ Tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling, which lead to the regulations of Arg I, were analyzed using Western blot assay.


Introduction
Depressive disorder (DD) is a common illness that severely limits psychosocial function and increases the risk of suicide [1]. According to a report by World Health Organization in 2017, there are about 322 million patients with depression worldwide. The high prevalence and risk of disability make the disease be a serious global health problem [2].
The pharmacotherapies for DD are primarily based on the enhancement of monoamine neurotransmission until the present. However, few drugs combine effectiveness and tolerability [3]. This is partly due to the unclear pathology of DD for the moment. Typically, more than the reductions of monoamine neurotransmitters, physiological disorders can be also found in other pathways in DD such as dysfunctions of the hypothalamic-pituitary-adrenal (HPA) axis, in ammation, inhibition of neurogenesis, loss of brain function, and genetic modi cations. Thus, multi-target medications may be a promising therapeutic strategy for DD treatment.
Emerging evidence shows that ethnomedicines, which are generally multi-targeted, especially Chinese traditional medicine (CTM), play roles as important complementary therapies in DD treatment [4]. Several inherited prescriptions from TCM have been reported to be useful to improve depressive symptoms [5][6][7][8].
It is worth noting that most herbal drugs show a common effect of serotonin enrichment, which is likely to be a common effect of most TCM prescriptions on DD [9]. According to the theory of TCM, DD is associated with the de cient circulation of blood and qi, invasion of detrimental qi, and organ weaknesses, which can be implicated with the depressive symptoms of anemia and malnutrition, in ammation, and impaired organ functions, respectively [10][11][12]. Interestingly, these conditions were reported to be improved by Tao-Hong-Si-Wu decoction (TSD), a classical formula recorded in TCM, which consists of Semen Prunus (Amygdalus persica L.), Flos Carthami (Carthamus tinctorius L.), Rhizoma Chuanxiong (Ligusticum chuanxiong Hort.), Radix Angelicae sinensis (Angelica sinensis (Oliv.) Diels), Radix Rehmanniae praeparata (Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey.), and Radix Paeoniae Alba (Paeonia lacti ora Pall.). The traditional use of TSD was to treat ischemic stroke [13]. In the past decades, bioactive compounds contained in these herbs such as ferulic acid, albi orin whose effects have been revealed in improving in ammation and depressive symptoms [14,15]. However, no previous studies focused on the antidepressant function of TSD. In the current study, our initial hypothesis was to investigate whether TSD is useful to improve DD.
It is challenging to clarify the mechanism behind the curative effect of a TCM formula because of the comprehensive ingredients contained in the mixture of various herbs. To this end, metabolomics is applied as a promising tool since the improvement of the disease-induced dysregulations can be witnessed sensitively and simultaneously by the restorations of metabolic disorders [16]. Also, variations in inherent metabolites can appear individual responses to treatments thereby providing evidence for personalized therapy. In addition, an overview of the regulations in the metabolome is following the theory of TCM which emphasizes restoring the integral balance and harmony for the internal environment of patients [17]. However, the metabolic pro ling is prone to be varied by environmental variations and individual differences. Further veri cations in the upstream proteins related to the drug effect-induced metabolic variations are necessary to ascertain the medicinal actions and corresponding drug targets [18].
Herein, we rst con rmed the antidepressant effect of TSD on the DD animal model by bodyweight measurements and behavioral tests. A series of serum metabolomic analyses were conducted subsequently to reveal discriminant metabolites and varied metabolic pathways associated with the CUMS modeling and the effect of TSD on DD. Feature metabolites-associated upstream proteins in serum and hippocampus were tested to explore the mechanism behind TSD effects on DD.

Experimental Animals
Forty-eight male SD rats weighing 200-250g were purchased from the Experimental Animal Center of Anhui Medical University (Hefei, China). Animals were housed while free access to food and water was allowed for 1 week.

