Name: Network Pharmacology and Validation of the Antidepressant Mechanisms of Qiangzhifang in a Chronic Restraint Stress-induced Depression Rat Model (Video) | JoVE Journal
Uploaded: 2025-06-06T15:25:00.000+00:00
Duration: 8 min 15 s
Description: Discover how Qiangzhifang exerts antidepressant effects in a chronic restraint stress rat model through network pharmacology and molecular docking analysis of HIF-1 and JAK-STAT pathways.

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The research was supported by the National Natural Science Foundation of China (82374311), the State Administration of Traditional Chinese Medicine High-Level Traditional Chinese Medicine (TCM) Basic Theory Key Discipline Construction Project (zyyzdxk-2023118), the National Traditional Chinese Medicine Experts Studio Construction Project (National Chinese Medicine Education Letter No.75) and the Natural Science Foundation of Shandong Province (ZR2022LZY016). QZF granules were prepared by the Department of Pharmaceutical Products, Affiliated Hospital of Shandong University of Traditional Chinese Medicine.

All experimental protocols were approved by the Animal Experiment Ethics Committee of Shandong University of Traditional Chinese Medicine (approval number: YYLW2023000327) and complied with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health. In this experiment, we used 40 healthy male Wistar rats, SPF grade, with an average body weight of (140 ± 10) g (Figure 1). See the Table of Materials for a list of all the materials, equipment, and software used in this protocol.
1. Rat depression model
<ol>
	<li>Animal housing and grouping
	<ol>
		<li>Enter the breeding room with the animals and number them using a tail marking instrument.</li>
		<li>House the rats individually in cages, maintaining a temperature of 21 ± 2 °C and a 12 h/12 h light/dark cycle.</li>
		<li>Acclimate the rats in the laboratory for 7 days, providing ad libitum access to food and water while handling them daily for adaptation.</li>
		<li>After the acclimation period, measure the body weight, and conduct the sucrose preference tests (SPT) and open field tests (OFT).</li>
		<li>Based on the experimental data, divide the rats into four groups, ensuring each group consists of 10 rats: the control (CON) group, the model (CRS) group, the QZF group, and the fluoxetine (F) group.</li>
	</ol>
	</li>
	<li>Establishment of a Chronic Restraint Stress (CRS) rat model
	<ol>
		<li>Construct the rat restraint device. Select a transparent plastic tube with a diameter and length suitable for the rat's size21, allowing the rat to stand and turn inside while preventing escape. Use an electric soldering iron hole puncher to create holes on the sides of the plastic tube and on the lid to ensure proper air circulation.</li>
		<li>Gently place the rats in the restraint devices (except for those in Group C) 1 h after daily intragastric administration of the drug, ensuring they are in a comfortable position.</li>
		<li>Deprive all groups of rats of food and water during the restraint period. After the restraint period ends, provide them with ample food and water uniformly. Fix the daily restraint duration at 6 h (from 9:30 h to 15:30 h) and maintain it for 28 consecutive days.</li>
	</ol>
	</li>
</ol>
2. Drug intervention
<ol>
	<li>Administer via gavage: 1 mL of solution/100 g of body weight, fluoxetine (2.7 mg·kg-1·day-1) and QZF (2 g·kg-1·day-1)22. Provide Groups C and CRS with equivalent normal saline for single-variable control. 
	NOTE: Daily drug administration was conducted at 08:00 h, initiating synchronously with model establishment and persisting throughout the 28-day modeling period.</li>
</ol>
3. Sucrose preference test (SPT)
<ol>
	<li>Deprive the rats of food and water for 24 h before the experiment begins.</li>
	<li>Prepare a 1% aqueous sucrose solution and fill the solution and pure water into the drinking bottles of the experimental animals for weighing. Measure the consumption of pure water and sucrose water by weighing the bottles before and after the experiment.</li>
	<li>Place one bottle of sucrose solution and one bottle of pure water at the water intake of each rat cage cover, one on the left and one on the right, for free access to drinking. To prevent rats from favoring one side for water intake, switch the positions of the water bottles on the left and right after 30 min into the experiment.</li>
	<li>After 1 h of the experiment, remove all water bottles, weigh them promptly, and record the consumption of sucrose solution and pure water. Calculate the weekly sucrose preference ratio using the formula: 
