We are grateful for the instrumentation and laboratory provided by the Mongolian medical faculty of the Inner Mongolian Medical University, China. This study was supported by the National Natural Sciences Foundation of China (81760762) and the Science and Technology Plan Project of the Health Commission of Inner Mongolia, China (202201300).

All the experimental protocols were approved by the Ethics of Animal Experiment Care Committee of Inner Mongolia Medical University and followed the guidelines of the National Institutes of Health on animal care and ethics. Male Sprague Dawley (SD) rats aged 8 weeks old (200 g &#177; 20 g) were housed in a room with a controlled temperature (22 &#176;C &#177; 2 &#176;C) and humidity (55% &#177; 15%) under a 12 h/12 h regulated light/dark cycle for 1 week. See Figure 1 for the workflow of the network pharmacology analysis.
1. Behavioral test in rats
<ol>
	<li>Establish a CUMS rat model
	<ol>
		<li>Apply the following stimuli combined with isolation for 28 days to all the rats, except for the controls: inversion of the light/dark cycle for 24 h, food deprivation for 24 h, water deprivation for 24 h, high-speed level shaking for 15 min (one time/s), tail clamp for 2 min, swimming in cold water (4 &#176;C) for 5 min, 45 &#176;C heat stimulation, and wet padding for 24 h (Table 1). Raise the rats in individual cages. 
		NOTE: Avoid repeating the same type of stimuli for consecutive days.&#160;&#160;The above procedures to establish a CUMS rat model have been approved by the animal ethics committee and described previously15.</li>
	</ol>
	</li>
	<li>Drug preparation
	<ol>
		<li>Pulverize the Zadi-5 pill in a grinder, and prepare a 1.16 g/mL solution in distilled water. Separately prepare a fluoxetine solution of 0.36 mg/mL in distilled water.</li>
	</ol>
	</li>
	<li>Drug administration
	<ol>
		<li>Divide the rats randomly into six groups (n = 10): control (CON), model (MOD), Zadi-5 group (Zadi-5, 1.6 g of Zadi-5/kg16), fluoxetine group (Fluoxetine, 3.6 mg of fluoxetine/kg). One time per day for 28 days, administer 1 mL/g per rat of the appropriate drug solution by gavage and treat the CON and MOD groups with an equal volume of distilled water. 
		NOTE: Gavage starts at the beginning of model establishment for all the groups.</li>
	</ol>
	</li>
	<li>Open-field test (OFT)
	<ol>
		<li>Divide a black box (50 cm x 50 cm x 30 cm) into nine square regions of equal area. Equip the box with a video tracking analysis system. One day after the last gavage, place the rat in the center square, and record its horizontal and vertical activities for 3 min.</li>
		<li>Score the number of squares crossed with all paws as a horizontal activity, and score standing and grooming as a vertical activity. After each test, clean the box with 75% alcohol to remove the smell of the rat for subsequent tests17.</li>
	</ol>
	</li>
	<li>Sucrose consumption test (SCT)
	<ol>
		<li>Weigh the respective bottles before and after consumption, and calculate the 60 min sucrose preference rates on day 0, day 7, day 14, day 21, and day 28 using equation (1): 
		&#8203;Sucrose consumption = <img alt="Equation 1" src="/files/ftp_upload/64832/64832eq01.jpg" style="margin: auto;" /> &#215; 100%&#160; &#160; &#160;(1)</li>
	</ol>
	</li>
	<li>Morris water maze (MWM)
	<ol>
		<li>Divide the pool into four quadrants. Order the quadrants from one to four, and place the hidden platform in the third quadrant, 1 cm below the water surface.</li>
		<li>Place the rat subject into the maze in different quadrants to look for the platform for 120 s, and record the latency time using the MWM video trail analysis system.</li>
		<li>Place the rat subject in a fixed position in the pool. If the subject cannot find the hidden platform in 120 s, record the latency as 120 s.</li>
		<li>Next, dislodge the platform, place the rat in the water, and record the number of zone-crossings for 120 s.</li>
		<li>Add milk to the pool for some level of opacity. Maintain the water temperature at 23 &#176;C &#177; 1 &#176;C during the experiment.</li>
	</ol>
	</li>
</ol>
2. Network pharmacological prediction
<ol>
	<li>Screen the active components in Zadi-5.
	<ol>
