1 Xiaoyao Pill maintains homeotasis of the intestnal flora via regulating Gut

2 Microbiota and Tryptophan Metabolic to improve rats with Depression

3 Induced by Chronic Stress

4 Ying Liu 1, a, b, Jie Shen a, b, Xing Zhang a, b, Fan Ping c, Kai Qu d, *, Xia Shen a, b, *

a
5 College of Pharmacy, Shaanxi University of Chinese Medicine, Shaanxi, Xi'an, China,712046

b
6 Shaanxi Qinling Application Development and Engineering Center of Chinese Herbal Medicine,
7 Xi'an New Area, Shaanxi, Xi'an, China,712046

c
8 Department of Pharmacy, The First Affiliated Hospital of Jiao Tong University, Shaanxi, Xi'an,
9 China,710061

d
10 Shaanxi Provincial Hospital of Chinese Medicine, Shaanxi, Xi'an, China, 710003

11 * Correspondence: Xia Shen, College of Pharmacy, Shaanxi University of Chinese Medicine,

12 Shaanxi, Xi'an, China; Tel: +86 13689250628; Fax: 86-29-38185165; email:

13 [email protected]. Kai Qu, Shaanxi Provincial Hospital of Chinese Medicine, Shaanxi,

14 Xi'an, China; Tel: +86 18792508567；Fax: 029 8725 2903; email: [email protected];

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16 Background: Depression is a growing global health concern, and the need for
17 effective prevention and intervention strategies is paramount. The fundamental
18 principles of traditional Chinese medicine offer a viable approach for early disease
19 intervention in the context of depression. The traditional Chinese medicine (TCM)
20 formula XYP has a millennia-old history of clinical use in treating depression in
21 China, which serves as evidence of its efficacy in treating both anxiety and
22 depression. However, its potential molecular mechanism is still unclear.
23 Methods: Depression was induced in rats through chronic unpredictable mild stress
24 (CUMS), and the effects of XYP were evaluated using various tests including open
25 field and sugar water preference experiments. Serum neurotransmitter and
26 inflammatory factor levels were measured, and histological analyses were performed.
27 Fecal samples were collected for macro-genomic sequencing for more precise
28 assessment of changes in gut flora composition, and blood samples were collected for
29 LC-MS to identify differential metabolites.
30 Results: XYP treatment significantly improved behavioral outcomes in CUMS rats,
31 restored hippocampal pathology, increased serum neurotransmitter levels, and reduced
32 inflammatory factors. XYP improved the species changes of Faecalibacterium _
33 prausnitzii and Lactobacillaceae _ bacterium, especially for the Parabacteroides
34 distasonis, which was found firstly in this study. Metabolomic analysis revealed
35 differential expression of tryptophan, kynurenine, and glutamine metabolites between
36 CUMS and XYP-treated rats. Pathway analyses highlighted alterations in amino acid
37 metabolism, specifically in the tryptophan pathway.
38 Conclusion: Our study elucidates the therapeutic potential of XYP in depression by
39 unveiling its ability to enhance nerve synthesis through the modulation of intestinal
40 flora homeostasis and the regulation of tryptophan metabolism. These findings not
41 only offer a novel perspective on the treatment of depression but also highlight the
42 intricate interplay between gut microbiota and neurotransmitter pathways in mental
43 health. By shedding light on these mechanisms, our research not only contributes to

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44 the advancement of depression treatment strategies but also underscores the
45 importance of exploring traditional remedies for mental health disorders.
46 Keywords: Xiaoyao Pill, depression, intestinal flora, tryptophan metabolism, Chronic
47 Stress
48

49 Abbreviations : XYP: Xiaoyao Pill; TCM: Traditional Chinese medicines;

50 CUMS:chronic unpredictable mild stress; OFT: Open field test; SPT: Sucrose
51 Preference Test; 5-HT, 5-Hydroxytryptamine; VIP, vasoactive; NE, norepinephrine;
52 DA Dopamine; HE, hematoxylin-eosin; IL6: Interleukin 6;TNF-α: Tumor Necrosis
53 Factor-alpha;L1β: Interleukin 1 beta ; LBP: lysaccharide-binding protein; 5-HTP:5-
54 Hydroxytryptophan; KYN：kynurenine;3-HK ：3-hydroxykynurenine; L-Kyn: L-
55 Kynurenine; KYNA: Kynurenic acid; QUIN: Quinolinic Acid.