Medication
All the herbs were purchased from Anhui Baixingyuan Pharmacy, Hefei, China. The herbs were mixed before decoction with the mass proportion of each herbal medicine as follows: Semen Prunus 12g, Flos Carthami 18g, Rhizoma Chuanxiong 18g, Radix Angelicae sinensis 18g, Radix Rehmanniae praeparata 18g, and Radix Paeoniae Alba 18g. according to the original record of "Yuji Weiyi", a Chinese classical medical book.

Preparation and the evaluation of the homogeneity of TSD
Herbs contained in TSD were soaked in double-distilled water (the mass ratio of medication on the water was equal to 1:8) for more than 2 hours. The mixture of medications was decocted for 50min. The resulting decoction was ltered with gauze and the ltrates were centrifugated at 3000r/min 4℃ for 10min. The supernatants of the two parts of decoctions were combined and then lyophilized into powder and stored at -80℃ before medication administrations.
To ensure the homogeneity of TSD used during the two-week intervention, the internal standard Lnorvaline (1mg/L, relative standard divergence (RSD) = 0.09) added in the prepared TSD samples was evaluated every three days before the next gavage (shown in Table S-1). The sample preparation and the determination of the internal standard based on gas chromatography coupled with mass spectrometry (GC-MS) have been described in the supplementary data. To authenticate all the herbs bought in the pharmacy, comparisons between the standard products of the six herbs included in TSD and the medications applied to feed the rats were done with the non-targeted metabolomic analyses. Figure S-1 shows an example of the comparison between the metabolic ngerprint of an extracted Radix Paeoniae Alba sample and that of the standard product. The instrumental conditions are also noted in the supplementary material.

Animal modeling and treatment
All the rats underwent an adaptive feeding one week before further treatments. They were randomly divided into 6 groups as follows: the blank control group (B), the model group (M); the model group treated with uoxetine (FX,10mg/kg), and the model group treated with TSD (2.5(TL) 5(TM) and 10g/kg (TH)). Except for the blank controls, the DD was imitated by the induction of chronic unpredictable mild stress (CUMS) stimulations [19] in modeled rats. The operations included water fasting, reversal of day and night, tilting, dampness, crowding, water fasting and dampness (24h), and ice water stimulation (4℃, 5min) in turn. The modeling groups were repeatedly and randomly stimulated for 35 days. Of the ve modeling groups, three groups of CUMS rats were treated with low (2.5 g/kg. D), medium (5 g/kg. D), and high (10 g/kg. D) doses of TSD, respectively, while the rats in the FX group were subject to the uoxetine (positive control, 10 g/kg. D). Prepared TSD powder and uoxetine were dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) suspension before medication administrations. The other two groups were meanwhile subject to saline injections.

Bodyweight measurements and behavioral tests
The bodyweight measurement and the behavioral tests were rst performed before medication interventions to verify the success of the model establishment. The same tests were repeated after a twoweek medication therapy to observe the effect of TSD on depression-like symptoms. Open-eld tests (OFT) were carried out with light alteration as described previously [20]. Brie y, the OFT apparatus was a black wooden box (100 cm long × 100 cm wide ×50 cm high). The oor of an arena was divided into 25 equal-size squares by white lines. Groups of rats were placed individually into the corner of the apparatus back to the wall and were allowed to explore freely for 5 min. A minimal amount of light (60 lx) was used to avoid anxiety behavior. The apparatus was cleaned with 90% ethanol and dried before the next test to remove the smell from the former animal. The number of rearing, grooming, and defecation, and standing in the center were manually recorded by trained observers blind to the experiment. Forced swimming tests (FST) were conducted according to our previous work [21]. All the rats were separated and individually put into a transparent glass cylinder (60 cm tall× 25 cm inner diameter) lled with water. The water temperature was maintained at 25 ± 1°C during the experiments. Immobility time was de ned as the period when there was a lack of body motion with only the small necessary movements such as taking a breath above the water surface. The test for each rat lasted 6 minutes, in which the immobility time was measured on the nal 5 min.

Sample collections from rats
Twenty-four hours after the nal behavioral tests, the animals were sacri ced immediately under anesthesia. Rat blood was sampled from the abdominal aorta. The serum was kept after centrifugation at 3000 r/min and 4°C for 15 min. Brain tissues of the rats were meanwhile retained in which the hippocampi were dissected and collected in an ice bath. Hippocampus samples were subsequently snapfrozen by liquid nitrogen and subsequently stored at -80°C until analysis.

