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...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+antagonists+augment+antidepressant-like+effects+of+lithium+in+the+mouse+forced+swimming+test.">NMDA receptor antagonists augment antidepressant-like effects of lithium in the mouse forced swimming test.</a> Journal of Psychopharmacology. 24 (4), 585-594 (2010).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tea+attenuates+chronic+unpredictable+mild+stress-induced+depressive-like+behavior+in+rats+via+the+gut-brain+axis.">tea attenuates chronic unpredictable mild stress-induced depressive-like behavior in rats via the gut-brain axis.</a> Nutrients. 14 (1), 99 (2021).</li><li>Kandola, A., Ashdown-Franks, G., Hendrikse, J., Sabiston, C. M., Stubbs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity+and+depression:+Towards+understanding+the+antidepressant+mechanisms+of+physical+activity.">Physical activity and depression: Towards understanding the antidepressant mechanisms of physical activity.</a> Neuroscience and Biobehavioral Reviews. 107, 525-539 (2019).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+butyricum+attenuates+chronic+unpredictable+mild+stress-induced+depressive-like+behavior+in+mice+via+the+gut-brain+axis.">Clostridium butyricum attenuates chronic unpredictable mild stress-induced depressive-like behavior in mice via the gut-brain axis.</a> Journal of Agricultural and Food Chemistry. 66 (31), 8415-8421 (2018).</li><li>Willner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+mild+stress+(CMS)+revisited:+Consistency+and+behavioural-neurobiological+concordance+in+the+effects+of+CMS.">Chronic mild stress (CMS) revisited: Consistency and behavioural-neurobiological concordance in the effects of CMS.</a> Neuropsychobiology. 52 (2), 90-110 (2005).</li><li>Albrakati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+efficiency+of+prodigiosins+conjugated+with+selenium+nanoparticles+in+rats+exposed+to+chronic+unpredictable+mild+stress+is+mediated+through+antioxidative,+anti-inflammatory,+anti-apoptotic,+and+neuromodulatory+activities.">Neuroprotective efficiency of prodigiosins conjugated with selenium nanoparticles in rats exposed to chronic unpredictable mild stress is mediated through antioxidative, anti-inflammatory, anti-apoptotic, and neuromodulatory activities.</a> International Journal of Nanomedicine. 16, 8447-8464 (2021).</li><li>Chan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+practice+in+reviewing+and+publishing+studies+on+herbal+medicine,+with+special+emphasis+on+traditional+Chinese+medicine+and+Chinese+materia+medica.">Good practice in reviewing and publishing studies on herbal medicine, with special emphasis on traditional Chinese medicine and Chinese materia medica.</a> Journal of Ethnopharmacology. 140 (3), 469-475 (2012).</li><li>Su, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+the+mechanism+of+Lianhua+Qingwen+in+treating+influenza+virus+pneumonia+and+new+coronavirus+pneumonia+with+the+concept+of+&amp;#34;different+diseases+with+the+same+treatment&amp;#34;+based+on+network+pharmacology.">Exploration of the mechanism of Lianhua Qingwen in treating influenza virus pneumonia and new coronavirus pneumonia with the concept of &amp;#34;different diseases with the same treatment&amp;#34; 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Redox Signaling. 18 (10), 1208-1246 (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salidroside,+a+phenyl+ethanol+glycoside+from+Rhodiola+crenulata,+orchestrates+hypoxic+mitochondrial+dynamics+homeostasis+by+stimulating+Sirt1/p53/Drp1+signaling.">Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.</a> Journal of Ethnopharmacology. 2022, 115278 (2022).</li></ol>

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 irri...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cytoscape software</td>
			<td></td>
			<td>&#160;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&#160;</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>&#160;SCXK (JING) 2016-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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 ...

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 ...