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57 Graphical Abstract

58 1. INTRODUCTION
59 Depression is a prevalent and chronic mental disorder that seriously affects
60 quality of life (Blier, 2016; McCarron et al., 2021). It is characterized by persistent
61 sadness and a loss of interest and pleasure (McCarron et al., 2021). The condition
62 arises from a complex interplay of genetic, environmental, and psychological factors,
63 leading to a range of cognitive and physical symptoms(Malhi and Mann, 2018;
64 McCarron et al., 2021). Chronic stress is specifically associated with depressive

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65 symptoms, often due to gut microbiota imbalances and metabolic disorders that may
66 lead to neuronal degeneration.(Varatharaj and Galea, 2017).
67 Evidence is Gut microbiota plays an essential role in maintaining the health of
68 host, and the crosstalk of gut and brain has become a research hotspot (Du et al.,
69 2020; Gao et al., 2020; Kelley and Reaux-Le, 2020; Morais et al., 2021). The
70 interaction between intestinal flora and tryptophan metabolism reveals the
71 communication between the intestine and the brain(Gheorghe et al., 2019; Kennedy et
72 al., 2017; Wang et al., 2021). Tryptophan is an essential amino acid for protein
73 synthesis, which affects the growth and health of animals and humans(Guo et al.,
74 2023; Wang et al., 2023b; Zhou et al., 2023). In addition to being a nutrient, there is
75 growing evidence that changes in the composition of the gut microbiota affect the
76 brain by regulating tryptophan metabolism(Zhou et al., 2023). Tryptophan can be
77 metabolized into kynurenine, tryptamine, and indole. These metabolites regulate
78 neuroendocrine and intestinal immune responses(Gao et al., 2018; Gheorghe et al.,
79 2019; Xu et al., 2023; Zhou et al., 2023). The impact of intestinal microorganisms on
80 tryptophan metabolism has emerged as a significant factor in regulating tryptophan
81 metabolism(Gao et al., 2018; Xu et al., 2023; Zhou et al., 2023). The intestinal
82 microbial community can influence tryptophan metabolism, and the resulting
83 metabolites can impact neurotransmitter synthesis, thereby affecting brain function
84 and emotion regulation(Colonna and Butovsky, 2017; Gao et al., 2023). The
85 occurrence and development of depression are closely related to the disorders of the
86 Intestinal flora and its metabolites(Du et al., 2020).
87 Xiaoyao Pill(XYP) is derived from 'Taiping Huimin Heji Jufang', which is used
88 in the treatment of mild depression (Jiao et al., 2024; Li et al., 2023), depressive-like
89 behavior, and other clinical conditions which caused by stress(He et al., 2022). It is
90 believed that the combination of increase amine neurotransmitters and anti-
91 inflammation effects is the main pharmacological result of the XYP used for the
92 treatment of depression(Fang et al., 2020; Yang et al., 2022b). The study suggested
93 that XYP exerts potential antidepressant effects by reversing the imbalance of

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 94 oxidative stress, increasing short-chain fatty acid production and anti-inflammatory
95 bacteria, and decreasing inflammatory and tryptophan metabolizing bacteria(Yang et
96 al., 2022b; Zhou et al., 2021).
97 In this study, we investigated the molecular mechanism of XYP against
98 depression-related symptoms using different omics approaches and an CUMS rats
99 model. To explore the molecular mechanism underlying stress Intestinal flora
100 disorders and monoamine neurotransmitters, we investigated the effects of CUMS by
101 regulatory Intestinal bacteria ( Firmicutes and Actinobactria, Proteobacteria,
102 Bacteroidetes ) on the activities of inflammatory stress, monoamine pathway
103 enzymes. We used LC-MS analysis to determine serum differential metabolites. And
104 used the Illumina sequencing to analyze gut microbial diversity of depression. We set
105 the stage for a detailed exploration of the potential molecular mechanisms through
106 which XYP may exert its antidepressant effects. This research will aim to provide
107 additional empirical more evidence supporting the effectiveness of XYP in managing

108 depression-related symptoms，and Provide new alternative strategies for clinical

109 treatment of depression.