Serum sample preparations
For the metabolomic analyses, samples were rst thawed at room temperature. A series of metabolite extraction and derivatization reactions were performed with 100µL of each serum sample from each rat following the same method applied in our previous work [22]. An equal volume of each collected sample was taken out to mix up the quality control (QC) pool. The QC samples were prepared in the same way as other real samples during the metabolite extraction and derivatization reactions. The QC samples were used to serve for balancing the instrument and for estimating the stability of the analyses. In the sequence of GC-MS analyses, the QC sample was randomly inserted after every 5 real samples.

GC-MS conditions
A Shimadzu GC-MS 2010 was used for the determination of the metabolic pro ling in each sample. The GC-MS condition referred to our previous work [22] except for the gradient temperature program. For this current work, the gradient temperature program was set as follows: the inlet temperature was set at 300°C, the interface transfer temperature was 280°C, and the ion source temperature was 230°C. The column ow was 1.2 mL•min − 1 , and the split ratio was set as 10:1. The event time was set to 0.2s. Ions with their mass-to-charge ratios ranging from 50 to 600 m/z were detected under full-scan mode. The injection volume was 1 µL for each data acquisition. The gradient temperature program was set as follows: initial column temperature was set to 70°C and was maintained for 3 min. The temperature was then increased to 200°C with a gradient of 3°C•min − 1 and was then held for 5 min. A gradient of 5℃•min − 1 was applied while the temperature raised to 300°C and the column temperature was nally maintained for 3 min.
2.10 Data processing and data analysis for the metabolic pro ling One of the initially obtained total ion chromatography diagrams has been shown in Figure S-2. The acquired data were rst processed with the steps of ion peak alignment, deconvolution, normalization as described in extenso previously [23]. Brie y, normalized peak areas were used for the relative quanti cation for each ion peak. The annotation was done with the NIST database (version 2019) and by comparing it to the spectra of the standard substance. The screening of data was conducted within the QC samples in light of the relative standard derivation (RSD) and the ratio of signal on noise (S/N) of each ion peak. Those metabolites with their S/N inferior to 3 or with their RSD superior to 30% across all the QC samples were removed. After the pre-processing and the screening, the data set was subject to Metaboanalyst software (Version 5.0, www.metaboanalyst.ca) for further analyses [24]. The online opensource software assumes the analyses such as principal component analysis (PCA), orthogonal projection to latent structures-discriminatory analysis (OPLS-DA), and metabolic pathway analysis. The pvalue of student's T-test between compared groups was calculated by Graphpad Prism software (version 9.0, San Diego, USA).

ELISA assays
Serum serotonin and Arg I were evaluated by ELISA kits produced by Abcam Company (Cambridge, UK) according to the instructions provided by the manufacturer. The concentration of serotonin was measured at 405nm while the concentration of serum Arg I in rats was measured at 450 nm.

Western Blot Assays
Tissue samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl,1 mM EDTA, 1 mM EGTA, and 1 mM The relative expression levels of proteins were determined using a densitometer and were normalized by referring to β-actin. Image J software (NIH, USA) was used to quantify relative protein expressions. Oneway analysis of variance (ANOVA) with the Tukey HSD correction was used to determine the signi cant differences of protein expressions between the groups.

Effect of TSD on depressive behavior in CUMS model rats
The animal experiment process has been displayed in Fig. 1a. Inhibited anabolism and malnutrition are common symptoms of DD. We, therefore, measured the weights of all the rats before and after medication interventions. General decreases of body weight were observed in CUMS model rats compared with the non-treated controls. Rats treated with TSD (2.5, 5, and 10g/kg) and uoxetine (FX, positive control, 10mg/kg) signi cantly increased their body weights compared with the model group (Fig. 1b). OFT and FST were also performed in accompany with the weight measurements. The rst behavioral tests aimed to con rm the success of the model establishment while the repeated actions after medication administrations served to visualize the effect of TSD on depression. Accordingly, model rats before pharmacotherapies presented extended periods of immobility compared with controls in the behavioral tests, showing that the models were successfully established. The episodes of immobility were signi cantly reduced in the groups with TSD or FX treatments, which con rmed the anti-depressant effect of TSD in rats as shown in Fig. 1c-e.