		<li>Browse the Traditional Chinese Medicine Systems Pharmacology (TCMSP, https://old.tcmsp-e.com/tcmsp.php), and input&#160;&#34;Myristicae Semen Seeds,&#34; &#34;Aucklandiae Radix roots,&#34; and &#34;Piperis Longi Fructus&#34; in the &#34;herb name&#34; section to obtain the names of chemicals. Set the pharmacokinetic index of oral bioavailability (OB) to be &#62;30% and the drug-like (DL) index to be &#62;0.18 (Supplementary File 1).</li>
		<li>Search &#34;Rou Dou Kou&#34; (Myristica fragrans Houtt), &#34;Tu Mu Xiang&#34; (Inula helenium L.), &#34;Mu Xiang&#34; (Aucklandia lappa Decne.), &#34;Guang Zao&#34; (Choerospondias axillaris Roxb. Burtt Hill), and &#34;Bi Ba&#34; (Piper longum L.) in the Chinese Medicine Pharmacopeia (http://www.zhongyaocai360.com/zhongguoyaodian/) to identify the chemical names of each component.</li>
		<li>Search the identified chemical names in PubChem (https://pubchem.ncbi.nlm.nih.gov/) to find the isomeric SMILES or InChIkey</li>
	</ol>
	</li>
	<li>Identify the target proteins of the active components in Zadi-5.
	<ol>
		<li>Identify the target proteins of the active components using SEA (http://sea.bkslab.org/), BATMAN (http://bionet.ncpsb.org.cn/batman-tcm/), and Swiss Target Prediction (http://www.swisstargetprediction.ch/) with isomeric SMILES or InChIkey, and find the overlapping proteins.</li>
		<li>Use the protein database UniProt (http://www.uniprot.org/uploadlists/) to convert the identified targets to unified gene names.</li>
	</ol>
	</li>
	<li>Search for the target proteins for depression.
	<ol>
		<li>Search and identify the potential protein targets for depression by using the keywords &#34;depression&#34; and &#34;depressive disorder&#34; in Genecards (https://www.genecards.org/), Disgenet (https://www.disgenet.org/), and Drugbank (https://www.drugbank.com/).</li>
	</ol>
	</li>
	<li>Find the target genes.
	<ol>
		<li>Browse the Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/), upload the targets of the active components of Zadi-5 in List-1, upload the targets for depression in List-2, and submit. Obtain the Venn diagram, and filter out the overlapping target candidates.</li>
	</ol>
	</li>
	<li>Construct the network.
	<ol>
		<li>Construct a spreadsheet called &#34;Type and Network&#34; (Supplementary File 2). &#34;Type&#34; is the network&#39;s signature, and &#34;Network&#34; illustrates the relationship between the signs.</li>
		<li>Export the &#34;Type and Network&#34; to Cytoscape v3.9.0 to construct the network &#34;Zadi-5 herbs-ingredients-disease targets.&#34;</li>
	</ol>
	</li>
	<li>Analyze the protein-protein interaction (PPI) network of the target candidates.
	<ol>
		<li>Set the common targets in the STRING database (https://cn.string- db.org/) to analyze their interactions. Set the&#160;protein type as &#34;homo sapiens.&#34; Set the interaction threshold value to 0.9, and select only the experimentally verified types. Do not display the lonely island nodes.</li>
	</ol>
	</li>
	<li>Conduct a Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of the target-related pathways.
	<ol>
		<li>Paste 86 potential antidepressant targets of Zadi-5 into the start analysis bracket in DAVID (https:// david.ncifcrf.gov/) to study the related signaling pathways by performing the Gene Ontology (GO) function-including biological process, cellular component, and molecular function-and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. 
		NOTE: KEGG is visualized in the bubble chart using an online IMageGP (http://www.ehbio.com /ImageGP/index.php/Home/Index/). The bubble size represents the number of targets enriched in the indicated pathway, and the bubble color represents the enrichment&#8217;s&#160;&#8203;P-value.</li>
	</ol>
	</li>
	<li>Construct the network to illustrate the active compounds in Zadi-5 that interact with the PI3K-AKT signaling pathway.
	<ol>
		<li>Download the KEGG pathway document, select the genes of the PI3K-AKT pathway from the enrichment analysis, and paste them on the spreadsheet to construct a &#34;Type and Network&#34; document.</li>
		<li>Export the &#34;Type and Network&#34; document to the Cytoscape to generate the &#34;PI3K-AKT visualized compounds-targets-pathways network&#34; (Supplementary File 3). 
		&#8203;NOTE: &#34;Type&#34; is the network&#39;s signature, and &#34;Network&#34; illustrates the relationship between the signs.</li>
	</ol>
	</li>
</ol>
3. Statistical analysis
<ol>