110 Materials and Methods
111 Chemicals and reagents
112 Xiao Yao Pills ( Taiji Group, Z50020438 )included Bupleurum chinense,
113 Angelica sinensis, Radix Paeoniae Rubra, Atractylodes macrocephala, Poria umbellus,
114 Radix glycyrrhizae, Zingiber officinale Rosc, Mentha haplocalyx. Sucrose reagent
115 ( Tianjin Komi Omi Chemical Reagent Co., Ltd. ), Experimental water was purified
116 by using Milli-Q water purification system ( Millipore Company, USA ). 4 %
117 paraformaldehyde tissue fixative ( Shaanxi Zhong Hui Hecai Biomedical Technology
118 Co., Ltd. ).
119 Animals
120 30 male SPF SD rats, aged 6-8 weeks with a body mass of 200 ± 20 g, were
121 procured from Chengdu Da shuo Experimental Animal Co., Ltd. (animal production

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122 license number: SCXK (Sichuan) 2020-030). The rats were housed at the Key
123 Laboratory of Pharmacodynamic Mechanism and Material Basis of Traditional
124 Chinese Medicine of Shaanxi Provincial Administration of Traditional Chinese
125 Medicine. The rats were maintained in an environment with a 12-hour light-dark cycle
126 (light period: 08:00-20:00) at a humidity of 45% and a temperature of 22-24 °C. This
127 study adhered to ethical standards for animal experiments and was approved by the
128 Experimental Animal Ethics Committee of Shaanxi University of Traditional Chinese
129 Medicine (batch number: SUCMDL20230515001).
130 CUMS protocol and treatments
131 After a one-week acclimatization period, the chronic unpredictable mild stress
132 (CUMS) protocol was initiated. The CUMS group was subjected to various stressors
133 for four weeks, as outlined in Table 1. Each day, two different types of stressors were
134 applied, with each stressor occurring non-concurrently. Non-stressed rats were kept in
135 undisturbed cages and fed a regular diet. After 4 week of the CUMS procedure, rats
136 developed depression symptoms, were randomly assigned to 2 groups (n = 10 in each
137 group): CUMS group, XYP ( 1.86g / kg -1 ) group，Control group ( Regular diet and
138 non-stressed mice served ). The dose was converted according to the body surface
139 area of the human and mouse. XYP was administered i.g. once daily for the following
140 4 weeks, while the CUMS stimulus were continued throughout the treatment period.
141 Rats in the control group were given an equal volume of water.
142 External Behavioural Representation
143 The body weight of each group of rats was measured weekly to observe the
144 effect of rat modeling on rats and whether the treatment drugs could regulate the body
145 weight of depressed rats. The Sucrose Preference Test ( SPT ) is used to measure the
146 sensitivity of rats to rewards as an indicator of depression levels. Rats were presented
147 with a bottle of pure water and a bottle of 1% sucrose solution simultaneously after 72
148 hours of training and 24 hours of food and water deprivation. The volume of the
149 remaining pure water and sucrose solution was recorded after 1 hour. SPT was
150 performed after CUMS induction (as a baseline measure) and after continuous

