This research was supported by grants from National Natural Science Foundation of China (No. 81630104&#65307;No. 81473597), and the Fundamental Research Funds for the Central Universities (No. 2018-JYBZZ-XS005). The protocol and results demonstrated in this paper originates from the article Involvement of Normalized Glial Fibrillary Acidic Protein Expression in the Hippocampi in Antidepressant-Like Effects of Xiaoyaosan on Chronically Stressed Mice by Dr. Xiu-Fang Ding, et al.

This protocol was approved by the Animal Ethics Committee of Beijing University of Chinese Medicine and was in accordance with all guidelines for the Care and Use of Laboratory Animals of China.&#160;There was no accidental death situation during the experimental procedure, and no animals needed to be euthanized in this study.
1. Preparing Drugs for the Experiment
NOTE: The Chinese herbs were purchased and then authenticated by Dr. B. Liu of the Beijing University of Chinese Medicine. Xiaoyaosan contains the following eight traditional Chinese drugs in a ratio of 6:6:6:6:3:6:2:2: Radix Angelicae Sinensis, Radix Paeoniae Alba, Poria, Radix Bupleuri, Radix Glycyrrhizae, Rhizoma Atractylodis Macrocephalae, Herba Menthae and Rhizoma Zingiberis Recens,.
<ol>
	<li>Prepare Xiaoyaosan as previously described16. 
	Note: We extracted these compounds in the Chinese medicine preparation room of the China-Japan Friendship Hospital, and the extraction yield was 18.8%.</li>
	<li>Verify the quality of Xiaoyaosan by high-performance liquid chromatography-mass spectrometry analysis (LC-MS/MS)17.</li>
</ol>
2. Preparing Mice for the Experimental Procedure
NOTE: For the experimental procedure, 60 healthy male C57BL/6J mice aged 12 weeks were selected, with each mouse weighing between 18 and 22 g.
<ol>
	<li>Keep mice individually in cages in a typical Specific Pathogen Free (SPF) laboratory animal room (22 &#177; 1 &#176;C, 12 h/12 h dark/light cycle, relative humidity: 30&#8211;40%) for one week before the CUMS procedure.</li>
	<li>Randomly divide all mice into a control group, a model group, a Xiaoyaosan treatment group, or a fluoxetine treatment group according to their body weight. Each group has 15 mice.</li>
	<li>Before initiation of the experiment, number the mice in each group on their tail using a marking pen.</li>
	<li>Test all mice using a five-day sucrose preference test (refer to step 5) and an open field test (refer to step 6).</li>
</ol>
3. Induction of Depression in Mice
NOTE: To induce depression, the mice in the model group and two treatment groups underwent CUMS for 21 days18. The plan can be changed according to the practical situation, but the principle that the same stressor cannot be used continuously should be followed.
<ol>
	<li>Expose mice daily to any two of the following seven stressors for 21 consecutive days19. Our modeling plan is designed as follows:
	<ol>
		<li>On Monday, expose mice to food deprivation (empty the feed of each cage for 24 h, 8:00 a.m. to 8:00 a.m.) and water deprivation (remove the drinking bottle of each cage for 24 h, 8:00 a.m. to 8:00 a.m.).</li>
		<li>On Tuesday, expose mice to restraint stress (restrain mice in a small plastic tub for 3 h, 8:00 a.m. to 11:00 a.m.) and empty cages (remove the padding in each cage for 11 h, 8:00 a.m. to 19:00 p.m.).</li>
		<li>On Wednesday, expose mice to wet and soiled cages (pour water into each cage and keep wet for 24 h, 8:00 a.m. to 8:00 a.m.) and crowded cages (put five mice in one cage for 24 h, 8:00 a.m. to 8:00 a.m.).</li>
		<li>On Thursday, expose mice to restraint stress and ice-cold swimming20,21 (force each mouse to swim in a clear glass aquarium with ice water for 5 min).</li>
		<li>On Friday, expose mice to wet and soiled cages and food deprivation.</li>
		<li>On Saturday, expose mice to restraint stress and crowded cages.</li>
		<li>On Sunday, expose mice to empty cages and ice-cold swimming.</li>
	</ol>
	</li>
	<li>Record the coat state and body weight weekly until the end of modeling (day 21). Calculate the total score of the coat state as the sum of scores from seven different body parts: head, neck, dorsal coat, ventral coat, forepaws, tail, and hind paws. A score of one indicates a well-groomed coat, and a score of zero indicates an unkempt coat22.</li>
	<li>Perform a five-day sucrose preference test (refer to step 5) and an open field test (refer to step 6) at the end of experiment.</li>
</ol>
4. Administration of Xiaoyaosan
NOTE: The dose of Xiaoyaosan (0.25 g/kg/day) was selected on the basis of its satisfactory efficacy as previously described9.
<ol>
	<li>During the experiment, use distilled water to prepare the Xiaoyaosan and fluoxetine suspensions, and make sure to use immediately.</li>
	<li>Give 0.5 mL of distilled water to the mice in the control group and model group by intragastric administration every day before modeling. Meanwhile, give Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day) to the mice in the two treatment groups.</li>
</ol>
5. Sucrose Preference Test
NOTE: No water or food deprivation was applied before the test. The protocol was performed as previously reported23.
<ol>
	<li>On the first two days, house the mice individually with two bottles filled with tap water.</li>
	<li>On the next two days, give the mice two bottles filled with 1% sucrose solution.</li>
	<li>On the last day, give the mice two bottles for 24 h, one with tap water and the other with 1% sucrose solution. To eliminate a side preference of the mice in drinking behavior, switch the position of the two bottles after 12 h.</li>
	<li>Record the volume (mL) of both the consumed sucrose solution and tap water. Calculate the sucrose preference as follows: sucrose preference (%) = sucrose consumption/total liquid consumption &#215; 100%.</li>
</ol>
6. Open Field Test
<ol>
	<li>Perform the open field test in a dimly lit and quiet room with a four-sided wooden black lusterless box. Divide the &#64258;oor into 25 equal squares by blue lines.</li>
	<li>Clean the open-field box with 5% alcohol prior to the access of each mouse. Then, place the mouse in the center of the open field gently.</li>
	<li>Suspend a high definition digital camera approximately 200 cm right above the field, and record the behavior of each mouse for 5 min.</li>
	<li>Evaluate the mice behavioral video by the video-tracking system software and record the data of the total running distance and number of entries into the center zone.</li>
	<li>Analyze the data of the total distance moved and number of entries into the center zone by statistical software.</li>
</ol>