	Sucrose preference value = <img alt="Sucrose-water consumption ratio formula; numerator and denominator explain solution and water use." src="/files/ftp_upload/68198/68198eq001.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 1"> × 100%</li>
</ol>
4. Body weight measurement
<ol>
	<li>Weigh the rats weekly upon their entry into the laboratory and set the fixed time for weighing at 7:00 AM.Establish this schedule to facilitate the observation of body weight changes.</li>
</ol>
5. Open-field test (OFT)
<ol>
	<li>Before the experiment starts, acclimate the rats to the behavioral room for 1 h and adjust the lighting in the open field box to ensure even distribution. Confirm that the rats are clearly visible in the tracking software.</li>
	<li>Utilize the video tracking and analysis system to divide the bottom surface of the open field box (50 cm x 50 cm x 50 cm) into nine equal-area square grids. Designate the eight grids adjacent to the walls as the peripheral area and the central grid as the central area.</li>
	<li>Place the rat in the central area of the open field box. Record the rat's movement for 5 min using the video tracking system.</li>
	<li>After testing each rat, clean the chamber with 75% ethanol to remove residual odor and prevent interference with the behavior of the next rat. Enter the total distance (mm) of open field activities and the number of entries in the central grid in the OFT records.</li>
</ol>
6. Forced swimming test (FST)
NOTE: The rat forced swimming experiment comprises a pre-experiment and a formal experiment. Conduct the pre-experiment 24 h prior to the formal experiment, following the same procedure, with the rat swimming for 15 min.
<ol>
	<li>Transport the experimental animals to the behavioral room at least 30 min before the experiment to allow them to acclimate to the environment.</li>
	<li>Prepare a transparent cylindrical plexiglass water cylinder (50 cm high, 20 cm diameter) and fill it with water at 23-25 °C. Adjust the water depth based on the animal's weight, ensuring the animal's tail remains a certain distance from the bottom of the cylinder.</li>
	<li>Slowly place the rats into the water cylinder and keep quiet throughout the experiment. Activate the camera and signal acquisition system. Observe and record the duration of floating immobility within 300 s. Immediately remove the rats from the water and dry them at the end of the experiment.</li>
	<li>After each session, replace the water to prevent any influence on the next rat.</li>
</ol>
7. Network pharmacological prediction
<ol>
	<li>Collection of QZF compounds and putative targets
	<ol>
		<li>Access the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://old.tcmsp-e.com/)23, the HERB database24, and the TCMID database (https://www.bidd.group/TCMID/). Use the eight TCM names in QZF, including GZ, YZ, YZR, BS, CBM, RS, HJT, and ZGC, as keywords to search for active compounds and targets of the herbs. Collect targets from the TCMSP and Swiss target prediction (http://www.swisstargetprediction.ch/). Set the filter value to Probability* &gt; 0. 
		NOTE: Typically, ingredients were included as active ingredients based on their pharmacokinetic characteristics: oral bioavailability (OB) ≥ 30% and drug-like characteristics (DL) ≥ 0.1825.</li>
	</ol>
	</li>
	<li>Prediction of disease targets
	<ol>
		<li>Search for the keyword "depression" in the GeneCards database (https://www.genecards.org/), obtain gene targets associated with depression, download the electronic spreadsheet of disease targets, filter the gene scores that are higher than the average value, and compile a list of depression targets26.</li>
	</ol>
	</li>
	<li>Drug-component-disease-target network
	<ol>
		<li>Create a new spreadsheet and populate it with depression-related targets and drug targets in the same column.Click on Start in the menu bar | Conditional Formatting | Highlight Cells Rules | Duplicate Values. Select a format (for example, "Light Red Fill") in the dialog box that appears27, click OK to view the results.</li>
		<li>Launch the network analysis software and import the spreadsheet file by clicking on File in the menu bar | Import | Network. Optimize the appearance of the network by adjusting the size and color of nodes in the Style panel located in the left control panel. Perform network topology analysis by clicking on Tools in the menu bar | Analyze Network27.</li>
	</ol>
	</li>
	<li>Protein-protein interaction (PPI) network
	<ol>