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151 intragastric administration. Open field test ( OFT ) was used to evaluate the anxiety
152 and exploratory behavior of rats. After 1 minute of adaptation in the open field, the
153 activity of the rats was recorded within the next 5 minutes. These parameters include
154 the time of the central area, the number of standing times and the distance through the
155 grid.
156 Enzyme-linked immunosorbent assay
157 Colon samples were collected for the detection of 5-HT and VIP, IL6 ,TNF-
158 α ,L1β ,LBP concentrations using a competitive enzyme-linked immunosorbent assay
159 (Meimian, Jiangsu, China). All tests were performed according to the manufacturer’s
160 instructions.
161 Pathological examination
162 Brain tissue fixed in 4 % paraformaldehyde was subjected to histopathological
163 observation. The fixed Brain tissue was embedded in paraffin and cut into 5 μm
164 sections. Then the sections were stained with hematoxylin-eosin (HE). Briefly,
165 paraffin-embedded sections of Brain tissue were prepared and incubated with
166 antibodies. Sections were counterstained with DAPI.
167 Metagenomics sequencing
168 The cecal contents were collected and stored at -80 °C. Metagenomic sequencing
169 was performed at Genedenovo. Genomic DNA was extracted using HiPure Bacterial
170 DNA Kits (Magen, Guangzhou, China) according to the manufacturer’s instructions.
171 The DNA quality was detected using Qubit (Thermo Fisher Scientific, Waltham, MA)
172 and Nanodrop (Thermo Fisher Scientific, Waltham, MA) accordingly.
173 Qualified genomic DNA was firstly fragmented by sonication to a size of 350bp,
174 and then end-repaired, A-tailed, and adaptor ligated using NEBNext® ΜLtra™ DNA
175 Library Prep Kit for Illumina (NEB, USA) according to the preparation protocol.
176 DNA fragments with length of 300-400 bp were enriched by PCR. At last, PCR
177 products were purified using AMPure XP system (Beckman Coulter, Brea, CA, USA)
178 and libraries were analysed for size distribution by 2100 Bioanalyzer (Agilent, Santa

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179 Clara, CA) and quantified using real-time PCR. Genome sequencing was performed
180 on the Illumina Novaseq 6000 sequencer using the pair-end technology (PE 150).
181 XYP treatment improves intestinal flora and affects serum metabolic
182 disorders
183 After separation, the collected blood samples were promptly frozen in liquid
184 nitrogen. Separation was performed on an Agilent 1290 Infinity LC Ultra High
185 Performance Liquid Chromatography ( UHPLC ) HILIC column. The samples
186 remained in an automatic sampler at 4°C throughout the analysis. In order to avoid the
187 influence of instrument detection signal fluctuation, the continuous analysis of
188 samples is carried out in random order. QC samples are inserted into the sample queue
189 to monitor and evaluate the stability of the system and the reliability of the
190 experimental data. The primary and secondary spectra of the samples were collected
191 by AB Triple TOF 6600 mass spectrometer.
192 Correlation analysis
193 Spearman correlation was used to assess the correlation between depression-
194 related behavioral parameters, different intestinal microbial species and metabolites in
195 the group. The data matrix of behavioral indicators and metabolites was imported into
196 the bioinformatics platform Genes cloud platform ( https://www.genescloud.cn/) ) for
197 correlation analysis(Fig.5). The correlation coefficient ( R ) and p-value described the
198 degree of correlation between depression and metabolites. The inter-layer correlation
199 between behavioral indicators and serum metabolites and between intestinal flora was
200 calculated. The data of behavior parameters are expressed as mean ± standard
201 deviation ( SD ). The significant differences in behavioral data and relative metabolite
202 concentrations between groups were analyzed by unpaired t-test. All statistical
203 analyses were performed using Prism 8 ( GraphPad ), and there was a significant
204 difference in P < 0.05. The data are shown as mean ± s.e.m. When the parameters of
205 the two experimental groups were tested, a two-sample unpaired t-test was
206 performed ; one-way analysis of variance ( ANOVA ) was performed when the
207 parameters of the three experimental groups were tested.

