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

<ol>
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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 ...

This method can help answer key questions in the field of depression, prevention and treatment using traditional Chinese medicine such as Xiaoyaosan, a widely used formula in the psychological disease for thousands of years in China. The main advantage of this technique is to provide an experimental support for the availability of Xiaoyaosan in the prevention and treatment of depression. The implications of this technique extend toward pre-clinic research on the treatment of depression because it provides a standardized protocol for sensing the anti-depressant effect using traditional Chinese medicine.Generally, individuals new to this method will struggle because the modeling process is very complicated and the important measurement parameters for depressive behaviors is not easy to master. To induce depression, expose mice daily to any 2 of the following 7 stressors for 21 consecutive days. Note administer medicine to the mice in the two treatment groups every day before modeling and provide 0.5 milliliters of distilled water to the mice in the control and model groups.Expose mice to food deprivation by emptying the feed of each cage. Or water deprivation by removing the water bottle for 24 hours. Expose mice to restraint stress, where the mouse is restrained in a small plastic tub for 3 hours.Or an empty cage with the padding removed. As another option, expose mice to wet and soiled cages by pouring water into each cage and keeping wet for 24 hours. Expose mice to crowded cages in which five mice are placed in one cage for 25 hours.For the last option, expose mice to ice cold swimming by forcing each mouse to swim in a clear glass aquarium with ice water for five minutes. Finally, record the coat state and body weight weekly until the end of modeling at day 21 and calculate the total score of the coat state. On the first two days of the sucrose preference test, house the mice individually with two bottles filled with tap water.For the next two days, give the mice two bottles filled with 1%sucrose solution. On the last day, give the mice two bottles for 24 hours, 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 hours.Record the volume of both the consumed sucrose solution and tap water. Lastly, calculate the sucrose preference. Begin by dividing the floor of a four sided wooden black lusterless box into 25 equal squares by blue lines.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. Suspend a high definition digital camera approximately 200 centimeters right above the field and record the behavior of each mouse for 5 minutes.Finally, evaluate and analyze 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. The coat state of mice was notably improved after continuous treatment with Xiaoyaosan or Fluoxetine compared to the model group. A significant reduction in body weight was manifested by the third week.At baseline, mice in all groups had a similar sucrose preference. However, a significant drop in sucrose preference was observed after 3 weeks of stress procedure in the model group. Further, all mice exhibited a similar behavioral state before stress exposure at baseline.However, after stress exposure for 3 weeks, the total distance moved in 5 minutes was significantly lower in the model group than the control group. The number of entries into the central zone of the model group significantly decreased as well compared with the control group. While attempting this procedure, it's important to remember to conduct this behavior test as well as administer the two medicine, 1 to 2 hours before daily modeling in order to confirm the effects by the medicine itself.Following this procedure, other methods like forced swimming test can be performed in order to answer additional questions like can Xiaoyaosan improve the desperate behavior of depressive mice in the forced swimming test? After it is development, this technique paved the way for researchers in the exploration of the effect and underlying mechanism of Chinese medicine in the field of prevention and treatment of depression.

Research

JoVE Journal

Behavior

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