Effect of TSD on the serum metabolome of CUMS rats
After the samplings of rat serum, we applied the GC-MS-based metabolomics method in the rats to study the metabolic characteristics of each group, especially to have an insight into the metabolic alterations caused by TSD administration. Before data analysis, an assessment of the stability of instrumental data acquisition was conducted with the QC samples. The QC samples were emerged in a PCA by differentiation from the real samples. A clear gather of the QC samples surrounded by the real samples in the PCA shown in Figure S-3, which proved a satisfying reproducibility for the analyses. Consequently, we identi ed 82 metabolites among the retained 166 ion peaks. MetaboAnalyst 5.0 software was employed to analyze the data corresponding to the re ned and normalized peak areas of metabolites. Figure 2 shows differences in the metabolic pro les between different groups by the score plots of PCA and OPLS-DA. Concretely, a PCA separating the blank controls from the CUMS model group was shown in Fig. 2a (PC1 = 18%, PC2 = 13.8%). The same separation based on OPLS-DA (two components, R 2 X = 0.511, R 2 Y = 0.993, Q 2 Y = 0.985) also showed clear discrimination between these two groups (Fig. 2b). Aiming to understand the effect of TSD on the metabolome of CUMS rats, another PCA was conducted between the model group (red dots illustrated in Fig. 2c, PC1 = 12.3%, PC2 = 9.7) and TSD treatment groups with different dosages (presented by blue, cyan, and green dots, respectively). Correspondingly, the model group samples in the PCA were shown to be separated from those of TSD-treated groups. However, unexpectedly, few metabolic differences were present for the model rats fed with different dosages of TSD. Clear discrimination of metabolome was otherwise con rmed between the CUMS rats and overall TSD-treated rats by another OPLS-DA (R 2 X = 0.075, R 2 Y = 0.811, Q 2 Y = 0.588, Fig. 2d). Variable permutation veri cations exhibited that the OPLS models were not over-tting (Figure S-4). Differential metabolites (p < 0.05) associated with CUMS modeling and with the effect of TSD intervention are shown in Table S overall TSD-treated rats are equally listed in the Venn diagram (Fig. 3a). Fifteen common discriminants were observed for both the separation of Blank vs. Model and the separation of Model vs TSD treated. Among these key metabolites, urea, and ornithine (Fig. 3b-c) signi cantly increased in the model group compared with the control group, but signi cantly decreased in the TSD treatment group. Other primary discriminants comprising four amino acids (AAs), phosphate, citrate acid, lactate, pseudouridine, Myoinositol, and 3-aminoisobutyric acid (3-AIBA). These molecules were found to be rifely decreased in the model rats, but restored in the treated groups, as shown in Fig. 3d-p. An enrichment of primarily varied metabolic pathways was performed along with the determination of metabolic features between groups. As shown in Fig. 3q-r, varied metabolic pathways associated with the depression (comparison between the M and S group) were similar to those associated with the effect of TSD on depression (comparison between the M and integrated groups of rats treated with TSD). Notably, arginine-related pathways were determined as primary characteristics in both pathway analyses.

Effects of TSD on serum levels of arginase I and serotonin
Arginase I (Arg I) is the enzyme that contributes to the conversion from arginine to urea and ornithine. We assessed Arg I in the serum of all the groups of rats since consistent enrichments were found in urea and ornithine in CUMS rats but such an effect was alleviated by TSD administration. Our data showed that Arg I was decreased in the model group but re-increased in the TSD-fed groups (Fig. 4a), which was in concert with the above two products of arginase degradation. Meanwhile, considering that increasing evidence shows the enhancement of serotonin by herbal medications, we also tested serum serotonin by another ELISA assay. As shown in Fig. 4b, an obvious decrement of serotonin was observed in the CUMS rats without treatments in comparison to the blank controls. The depletion of serotonin was attenuated in the rats with TSD interventions. Such effect of TSD on serotonin was found to be dose-dependent. There was no surprise even a more adequate recovery of serum serotonin was found in the CUMS rats treated with FX since 5-HT receptors are known targets of FX.