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209 3.Results
210 XYP improved anxiety and depression-like behavior in CUMS rats.
211 XYP (1.86 g· kg-1) (Ji et al., 2021; Shi et al., 2019; Xie et al., 2023)was administered
212 by gavage (once daily) to CUMS-fed rats for 4 weeks (Fig. 2A) .We found that XYP
213 interfered with anhedonia, social behavior, and despair behavior of mice in various
214 behaviors tests (Fig.2 BCD). In the SPT, XYP modulated the preference for sugar
215 water in anxiously depressed mice in the OFT, XYP also improved anxious behavior,
216 the immobile time in OFT of XYP were higher than Model group. And also increase
217 the total distance and the time spent in the center zone of OFT (Fig2. B). We observed
218 that the anxiety and depression-like behaviours of CUMS rats were almost completely
219 rescued by XYP treatment (Fig2.B) XYP did not significantly change the body weight
220 of CUMS rats during treatment (Fig2. E).
221 Pathological changes in tissues of CUMS rats and detection of biochemical
222 indices
223 We determined levels of 5-HT, VIP, IL-1β, LBP, TNF-α and IL6 in the peripheral
224 blood by ELISA to investigate whether XYP treatment could alter neurotransmitters,
225 pro-inflammatory cytokines in anxious depression rat (Fig2.F). XYP significantly
226 reduced levels of 2 neurotransmitters (5-HT, VIP) and and three proinflammatory
227 factors (IL-1β /IL6 and TNF-α) in peripheral blood (both p < 0.001). XYP could
228 significantly reduce levels of these substances. After treated with XYP, levels of LBP,
229 IL-1β, IL6 and TNF-α (P*** < 0.001) were reduced. XYP also increased levels of 5-
230 HT, VIP (P *< 0.05) (Fig.2 F).
231 XYP treatment improved the pathological changes of brain tissue.
232 Hematoxylin-eosin staining results showed that XYP significantly improved
233 neuronal sparing and neuronal degeneration in the hippocampal region after
234 prophylactic tablet administration. Collectively, these results suggest that XYP
235 treatment significantly reduced anxiety- and depression-related neurons in CUMS rats
236 (Fig2.G).

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237 Immunohistochemistry was used to verify the protein expression level of 5-HT
238 in normal brain tissue. The results showed that 5-HT was moderately expressed and
239 strongly stained in normal brain tissue, but weakly expressed and stained in CUMS rat
240 brain tissue. The expression of 5-HT was improved in XYP group (Fig2.H).
241 XYP treatments remodel the gut microbiota in CUMS rats
242 To determine whether XYP affects anxiety and depression behaviour in CUMS
243 rats by regulating the gut microbiome, we analysed the rats' gut microbiome using
244 metagenomic sequencing. In addition to the rich features of functional analysis,
245 metagenomics is pushing species research to a more detailed level. We showed
246 Chao1, ACE, Shannon, Simpson, and we performed a sample relationship analysis
247 from species abundance (Fig .3B.C.E.F.I ). The relative abundance of the top 10
248 bacterial phyla, genera and species also changed significantly after XYP treatment
249 (Fig .3A. D. G). The difference in alpha diversity was not obvious. The alpha
250 diversity index, including Shannon's index and Simpson's index, increased in XYP-
251 treated rats, but the beta diversity in the XYP group was significantly different from
252 that in the CUMS group and the control group (P* < 0.05). The species differences
253 between the CUMS group and the XYP group. First, we can show the differences
254 between the different groups at the species level using the Venn diagram as shown
255 below: There are 3604 species between the three groups, and there are 3923 different
256 species between the CUMS group and the XYP group (Fig.3H).
257 The study evaluated the differences in microbial structure at the genus level
258 between the XYP and CUMS groups (Fig.2J ). After 28 days of Xiaoyao Pill
259 intervention, the XYP group showed an increase in Lactobacillus, Clostridium,
260 Methanosphaera and Ruminococcus, and a decrease in Escherichia, Bifidobacterium,
261 Allobaculum, Faecalibaculum, Romboutsia and Corynebacterium compared to the
262 model group. At the species level, the study found an increase in Lactobacillus _
263 reuteri, Lactobacillus _ murinus, Candidatus _ Saccharibacteria _ bacterium _ TM7 _
264 G3 _ 2 _ Rum _ HOT _ 351B, Lactobacillus _ johnsonii, and bacterium _ 1xD42-87.
265 Additionally, there was a decrease in Escherichia _ coli, Bifidobacterium _ animalis,