		<li>Access the Jvenn (https://jvenn.toulouse.inrae.fr/app/example.html) tool, upload the compound targets and disease targets separately, plot the Overlapping Genes (OGE) between the compound presumed targets and disease targets.Click on the numbers in the image and copy them into a spreadsheet and download the Venn diagram image.</li>
		<li>Access the STRING database (https://stringdb.org/)28, and enter the OGEs from the spreadsheet into the database. Specifically, paste the QZF anti-depression overlapping target list into the List of Names dialog box. Select Homo sapiens in the Organisms section and click on SEARCH | CONTINUE. Select the Exports option from the title bar and download the summary table of the PPI network in both PNG and TSV formats29.</li>
	</ol>
	</li>
	<li>Screening of core proteins
	<ol>
		<li>Start the network analysis software (https://cytoscape.org/). Then, in the menu bar, click on File | Import | Network | File to import the TSV format file generated in the previous step30.</li>
		<li>Select Analyze Network in the menu bar and click the Analyze button. Then, view the analysis results and understand the overall structural characteristics of the network, such as the number of nodes, the number of edges, and the average degree.</li>
		<li>Select Apps | App Manager in the menu bar. Search for MCODE, install the plug-in, and run the plug-in to get the hub target. Then, search for CytoNCA, install the plug-in, and focus on the three parameter values of Degree, Closeness centrality (CC), and Betweenness centrality (BC). According to the values of these parameters, screen Nodes with higher Degree, CC, and BC, which are generally considered core proteins31.</li>
	</ol>
	</li>
	<li>Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
	<ol>
		<li>Open the bioinformatics platform (https://www.omicshare.com/). Click on the Tools menu, find the ID gene conversion tool, and click on it. Then, click the upload file button, select the generated step on the core of the target genes, and download the converted file ID list.</li>
		<li>In the Tools menu, click on the Dynamic KEGG enrichment Analysis tool. Upload the gene ID list. In the Species option, select Homo sapiens and click the Submit button.</li>
		<li>In the Tools menu, click on the Dynamic GO enrichment Analysis tool | Gene option | Upload file option. Select the gene ID list and in the Species option, select Homo sapiens. Select the GO type for analysis, including Biological Process, Molecular Function, and Cellular Component.</li>
		<li>For the results of KEGG and GO enrichment analysis, set the filtering threshold as p &lt; 0.05. Arrange the counts in descending order.</li>
	</ol>
	</li>
</ol>
8. Molecular docking verification
<ol>
	<li>Visit the PubChem (https://pubchem.ncbi.nlm.nih.gov/) website. Enter the target compounds in the search bar. Click on the 2D Structure and download it.</li>
	<li>Open the PDB (https://www.Rcsb.org/) database. Select the crystal structure with high resolution and containing the original ligand. Download the PDB file.</li>
	<li>For optimizing the protein structure, open the molecular visualization software. Load the downloaded PDB file. Remove water molecules and save the optimized PDB file.</li>
	<li>Open the molecular docking software and import the optimized PDB file. In AutoDockTools, click on Edit | Delete Water to delete water molecules. Click on Edit | Add Hydrogens | Add to add hydrogen atoms to the protein and ligand29.</li>
	<li>In AutoDockTools, set the receptor bar to receptor.pdbqt and the ligand bar to ligand.pdbqt. Open the pdbqt. file to view the binding sites of the protein and ligand and set the size and position of the docking box to ensure that it can completely enclose the receptor protein and the ligand compound. In AutoDockTools, click on Grid | Define Grid Box to set the center coordinates and dimensions of the box and use default values for molecular docking. The docking frames will be automatically sorted in descending order of binding energies.</li>
	<li>Open the result file and record the optimal binding energy value. Lower binding energies indicate more stable binding. Use the molecular visualization software to load the result file. Adjust the view and color to clearly display the ligand-receptor interaction.</li>
</ol>
9. Statistical analysis
<ol>
	<li>Conduct statistical analysis in the scientific data analysis and visualization software and represent all data as the mean ± SEM. Use repeated measures two-way ANOVA for comparisons between groups before and after drug administration. Employ one-way ANOVA for comparisons among more than two groups.</li>
	<li>Take the value of P &lt; 0.05 as statistically significant.</li>
</ol>