	<li>Use a one-way analysis of variance (ANOVA), followed by Duncan&#39;s post hoc test, to determine the significant differences in biochemical and gene expression parameters. Calculate the mean &#177; standard deviation (SD), and visualize the data. Consider P &#60; 0.05 as statistically significant.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Depression is a mental disease characterized by low mood, anhedonia, and a lack of energy. This disorder is accompanied by distraction, cognitive dysfunction, social withdrawal, insomnia, sexual dysfunction, and gastrointestinal diseases18,19. In the study of depression, establishing an animal model is crucial for understanding the pathological mechanisms and effects of new drugs. In this study, a CUMS-induced rat depression model was established through the irritations described in protocol section 2 and Table 1 to simulate stress, attack, and frustration from sources related to society, family, and work. The appropriate sequence and adjustment of the intensities of the stimuli are critical to establishing this depression model. In addition, after each rat test of OFT, the box must be cleaned immediately to avoid the next rat&#39;s track being influenced. CUMS combined with isolation effectively creates a reliable rat depression model20&#160;that simulates the development of depressive symptoms and biochemical signs in human patients. This model has been widely used to explore the pathophysiological mechanisms of depression and evaluate antidepressant drugs under development21,22. Tests such as the OFT, SCT, and MWM are simple, objective, and reasonable ways to assess the rat subjects&#39; appetite, absence of motivation, learning ability, and memory. These tests are recognized as the gold standard for testing animal models of neurodisorders.
In this study, the network pharmacology was based on the TCMSP database of online sites, which collects, stores, and handles data from many available sources, such as websites, books, and academic journals23. It effectively supports the exploration of traditional medicines that have complex mechanisms of action by reducing the time and costs needed for the investigation24,25. In network pharmacology, data are copied to the excel files and clearly classified based on each drug and database. Traditional Mongolian medicine is different from traditional Chinese medicine in that the components of each drug cannot be searched. In this regard, protocol step 2.2.1 lists a valuable database for identifying effective chemical components in this study. As described in protocol step 2.4, determining the overlapping target proteins of Zadi-5 and depression based on three distinct databases could ensure the accuracy and efficiency of the investigation. This represents a key modification of the experiment that is essential for improving the accuracy of the results. The PPI interaction, KEGGs, and GO functional enrichment analyses indicated that the PI3K-AKT signaling pathway plays a vital role in the therapeutic effects of Zadi-5 against depression. The pro-survival kinase-signaling cascade PI3K/AKT pathway is an intracellular signal transduction pathway that exerts a pivotal role in neuron injury through anti-oxidative stress and anti-apoptotic effects26. The PI3K-AKT signaling pathway is closely associated with fundamental cellular functions such as proliferation, survival, differentiation, and protein translation. Additionally, it plays a dominant role in the metabolism of cells in specific organs such as the heart and brain27. It has been reported that the activation of the PI3K-AKT pathway can control neuron functions and protect neurons from oxidative damage by regulating anti-oxidative Bcl-2 proteins to inhibit the generation of reactive oxygen species (ROS)28,29.
This study utilized a behavioral test and network pharmacological prediction to estimate the effects of Zadi-5. In the future, network pharmacology can be used for predicting the bioactive constituents of herbal medicines and their target proteins. Pathological assessment and key molecule tests will be undertaken to characterize the bioactive components of Zadi-5. Behavioral tests will be applied to validate the animals&#39; motivation, appetite, and memory. To ensure the quality of validation, the prediction of the components of Zadi-5 based on online databases will be analyzed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The target proteins will be analyzed by western blot and quantitative polymerase chain reaction (qPCR) analyses30.