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266 Allobaculum _ stercoricanis, Coriobacteriaceae _ bacterium, and Faecalibaculum _
267 rodentium. The alterations in the structure of the bacterial community are closely
268 linked to changes in the physiological state and metabolic function of the intestine.
269 XYP treatment affects serum metabolic disorders in CUMS rats
270 Firstly, principal component analysis (PCA) of unsupervised pattern recognition
271 was used to visually display the differences of serum samples in Control group,
272 CUMS group and XYP group ( Fig.4A ). We combined the VIP value of multivariate
273 statistical analysis OPLS-DA and the P value of univariate statistical analysis T test to
274 screen the differential metabolites between different comparison groups. The
275 threshold of the difference was VIP ≥ 1 and T-test P < 0.05 in the OPLS-DA model
276 (Fig.4C). It was considered that the difference was significant, and the differential
277 metabolites of XYP regulating CUMS rats were screened out. In the positive and
278 negative ion mode, the differential metabolites of the blank group, the model group
279 and the XYP group were identified respectively. In the positive ion mode, compared
280 with the model group, 308 metabolites changed in the XYP group, and 293
281 metabolites changed in the negative ion mode. There were 74 intersection differential
282 metabolites, of which 63 differential metabolites showed opposite trend changes in
283 the blank group and the model group, the model group and the XYP group( Fig.4E ).
284 The VIP ( Variable Importance in Projection ) diagram of OPLS-DA is used to
285 represent the importance of variables and their contribution to sample differentiation.
286 The following figure is the VIP results ( Fig .4F )corresponding to the differential
287 metabolites ( MS2 levels ) with the top 15 different VIP values in each comparison
288 group. In the positive ion mode, DL-lactic acid, isocitric acid, sodium
289 taurochenodeoxycholate, taurocholate, azelaic acid, DL-vanillin mandelic acid,
290 indoxyl sulfate, isogentisin, cholic acid, estrone glucuronide, DL-glutamine,
291 arachidonic acid, and sarcosine in the model group and XYP in the negative ion
292 mode. DL-arginine, glycerophosphocholine, acetylcarnitine, Lpc18 : 2,1-stearoyl-2-
293 hydroxy-Sn-glycero-3-phosphocholine, DL-proline, betaine, 3-methyl-L-histidine,
294 creatine, creatinine, tetrahydropyrimidine, Lpc18 : 1, D-glutamine.

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295 The differential metabolites were analyzed by metabolic pathway enrichment
296 analysis. Differential metabolites were significantly enriched in the anabolism of
297 amino acids, the metabolic pathways of secondary bile acid biosynthesis, nicotinic
298 acid and nicotinamide metabolism, ABC transporters, regulation of HIF-1 signaling
299 pathway, etc.
300

301

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302 Disscussion
303 Xiaoyao Pill is mainly used for depression like behavior caused by long-term
304 stress in clinical practice(He et al., 2022; Tong et al., 2023; Wang et al., 2023a). XYP
305 can alleviate liver depression and qi stagnation caused by long-term stress，and also
306 impact the nervous system, potentially resulting in neurocognitive disorders such as
307 anxiety, depression, dizziness, slow thinking, memory loss, and inattention(Morais et
308 al., 2021; Yang et al., 2022a). Our findings suggest that XYP is effective in both
309 preventing and treating stress-induced depressive symptoms by alleviating behavior
310 indicative of depression under stressful conditions, while also rectifying imbalances in
311 gut microbiota predisposed to depression and amino acid metabolic pathways.
312 In previous studies, depression and inflammation are inseparable. (Rahn and
313 Liston, 2023; Wang et al., 2024). LBP may be a key molecule in regulating
314 inflammation-monoamine, thereby inhibiting the biosynthesis of 5-HT, VIP and
315 NE(Drevets et al., 2022; Varatharaj and Galea, 2017).LBP is up-regulated by NF-κB
316 signal transduction in inflammation, environmental stress and stress factors
317 ( including TNF-α, IL-6, NE ), which inhibits the biosynthesis of 5-HT, NE and NA in
318 the brain(Wu et al., 2022). IL1 b is a crucial pro-inflammatory cytokine that triggers
319 depression-like behaviour under chronic stress. XYP inhibits inflammation through
320 NF-kb, mediates the immune system, TNFα polymorphism, and sleep abnormalities,
321 fatigue, and mood disorders.
322 Notably, we found that XYP improved the species abundance of P.distasonis. The
323 study discovered that Parabacteroides distasonis can cause depression (Cheng et al.,
324 2024; Gomez-Nguyen et al., 2021; Ma et al., 2019; Taglialegna, 2024). And, XYP can
325 regulate P.distasonis to improve intestinal inflammation and the nervous system(Fang
326 et al., 2020; Jiao et al., 2024). XYP can improve the effects of F.rodentium on
327 microglia activation and neuroinflammation(Fang et al., 2020; Ji et al., 2021; Yang et
328 al., 2022b). In the XYP group rats abundance of Faecalibacterium, Eubacteria,
329 Ruminococcus, Lachnospira, and Roseburia decreased, while the abundance of
330 Enterococcus, Rotabacter, and Lactobacillus increased.