The authors have no conflicts of interest to declare.

CRS is a widely used method for establishing animal models of depression. This model mimics the chronic psychological stress encountered in human life and induces depression-like behaviors in rats35. In this study, the rat restraint tube was constructed from transparent plastic, ensuring animal safety while enabling clear observation during the experiment. The transparent tube measured approximately 18 cm in length and 6 cm in diameter and featured multiple ventilation holes, each with a diameter ...

Depression, a pervasive global health challenge, is characterized by a persistent low mood, reduced interest and pleasure, and cognitive and neurological impairments1. As reported by the World Health Organization, depression impacts approximately 380 million people worldwide, and this figure is expected to increase2. As a complex, multifactorial mental disorder, depression affects patients&#39; quality of life and poses a considerable economic and medical burden on society, characterized by high incidence, recurrence rates, and disability rates3.
The etiology of depression is complex, with the precise mechanisms not yet fully understood. As research in this field progresses, factors such as neuroinflammation, oxidative stress, and apoptosis have garnered significant attention. Studies indicate that patients with depression exhibit elevated levels of pro-inflammatory cytokines like TNF and interleukin-1&#946; compared to healthy individuals, and a higher prevalence of depression is observed in those with inflammatory conditions4. In oxidative stress, reactive oxygen species (ROS) are overproduced in response to harmful stimuli, overwhelming the body&#39;s antioxidant defenses and leading to an imbalance between oxidative and antioxidant systems, thereby causing tissue damage. Elevated oxidative stress in depression can enhance lipid peroxidation and exacerbate damage to cellular genes and proteins, impacting neuronal function and contributing to neuronal degeneration, apoptosis, and impaired plasticity5. Additionally, the alterations observed in clinical presentations, biochemical markers, and brain structures in patients with depression are linked to apoptosis. Imaging studies reveal reduced hippocampal volume and atrophy in patients with depression, with neuronal apoptosis potentially playing a pivotal role in these changes6.
Currently, drug treatment is the primary approach for managing depression, with selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors (NRIs) being frequently employed in clinical practice7. However, these drugs are accompanied by significant adverse effects. In addition to central nervous system symptoms like headache and insomnia, most antidepressants also commonly exhibit gastrointestinal side effects, including nausea and diarrhea8,9. Some antidepressants can also cause sexual dysfunction10, which severely impacts treatment outcomes and reduces medication adherence among patients with depression11. Moreover, the efficacy of these drugs is limited for some patients. Recent metabolomics studies have indicated that individual differences in gut microbiota may influence drug efficacy12. Therefore, the development of safer and more effective treatments remains a critical focus in depression research.
Traditional Chinese medicine (TCM) formulations have demonstrated significant potential in treating depression, attributed to their synergistic effects involving multiple components, targets, and pathways13. TCM posits that vigorous Yang qi is essential for maintaining the body&#39;s vitality. Therefore, Professor Yuanqing Ding, leveraging the unique principles of TCM diagnosis and treatment and extensive clinical experience, proposed that &#34;yang yu shen tui&#34; is the fundamental pathogenesis of depression. Based on this concept, he developed Qiangzhifang (QZF) to specifically address this pathogenesis14. The clinical application of QZF in treating depression has demonstrated significant efficacy, with a total effective rate of 71.43%15. QZF is composed of various traditional Chinese medicinal materials, including Ramulus cinnamomi (gui zhi, GZ), Polygala tenuifolia (yuan zhi, YZ), Alpinia oxyphylla miq (yi zhi ren, YZR), Paeonia lactiflora (bai shao, BS), Fritillariae cirrhosae bulbus (chuan bei mu, CBM), Panax ginseng (ren shen, RS), Rhodiola rosea L (hong jing tian, HJT), and licorice (gan cao, GC) (Supplemental File 1). Studies have shown that Polygala tenuifolia is rich in saponins andexhibits neuroprotective effects16. Similarly, the Ramulus Cinnamomi-Paeonia lactiflora herb pair demonstrates potential efficacy in alleviating pain and depression17. Additionally, ginseng&#39;s total saponins can reduce hippocampal proinflammatory cytokine levels, improve depressive behavior, and attenuate hippocampal nerve damage in rats18. Licorice mainly contains triterpenoids and flavonoids. Licorice&#39;s total flavonoids (LF) can play an antidepressant role by improving depressive behavior, modulating the BDNF/TrkB signaling pathway, and enhancing synaptic plasticity19. However, the specific mechanisms underlying the antidepressant effects of QZF remain unclear, thereby limiting its widespread application.
Therefore, our study aims to establish a CRS depression rat model, demonstrate the therapeutic effect of QZF on depression in rats through behavioral experiments, and systematically evaluate the antidepressant mechanism of QZF using network pharmacology and molecular docking technology20. By clarifying the active components and potential targets of QZF, the core targets of depression can be accurately located. We believe that by deeply exploring the mechanism of action of QZF, we can not only provide safer and more effective treatment options for patients with depression but also provide a scientific basis for the application of TCM in the treatment of depression.