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The authors have nothing to disclose.

The CUMS model used in this study is a common method to establish the stress diathesis model of depression24. Most manifestations of CUMS can be restored by antidepressant drugs, which indicated that this model method has a strong predictive validity25. By this method, animals receive chronic unpredictable stressful stimuli and develop core symptoms of major depression, including decreased place preference conditioning, anhedonia, and impaired emotion-like behaviors<sup cla...

MDD is a chronic, highly recurrent mental disease that presents with persistent low mood, depression, low self-esteem, mental retardation, loss of interest, sleep disorders, profoundly pessimistic or suicidal tendencies; it has caused great harm to human health and social stabilization1. It has become one of the world&#39;s primary cause of disability, and it may be one of the main leading causes of disease burden by 2030 according to the World Health Organization2,3. Currently, antidepressant medications and psychotherapy are considered to be the first choice to treat MDD. Nevertheless, an enormous number of patients diagnosed with MDD do not respond to continuous medication or therapy, and they are listed as having treatment-resistant depression4.
Xiaoyaosan is a classic Chinese compound5 that has been widely used to treat psychological diseases for thousands of years since the Song dynasty in China6. It is composed of 8 Chinese herbs: Radix Bupleuri, Radix Paeoniae Alba, Radix Angelicae Sinensis, Poria, Rhizoma Atractylodis Macrocephalae, Radix Glycyrrhizae, Rhizoma Zingiberis Recens, and Herba Menthae. Xiaoyaosan contains multiple constituents, has multiple targets, and utilizes multiple pathways7. Xiaoyaosan has been commonly shown to be a safe and effective formula for treating various diseases related to mental disorders, and modern studies have also focused on the anti-depression mechanism of this compound8,9,10. The pathogenesis of MDD is yet unclear, even though there are multifarious hypotheses, including the monoamine hypothesis, the neurotransmitter receptor hypothesis, the neuroendocrine function change hypothesis, the immune system abnormality hypothesis, etc. Several studies have shown that the antidepressant effect of Xiaoyaosan has a connection with the material bases of the above hypotheses11,12, and its therapeutic action manifests as an improvement to depressive-like behaviors13.
Moreover, with the prevalence of depression, modern society has suffered from reductions in the work force, early retirements, and expensive treatments14. Expensive treatment needs strict medical attention, which means a range of increased medical costs and precise anticipation of precise undesirable outcomes as well as side effects15. The Chinese herb Xiaoyaosan has accessible components, unique effects, and slight side effects. Its application can play a role in reducing the burden of modern medical treatment. Therefore, to provide a more theoretical basis for the application of Xiaoyaosan, an alternative clinical therapy should be offered for the treatment of depression, the incidence of which increases year by year, and should improve human health at the same time. This paper describes a protocol involving the use of Xiaoyaosan to prevent the depressive-like behaviors by establishing the depressive model of the mouse. The open field test and sucrose preference test were used to verify the antidepressant effects of Xiaoyaosan.