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331 XYP balances serotonin production and tryptophan catabolism pathways through
332 regulation of intestinal microbial tryptophan metabolism. Probiotics, such as
333 Lactobacillus and Bifidobacterium, have been reported to have beneficial effects on
334 tryptophan metabolism(Li et al., 2023). On the one hand, as previously mentioned,
335 probiotics, such as species in Lactobacillus and Bifidobacterium, can directly convert
336 tryptophan to serotonin(Cervantes-Barragan et al., 2017). On the other hand, some
337 probiotic strains, such as Lactobacillus casei 327, can indirectly promote the synthesis
338 of colonic 5-HT by increasing the expression of TPH1(Zelante et al., 2013). All these
339 pathways affect the peripheral 5-hydroxytryptamine pool. These studies have shown
340 that XYP can regulate the probiotics of Lactobacillus and Bifidobacterium to inhibit
341 the kynurenine pathway to change the host 's tryptophan metabolism.
342 Our discovery revealed that the peripheral blood of CUMS rats exhibited
343 disrupted amino acid metabolism, with notable changes in tryptophan levels. After
344 XYP treatment, the rats displayed elevated serum kynurenine levels and increased
345 tryptophan content, indicating a shift in tryptophan metabolism pathways(Park and
346 Kim, 2021).Previous studies have shown Tryptophan, an essential amino acid linked
347 to regulating depression-like behavior, showed significant alterations. Tryptophan can
348 be metabolized into 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT)
349 in various cells.5-HTP is converted into 5-HT to increase the level of serotonin in the
350 brain, which helps to improve mood and reduce depressive symptoms and increased
351 expression of BDNF in the brain(Wang et al., 2022). The main tryptophan metabolic
352 pathways include kynurenine (Kyn), 5-hydroxytryptamine (HT), and indole pathways,
353 leading to the production of neuroprotective and neurotoxic metabolites (Lovelace et
354 al., 2017; Pu et al., 2021). Enzymatic activity in the kynurenine pathway is influenced
355 by pro-inflammatory cytokines, potentially impacting the neuroprotective effects of
356 kynurenine(Clarke et al., 2017). XYP can improve the tryptophan pathway mediated
357 by intestinal flora imbalance of CUMS-treated rats.
358 The relationship between intestinal flora and tryptophan is
359 bidirectional(Krautkramer et al., 2021; Lai et al., 2021; Sheibani et al., 2023). The

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360 influence of the gut microbiota on tryptophan metabolism holds great significance.
361 Intestinal flora affects the synthesis and metabolism of tryptophan, and tryptophan
362 affects the synthesis of nervous system and 5-HT. Therefore, XYP plays an important
363 role in regulating the two-way communication between the intestine and the brain.
364 More and more evidence shows that the imbalance of intestinal microbial species
365 abundance may be related to a series of neurological diseases, including anxiety and
366 depression. XYP can also improve the symptoms of neurocognitive disorders, such as
367 our long-term covid-19-induced ' brain fog '(Agirman and Hsiao, 2021; Boldrini et al.,
368 2021; Clarke et al., 2017; Del et al., 2020; Matheson and Lehner, 2020; Zhang et al.,
369 2023).As a new way of thinking, XYP provides new barrier protection for our brain,
370 can provide ideas and clinical medication advice.
371