Depression, also known as major depressive disorder (MDD), is a severe neuropsychiatric disease responsible for growing medical and economic burdens on society. Due to the associated complexity, morbidity, and mortality rates, a significant amount of research has been conducted to find remedies for the disorder1,2. According to a mental health survey by the World Health Organization, around 350 million people currently suffer from depression and its associated symptoms worldwide. It is predicted that depression will overtake cancer and cardiovascular diseases as the leading cause of disease burden globally by 2030. Thus, the prevention and treatment of depression will become a global priority in the near future3. The pathogenesis of MDD has not yet been elucidated. Still, it is commonly attributed to the following factors: genetic predisposition, dysfunction of the hypothalamus-pituitary-adrenal axis, reductions in neurotransmitter secretion, neuroimmune dysregulation-induced neuroinflammation, cell apoptosis, and reduced cell proliferation4,5. 
Among these factors, neuroimmune dysregulation-induced neuroinflammation and altered secretion of neurotrophic factors have received particular attention for their roles in the development of depression and many other psychiatric diseases6. In the past decade, scholars have demonstrated that the hippocampus is the dominant site for regenerative nerve functions and is involved in regulating emotion and cognition. In this regard, the hippocampal neurons are recognized as novel therapeutic targets for antidepressant medicines under development7,8. Moreover, the hippocampus is also reported to be involved in short-term and long-term memory in learning and consolidating memories. Specifically, the shortage of pyramidal neurons in the CA1 region of the hippocampus causes retrograde and anterograde amnesia9. A typical antidepressant therapeutic strategy aims to enhance cell proliferation and neurogenesis in the dentate gyrus of the hippocampus. Natural product-derived compounds and small molecules synthesized based on medicinal chemistry techniques are considered the primary sources of innovative therapeutic agents for various neuropsychiatric conditions. 
Traditional Mongolian medicines, which have a long history and a well-supported theoretical medical system, have descended from the nomads of the Mongolian plateau These medicines display multi-target and multi-pathway effects due to the various medicinal components that act in concert to generate synergistic functions. Zadi-5 is a well-established formulation among such drugs and was first recorded in "Clinical Experience of Dr. Gao Shi," written by an outstanding Mongolian clinician called Dr. Gao Shi (1804-1876). It has been clinical practice for a long time in Mongolia to use these pills to treat the symptoms of distress, palpitation, irritation, and cardiac stabbing pain10,11. Moreover, Zadi-5 has proven effects on alleviating post-stroke depression in affected patients12. The recent experimental research on CUMS has revealed that the Zadi-5 formulation alleviates depression by regulating the central neurotransmitters13; indeed, with Zadi-5, increased levels of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) have been detected and correlated with improved learning and memory in a rat model of depression14. However, the exact mechanism of action of Zadi-5 for such alleviation of depression has not been elucidated.
This study aimed to demonstrate the therapeutic effects of Zadi-5 against depression in rats using a behavioral test and identify the components of Zadi-5 using Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Swiss Target Prediction to predict the potential mechanisms underlying the efficacy of Zadi-5, a traditional Mongolian medicine, in treating depression.