Effects of TSD on BDNF, TrkB, ERK, PERK, and CREB protein expression in the hippocampus
It was previously reviewed by Caldwell et al. that cAMP response element-binding protein (CREB) was involved in the transcription of Arg I [25]. CREB is modulated by the BDNF-TrkB-ERK signaling cascade. Strikingly, we noticed that the BDNF-CREB axis was tightly related to the pathology of depression [26]. Therefore, to determine whether the effect of TSD on Arg I release was due to the regulation on the BDNF-CREB axis, we evaluated the related target proteins in the hippocampus of rats by Western blot analysis (Fig. 5a). The results displayed the expression levels of BDNF and TrkB proteins were signi cantly downregulated compared with the normal group (BDNF:(F (5,17) = 5.025, P < 0.05); TrkB:(F (5,17) = 33.093, P < 0.001); CREB:(F (5,17) = 35.045, P < 0.001)), and the effect was effectively reversed by TSD (2.5, 5 and 10g/kg) treatment. Also, the expression of ERK1/2 were decreased by CUMS model (F (5, 17) = 15.190, P < 0.01), TSD treatment can increase the phosphorylation level of hippocampal ERK1/2. These results suggested that the TSD might exert its antidepressant effect by inhibiting arginase, which was related to the up-regulation of the BDNF/ERK/CREB signaling pathway. Together, a schema summarizing possible protein targets of TSD belonging to the BDNF-CREB-Arg I axis is shown in Fig. 5b.