The therapeutic efficacy of Zadi-5 in treating depression can be established through behavioral tests, while network pharmacology can predict the potential mechanisms that underlie its potency. Network pharmacology can support the exploration of traditional medicines that display intricate mechanisms of action and further minimize the time and expenses required for investigation. Tests such as the open field test, sucrose consumption test, and the Morris Water Maze are the gold standard for evaluating appetite, motivation levels, learning ability, and memory, in animal models of neurological disorders.These tests are simple, objective, and reasonable methods for assessment. Establishing an animal model is crucial for comprehending new drug's pathological mechanisms and effects in a depressive disorder study. To establish a good depression model, researchers must appropriately sequence and adjust the stimuli intensities.To begin, apply the selected depression stimuli, combined with isolation, for 28 days to all the rats, except for the controls, and raise the rats in individual cages. To perform the open field test, divide a black box 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 three minutes. Then score the number of squares crossed with all pause 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 tests.Next, perform the sucrose consumption test by weighing the respective bottles before and after consumption. And calculate the 60 minute sucrose preference rates on days zero, seven, 14, 21, and 28 using this equation. To perform the Morris Water Maze Test, divide the pool into four quadrants.Order the quadrants from one to four, and place the hidden platform in the third quadrant, one centimeter below the water surface. Add milk to the pool for some level of opacity. Then place the rat subject into the maze in different quadrants to look for the platform for 120 seconds.And record the latency time using the Morris Water Maze video trail analysis system. Place the rat subject in a fixed position in the pool. If the subject cannot find the hidden platform in 120 seconds, record the latency as 120 seconds.Next, dislodge the platform, place the rat in the water, and record the number of zone crossings for 120 seconds. Browse the traditional Chinese medicine system's pharmacology webpage and input myristicae semen seeds, al-glandia-radiks roots, and piperis longi fructus in the herb name section to obtain the names of chemicals. Set the pharmacokinetic index of oral bioavailability, or OB, to be greater than 30%and the drug-like index to be greater than 0.18.Search the active components in the Chinese medicine pharmacopeia to identify the chemical names of each component. Next, search the identified chemical names in Pub Chem to find the isomeric smiles, or inky. Identify the target proteins of the active components using SEA, Batman, and Swiss target prediction with isomeric smiles or inky, and find the overlapping proteins.Copy the data to an Excel file. Next search and identify the potential protein targets for depression by using the keywords depression and depressive disorder in GeneCards, DisGeNET, and DrugBank. Copy the data to an Excel file.Browse the Venn diagram and upload the targets of depression in list one. Then upload the targets for the active components of Zadi-5 in list two and submit. Obtain the Venn diagram and filter out the overlapping target candidates.Construct a spreadsheet, called network. Also construct a spreadsheet, called type. Export the sheet to Cytoscape to construct the network, Zadi-5 herbs ingredients disease targets.Set the common targets in the string database to analyze their interactions. Set the protein type as homosapiens. Set the interaction threshold value to 0.9 to select only the experimentally verified types, and do not display the lonely island nodes.Open the DAVID tool and paste the 86 potential antidepressant targets of Zadi-5 into the start analysis bracket to study the related signaling pathways. Download the keg pathway document. Select the genes in the PI3k-AKT pathway from the enrichment analysis and paste them on the spreadsheet to construct a type and network document.Export the type of network document to Cytoscape to generate the PI3K-AKT visualized compound targets pathways network. The tested animal group showed no significant differences before chronic unpredicted mild stress or CUMS stimulation. After establishing the CUMS model, the group's vertical and horizontal scores were lower than the control.Compared with the model group, the vertical and horizontal scores of the positive control and Zadi-5 groups were significantly higher. On day zero, the tested groups had no significant difference in sucrose consumption. The Zadi-5 and positive control groups were higher than the model group on day 28.In the Morris Water Maze, the latency of the model group to find the platform was significantly higher when compared with the control. The latency of the positive control and Zadi-5 groups was lower than in the model group. Regarding the zone crossing counts, the model group showed fewer crossings than the control.The positive control and Zadi-5 groups showed more crossings than the model group. The Venn diagram analysis revealed 86 overlapping targets as the critical depression related targets of Zadi-5 of the 1000 depression-related protein targets. Based on these findings, Zadi-5 herbs ingredients disease targets, and a protein-protein interaction network analysis of the target candidates were constructed.According to the KEG pathway analysis, the PI3K-AKT signaling pathway was ranked seventh. It was associated with many signaling pathways. Accordingly, the PI3K-AKT pathway was considered relatively more important than the other enrichment pathways.While attempting this procedure, it's crucial to refrain from repeating the same type of stimuli for consecutive days. Following the analyses of the components of Zadi-5 in the online databases, the quality of validation can be insured by liquid chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy.

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JoVE Journal

Medicine

361 visualizações.  Inner Mongolia Medical University. A eficácia terapêutica do Zadi-5 no tratamento da depressão pode ser estabelecida através de testes comportamentais, enquanto a farmacologia de rede pode prever os potenciais mecanismos subjacentes à sua potência. A farmacologia em rede pode apoiar a exploração de medicamentos tradicionais que apresentam intrincados mecanismos de ação e minimizar ainda mais o tempo e os gastos necessários para a investigação. Testes como o teste de campo aberto, o teste de consumo de sacarose e o...