<table><tbody><tr><td>Cytoscape software</td><td></td><td>&amp;nbsp;version 3.7.0</td><td></td></tr><tr><td>Fluoxetine</td><td>Lilly Suzhou Pharmaceutical Co., Ltd</td><td>J20160029</td><td></td></tr><tr><td>Morris water maze video trail analysing system&amp;nbsp;</td><td>Tai Meng Tech Co., Ltd</td><td>WMT-200</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Beijing Biotechnology Co., Ltd, China</td><td>&amp;nbsp;SCXK (JING) 2016-0002</td><td></td></tr><tr><td>&amp;nbsp;video tracking system</td><td>Tai Meng Tech Co., Ltd</td><td>ZH-ZFT</td><td></td></tr><tr><td>Zadi-5 pill</td><td>Pharmaceutical Preparation Center of International Mongolian Hospital, Inner Mongolia, China</td><td>M1301006</td><td></td></tr></tbody></table>

behavioral and network pharmacology-based analyses for the traditional mongolian medicine zadi-5 in a rat model of depression

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic effects of Zadi-5 against depression have been indicated in previously reported clinical studies, the identity and impact of the active pharmaceutical compounds present in the drug have not been fully elucidated.&#160;This study used network pharmacology to predict the drug composition and identify the therapeutically active compounds in Zadi-5 pills. Here, we established a rat model of chronic unpredicted mild stress (CUMS) and conducted an open field test (OFT), Morris water maze (MWM) analysis, and sucrose consumption test (SCT) to investigate the potential therapeutic efficacy of Zadi-5 in depression. This study aimed to demonstrate Zadi-5&#39;s therapeutic effects for depression and predict the critical pathway of the action of Zadi-5 against the disorder. The vertical and horizontal scores (OFT), SCT, and zone crossing numbers of the fluoxetine (positive control) and Zadi-5 groups were significantly higher (P &#60; 0.05) than those of the CUMS group rats without treatment. According to the results of network pharmacology analysis, the PI3K-AKT pathway was found to be essential for the antidepressant effect of Zadi-5.

Behavioral test in animals 
Results of the behavioral tests in the CUMS-induced rat depression model 
No significant differences between the tested groups were found for the OFT score, sucrose consumption, and MWM analysis before CUMS stimulation. After establishing the CUMS model, the MOD group&#39;s vertical and horizontal scores were lower than those of the CON group (P &#60; 0.05). Compared with the MOD group, the vertical and horizontal scores of the POS and Zadi-5 groups were significantly higher (P &#60; 0.05) (Figures 2A,B).
On day 0, the tested groups had no significant difference in sucrose consumption (%). The sucrose consumption (%) of the Zadi-5 and POS groups was higher than that of the MOD group on day 28 (Figure 2C).
Compared with the CON group, the latency of the MOD group to find the platform in the MWM was significantly higher (P &#60; 0.01). The latency in the POS group was markedly lower than in the MOD group. The latencies of the rats in the Zadi-5 group were lower than in the MOD group but not to a significant degree. Regarding the zone-crossing counts, the MOD group showed fewer crossings than the CON group. The POS and Zadi-5 groups showed more crossings than the MOD group (Figure 2D,E).
Network pharmacology prediction 
There were 134 active components in Zadi-5, and 220 target protein candidates were retrieved for these components. Moreover, 1,000 depression-related protein targets were predicted. According to the Venn diagram analysis, 86 overlapping targets were identified as the critical depression-related targets of Zadi-5 (Figure 3). Based on these findings, &#34;Zadi-5 herbs-ingredients-disease targets&#34; and a PPI network analysis of the target candidates were constructed (Figure 4A,B, Figure 5, and Table 2). The PI3K-AKT pathway showed relatively irregular edges, indicating that this pathway is essential for the antidepressant effect of Zadi-5. According to the KEGG pathway analysis, the PI3K-AKT signaling pathway was ranked seventh (Figure 6) and was associated with many signaling pathways. Accordingly, it was considered relatively more important than the other enrichment pathways (Figure 7A-C and Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64832/64832fig01.jpg" /> 
Figure 1: Workflow of the network pharmacology analysis for in vivo validation. Abbreviations: PPI = protein-protein interaction; KEGG = Kyoto Encyclopedia of Genes and Genomes; GO = Gene Ontology; OFT = open-field test; MWM = Morris Water Maze; SCT = sucrose consumption test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64832/64832fig02.jpg" /> 
Figure 2: Effects of Zadi-5 on the CUMS-induced rat depression model. (A) OFT vertical scores. (B) OFT horizontal scores. (C) Sucrose consumption level (%) on day 0 and day 28. (D) Latency time for the MWM test. (E) Zone-crossing numbers for the MWM test. ##P &#60; 0.01 indicates that the CUMS MOD group showed significant differences compared with the CON group. *P &#60; 0.05 indicates that the POS group and Zadi-5 group showed significant differences compared with the MOD group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64832/64832fig03.jpg" /> 
Figure 3: Venn diagram of the protein targets of Zadi-5 and depression-associated proteins. The red circle represents the targets of the active components in Zadi-5, while the blue circle represents the proteins associated with depression. The intersection of the two colors represents the overlapping proteins that can be identified as the therapeutic targets for the alleviation of depression by Zadi-5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64832/64832fig04.jpg" /> 
Figure 4: Construction of the &#34;Zadi-5 herbs-ingredients-disease targets network.&#34; The red parallelograms represent Zadi-5 and its herbs, while the pink, orange, yellow, green, and purple circles represent the components of each herb. The blue diamonds represent the proteins associated with depression, which is the green hexagon. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64832/64832fig05.jpg" /> 
Figure 5: PPI of the overlapping protein targets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64832/64832fig06.jpg" /> 
Figure 6: The bubble chart for the top 20 KEGGs-enriched terms based on the 86 overlapping protein targets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64832/64832fig07.jpg" /> 
Figure 7: The key bioinformatics results for the Zadi-5 components and depression-associated protein targets. (A) The top 20 overlapping molecular functions GO terms of Zadi-5 and depression. (B) The top 20 overlapping cellular components GO terms of Zadi-5 and depression. (C) The top 20 overlapping biological processes GO terms of Zadi-5 and depression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64832/64832fig08.jpg" /> 
Figure 8: The Zadi-5 ingredients-PI3K-AKT signaling pathway enrichment targets network for the treatment of depression. The circle represents the ingredients of Zadi-5, the hexagons indicate each drug of Zadi-5, and the green diamonds represent the enriched targets of the PI3K-AKT signaling pathway. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequence and methods of application of stress stimuli for the induction of CUMS in rat subjects. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: The ID codes used in Figure 2. Abbreviations: MF = Myristica Fragrans Inula IH = Innula helenium L.; FC = Fructus Choerospondiatis; AL = Aucklandia lappa Decne.; PL = Piper Longum L. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Screenshot images of the network pharmacology protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832_supplementary file1.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Zadi-5 components-proteins-depression network. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832_supplementary2 network.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Type of network. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64832/64832_supplementary3 type.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression at JoVE.com