Discussion
The incidence of DD is increasing globally in recent years, which has been a worldwide public health problem. Safe and effective anti-depression drug development is always required. Recent studies have shown that ethnomedicine is a great treasure for the development of effective drugs treating depression [27]. In particular, some formulas of CTM have been reported to be useful to improve DD [28]. To our best known, here we rst report the effect of TSD on depression.
In this current study, depression-like symptoms were induced with CUMS that is a modeling method well imitating the occurrence and pathogenesis of human depression [29]. After the modeling within rats, results of behavioral experiments exhibit that such a modeling method is reliable. Also, those rats treated with TSD emerge signi cant increases of time of immobility in the behavioral experiments. The improvements of depression-like symptoms in rats are to some extent even better than the effect of uoxetine according to our data. A further investigation based on metabolomic techniques is conducted to provide a better understanding of aberrant metabolic variations associated with CUMS and of the pharmacological effect of TSD on DD. Dysregulations of two urea cycle intermediates, urea, and ornithine, are observed to be primary differential factors for the discrimination between rats with and without TSD gavage, and between the blank group and the model group. The increases of these two metabolites in the serum of model group rats indicate enhanced arginase degradation, which is found to be ameliorated after the intervention of TSD administration. The effect is further demonstrated by the results of serum Arg I, which shows that the variations of serum arginase I are in line with those of urea and ornithine. It is documented previously that the increase of arginase in the blood is associated with the occurrence of depression [30]. Interestingly, the level of arginase I is also regarded to be predicable for liver and heart injury [31]. Correspondingly, TSD was widely used for the treatment of heart diseases and was documented to be effective for improving liver injury [32,33]. Therefore, the restoration of arginase after the dosing of TSD may also indicate reversals of organ injuries caused by the CUMS modeling. In addition, the up-regulation of arginase is also related to immune defection and aberrant responses to in ammation [34]. The reversal of arginase disorder within TSD-treated rats may be partly owing to the anti-in ammatory effect of TSD, which is documented in another study [35].
General declines of AAs (including glycine, alanine, proline, phenylalanine, glutamate, lysine, and tyrosine) in the serum are remarkable features of CUMS. After treated with TSD, levels of most AAs recover in the serum of rats. First, decreases in AAs suggest a malnutrition situation of CUMS rats, which is reported previously [36]. Such a symptom is evidenced by the weight losses (Fig. 1a) in the model group. The impaired anabolic metabolism can be partly re ected by the lower levels of energetic metabolites such as citrate and lactate in CUMS rats, compared to other groups. Notably, common discriminants between M vs. B and M vs. TSD-treated like alanine and glutamate are AAs involved in the anaplerotic reactions from AA to the TCA cycle, which is the core metabolic pathway of energy supply. Second, ameliorations in the defective Tyr/Phe pathway are also found in the TSD-treated rats which is in accordance with the study by Liu et al [37]. Finally, glycine as well as glutamate which can be converted into GABA are associated with neurotransmitters. Thus, besides the diminished secretion of dopamine and 5-HT, depressive appearances of CUMS rats can be strongly attributed to the inhibited transmitter synthesis.
Other common discriminant metabolites between M vs. B and M vs. TSD include 3-AIB, pseudouridine, and 3-HB. 3-AIB is known as a critical synthetic precursor of AAs, its variation pattern is in line with most serum AAs in rats. Peseudouridine was reported recently as a potential biomarker of post-stroke depression [38]. 3-HB is an end-product of beta-oxidation in fatty acids. Decrease 3-HB in the CUMS rats is consistent with the diminished serum fatty acids such as propanoic acid in comparison with the blank controls, and with lower levels of arachidic acid found in the comparison between CUMS and non-treated rats.
Our data also suggest that the dramatic dysregulation of Arg I in depressive rats is involved in the inhibited BDNF/CREB signaling and is reversible after the intervention of TSD. Evidence support that BDNF as well as its membranal receptor in the brain, TrkB, in the hippocampus is implicated in both the pathogenesis of depression and antidepressant actions [39]. Several studies have shown that medications targeting the BDNF-TrkB-CREB axis improve depression-like symptoms in animal models [40]. Interestingly, a putative compound hydroxysa or A issued from sa ower is reported to up-regulate BDNF expression [41]. Similar results are available in another recent work showing the effect of sa ower yellow, a drug extracted from sa ower, on the activation of BDNF/TrkB/ERK signaling [42]. Alternatively, ethanol extract of Radix Rehmanniae praeparata also helps to enhance BDNF levels in the serum of CUMS rats [43]. Other potential compounds contained in TSD involving depression therapy encompass albi orin from Radix Paeoniae alba [44], ferulic acid from both Rhizoma Chuanxiong and Angelica Sinensis [45], and ligustrazine from Rhizoma Chuanxiong. Remarkably, ferulic acid can not only increase BNDF expression but also reduce oxidative stress induced by chronic corticosterone stimulation in mice [46]. Given that Arg I is a pivotal marker of NO production, with hints that the effect of TSD on depression may be due to the anti-in ammatory effect. Ligustrazine was also reported to attenuate in ammation and oxidative stress in rats recently [47]. Furthermore, our ndings support that the effect of TSD on DD is linked to the regulation of serotonin secretion. Emerging data show close interactions between BDNF and serotonin. Some studies suggested that BDNF was the target of serotonin [48]. However, the reverse regulation on serotonin receptor 5-HT1A by BDNF expression was also reported by Homberg et al. [49]. Together, our data imply that the actions of TSD on BDNF and serotonin are interdependent.
Our work does have some limitations. First, it remains unclear that what is the most effective composition or compound in TSD improving CUMS-induced depressive symptoms. Second, even though obvious metabolic differences and key discriminants are revealed between CUMS rats and TSD-treated ones, the variation patterns of the discriminants are not dose-dependent according to our data. This issue is supposed to be ascribed to the imitated sample size. Further study will be an investigation of antidepressant effects of the six component medications of TSD within a larger number of samples.
To summarize, our ndings display that the TCM formula TSD can be a useful complementary therapy for depression management. The underlying mechanism behind the e cacy of TSD on DD should be multi-targeted, which can be related to the enhancement of neural transmitters such as glycine and serotonin, as well as to the improvement of disorders on the BNDF-CREB-Arg I axis.