<table>
	<tbody>
		<tr>
			<td>Chinese Herbs</td>
			<td>Tongrentang (Bozhou, AnHui) Decoction Pieces Limited Company</td>
			<td>-</td>
			<td>The drugs were extracted by the Chinese medicine preparation room of the China-Japan Friendship Hospital, distilled water was used to prepare the suspension of Xiaoyaosan, and the dose was 0.25 g/kg/d.</td>
		</tr>
		<tr>
			<td>Fluoxetine</td>
			<td>PATHEON FRANCE</td>
			<td>5545A</td>
			<td>The distilled water was used to prepare the suspension of fluoxetine, and the dose 2.6 mg/kg/d.</td>
		</tr>
		<tr>
			<td>C57BL/6j mice</td>
			<td>Beijing Vital River of the Charles River Company</td>
			<td>SCXK 2011-0004</td>
			<td>60 healthy male C57BL/6J mice aged 12 weeks were purchased from Beijing Vital River of the Charles River Company.</td>
		</tr>
		<tr>
			<td>Mouse cage</td>
			<td>Styling Biotechnology</td>
			<td>34853016</td>
			<td>318&#215;202&#215;135 mm</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>-</td>
			<td>-</td>
			<td>The water was used througout the experiment.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>-</td>
			<td>The sucrose was used in the sucrose preference test to prepare 1% sucrose solution.</td>
		</tr>
		<tr>
			<td>ACS-2EAS Electronic Balance</td>
			<td>Beijing Hengzhongheng Electronic Technology Co., Ltd.</td>
			<td>-</td>
			<td>It was used to weigh the avoirdupois of mice at different stages.</td>
		</tr>
		<tr>
			<td>Wooden Black Lusterless Box</td>
			<td>-</td>
			<td>-</td>
			<td>(40 cm x 40 cm x 15 cm), divided into 25 equal squares by blue lines.</td>
		</tr>
		<tr>
			<td>WV-CP470 Camera</td>
			<td>Panasonic, Japan</td>
			<td>-</td>
			<td>A HD digital camera which was used for recording the whole process of the open field test.</td>
		</tr>
		<tr>
			<td>Etho Vision 3.0 Software</td>
			<td>Noldus, Wageningen, Holland</td>
			<td>-</td>
			<td>A video-tracking system which was used to evaluate the locomotor function and emotionality of mice.</td>
		</tr>
		<tr>
			<td>SPSS Statistics 21.0 Software</td>
			<td>IBM</td>
			<td>-</td>
			<td>SPSS 21.0 software was used to analyze the data, one-way ANOVA or non-parametric test was used for general data based on normality test and homogeneity test for variance, and LSD method was adopted for the comparisons between groups.</td>
		</tr>
		<tr>
			<td>Graphpad Prism 6.0 Software</td>
			<td>Graphpad Software,Inc.USA</td>
			<td>-</td>
			<td>Graphpad Prism 6.0 software was used to draw statistical graphs.</td>
		</tr>
	</tbody>
</table>

evaluating the anti-depression effect of xiaoyaosan on chronically-stressed mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Note that the results have been previously published by Dr. Xiu-Fang Ding, et al.18
Data were expressed as the mean &#177; standard error of the mean (SEM). A one-way ANOVA or a non-parametric test was used for general data based on the normality test and the test for homogeneity of variance. The LSD test was used for the comparisons between groups. In addition, the repeated measurement process o...

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Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

evaluating-anti-depression-effect-xiaoyaosan-on-chronically-stressed

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Pharmacotherapy of Depression and Anxiety Disorders

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9.1K Views.  Beijing University of Chinese Medicine. Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.