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

The present protocol describes a method for the behavioral test validation and bioinformatical prediction of the therapeutic efficacy of Zadi-5, a traditional Mongolian medicine, in depression.

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic ...

behavioral-network-pharmacology-based-analyses-for-traditional

Nanyang Technological University

Research

JoVE Journal

Medicine

448 Views.  Inner Mongolia Medical University. De therapeutische werkzaamheid van Zadi-5 bij de behandeling van depressie kan worden vastgesteld door middel van gedragstests, terwijl netwerkfarmacologie de mogelijke mechanismen kan voorspellen die ten grondslag liggen aan de potentie ervan. Netwerkfarmacologie kan de verkenning van traditionele geneesmiddelen ondersteunen die ingewikkelde werkingsmechanismen vertonen en de tijd en kosten die nodig zijn voor onderzoek verder minimaliseren. Tests zoals de open veldtest, sucroseconsumptietes...

Video: Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

Behavior

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

Neuroscience

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

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.

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 ...

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...

Developing a Rat Model for Bipolar Disorder

Bipolar disorder is a mental health condition characterized by extreme mood swings, including periods of emotional highs (mania) and lows (depression). While the exact underlying neurobiology is not yet fully understood, imbalances in neurotransmitter systems, particularly dopamine, appear to play a central role. For this reason, manipulations of dopaminergic pathways have been used to model mania or depression in rodents. However, models that accurately represent the typical switch between these two episodes are rare, limiting face validity. In a unique model, modern techniques are used to temporarily increase dopamine D1 receptor expression, which has been implicated in the pathology of bipolar disorder. A tetracycline-inducible lentiviral construct that expresses the dopamine D1 receptor under the control of the calmodulin kinase II alpha promoter is stereotactically injected into the medial prefrontal cortex of adult rats. Dopamine D1 receptor overexpression is achieved by adding the tetracycline analog doxycycline to the animals&#39; drinking water, leading to an increase in reward-related, impulsive, and risk-taking behaviors and a decrease in anxiety. These behaviors resemble a mania-like phenotype. By removing doxycycline from the drinking water, a depressive-like phenotype, characterized by increased helplessness and anhedonia, can be induced within the same animal. This article provides a step-by-step protocol for performing the surgery, as well as procedures for inducing the bipolar disorder-like phenotype. Additionally, considerations for assessing behavioral changes associated with mania-like and depressive-like behavior are described. This promising model, which demonstrates good construct and face validity, offers a valuable tool for further investigating the pathophysiological mechanisms of bipolar disorder.

This article presents a protocol for the induction of a unique rat model of bipolar disorder that captures both mania-like and depressive-like behavior.

Bipolar disorder is a mental health condition characterized by extreme mood swings, including periods of emotional highs (mania) and lows (depression). ...

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

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