372 Conclusion
373 In conclusion, our study reveals the significant impact of Xiaoyao Pill (XYP) in
374 alleviating depression by modulating intestinal flora and tryptophan metabolism. The
375 findings underscore the potential of XYP as a novel therapeutic approach for
376 depression, offering insights into the complex interplay between gut microbiota,
377 neurotransmitter levels, and inflammatory factors in the context of chronic stress. This
378 research not only sheds light on the molecular mechanisms underlying XYP's efficacy
379 but also highlights the importance of targeting gut-brain interactions in mental health
380 interventions. This study found the molecular mechanism of XYP in the treatment of
381 depression, which provided useful insights for the development of new therapeutic
382 drugs, and laid a theoretical foundation for clinical science and safe medication.
383

384 CRediT authorship contribution statement

385 Ying Liu: Conceptualization, data curation, formal analysis, methodology, writing
386 original draft, writing review& editing. Jie Shen: writing original draft, visualization.
387 Xing Zhang: data curation, methodology. Fan Ping : Formal analysis, methodology.
388 Kai Qu, Xia Shen: Conceptualization, resources, writing review and editing,

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389 supervision, funding acquisition.
390

391 Funding
392 This work was supported by the Special Scientific Research for Traditional Chinese
393 Medicine of State Administration of Traditional Chinese Medicine of China
394 (No.GZY-KJS-2021-005), Natural Science Basic Research Plan of the Shaanxi
395 Province (2022JM-538)，Shaanxi Provincial Administration of Traditional Chinese
396 Medicine “Dual-chain Integration” Young and Middle-aged Scientific Research
397 Innovation Team Project (2022-SLRH-LJ-003) .
398

399 Declaration of Competing Interest

400 The authors declare that they have no known competing financial interests or personal
401 relationships that could have appeared to influence the work reported in this paper.
402

403 Data availability

404 Data will be made available on request.
405

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549

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550 Figure 1: XYP improved anxiety and depression-like behavior in CUMS rats:(A)
551 CUMS model establishment and arrangement of behavioural experiments;(B) Open
552 Field Test; immobility time; central activity time; edge activity distances; (C) Sucrose
553 Preference;(D) Behavioural absconding experiments behavioural trajectory diagrams ;
554 (E) Figure of body weight change in rats;(F) Comparison of inflammatory factors
555 detected by ELISA ;(G) HE hippocampal staining results diagrams ;(H)
556 Immunohistochemical staining of 5-HT in rat brain. P*<0.05; P*<0.01; P*<0.001.
557 Figure 2: XYP treatments remodel the gut microbiota in CUMS rats
558 We show the top 10 highly abundant species (phylum, genus, species) in terms of

559 total abundance(A、D、G). Alpha Diversity shows Chao1, ACE (Alternating

560 Conditional Expectation), Shannon, Simpson, the four indices, and the results of the

561 correlation analysis. (B、C、E、F)；Species differencesLEfSe analysis of

562 differences in flora between groups allows the identification of major flora specific to
563 each group (J);XYP-depression differences in species (P<0.001) Species
564 differences(K).
565 Figure 3: XYP treatment affects serum metabolic disorders in CUMS rats. A
566 Principal component analysis (PCA); B Orthogonal partial least squares-discriminant
567 analysis (OPLS-DA) score plot; C Orthogonal partial least squares-discriminant
568 analysis (OPLS-DA) permutation test plot; D Difference metabolite basic analysis
569 volcano plot (the threshold of difference is: VIP ≥1 in the OPLS-DA model and T-test
570 P<0.05); E Multi-group difference scatter plot; F Difference metabolite VIP plots
571 (P<0.05,VIP ranked top 20); Differential metabolite KEGG bubble plots. (P<0.05)
572 Figure4: Correlation analysis. Spearman correlations between gut microbiota at
573 genus level, anxiety and depression-like behaviors, digestive system indicators,
574 neurotransmitters, inflammatory factors, and core differential mRNAs of rats treated
575 with XYP.

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576 Figure 5: Key metabolites between the gut. A The relative contents of
577 Parabacteroides _ distasonis, Faecalibacterium _ prausnitzi and Lactobacillaceae _
578 bacterium between XYP group and CUMS group ,control group were compared；B
579 The relative content of 3-hydroxykynurenine, 5-hydroxy-l-tryptophan and L-
580 kynurenine between XYP group、model group and control group was compared with
581 histogram.
582

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