<table><tbody><tr><td>Animal behavior analysis system</td><td>Shanghai Xinsoft Information Technology Co., LTD</td><td>XR-SuperMaze</td><td></td></tr><tr><td>AutoDockTools</td><td>The Scripps Research Institute</td><td></td><td></td></tr><tr><td>Cytoscape&amp;nbsp; software</td><td>Cytoscape Consortium</td><td>version 3.7.2</td><td></td></tr><tr><td>Electric soldering iron hole puncher</td><td>Nanjing Naiwei Technology Co., Ltd.</td><td></td><td></td></tr><tr><td>Fluoxetine</td><td>Lilly Suzhou Pharmaceutical Co., LTD</td><td></td><td></td></tr><tr><td>Open field experimental system</td><td>Shanghai Xinsoft Information Technology Co., LTD</td><td>XR-XZ301</td><td></td></tr><tr><td>PyMol</td><td>Schr&amp;ouml;dinger</td><td></td><td></td></tr><tr><td>Qiangzhifang&amp;nbsp;</td><td>Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China</td><td></td><td></td></tr><tr><td>Transparent plastic tube&amp;nbsp;</td><td>Nantong Baiyang Plastic Products Co., Ltd.&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

network pharmacology and validation of the antidepressant mechanisms of qiangzhifang in a chronic restraint stress-induced depression rat model

Network Pharmacology and Validation of the Antidepressant Mechanisms of Qiangzhifang in a Chronic Restraint Stress-induced Depression Rat Model

Depression is a complex psychiatric disorder that poses significant treatment challenges.Qiangzhifang (QZF), a compound used in traditional Chinese medicine, demonstrates potential clinical efficacy in treating depression.However, the mechanisms of action and active ingredients of QZF have not been fully elucidated.The primary aim of this study was to elucidate the effective active ingredients and potential molecular mechanisms of QZF for the alleviation of depression by integrating network pharmacology predictions with experimental validations.
We adopted a chronic restraint stress (CRS) rat model and conducted behavioral tests such as the open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST) to evaluate the therapeutic effects of QZF on depression. Regarding behavioral parameters, the QZF group exhibited significantly higher body mass, sucrose preference ratio, and central zone residence time compared to the model group (P &#60; 0.01, P &#60; 0.01, P &#60; 0.01), and a significantly reduced immobilization time in the forced swimming test (P &#60; 0.001).Network pharmacology and molecular docking studies suggest that QZF may have antidepressant effects by modulating the HIF-1 and JAK-STAT pathways, with key target genes including AKT1, IL-6, MTOR, and TP53, implicated in inflammation, neuroprotection, and apoptosis.In conclusion, this study offers new insights into the modernization and development of Chinese medicine compounds for the comprehensive treatment of depression.

Behavioral test results in the CRS-induced rat depression model 
Sucrose preference test results 
At baseline, there was no difference in the sucrose preference coefficient among the groups (P &gt; 0.05). Following 28 days of intervention, the sucrose preference coefficient of the CRS group was significantly lower than that of the CON group (P &lt; 0.05), while the F and QZF groups showed significantly higher coefficients compared to the C...

This study evaluated the antidepressant efficacy of Qiangzhifang in a rat model of chronic restraint stress-induced depression and elucidated its regulatory effect on HIF-1 and JAK-STAT pathways by network pharmacology and molecular docking analysis.

Depression is a complex psychiatric disorder that poses significant treatment challenges.Qiangzhifang (QZF), a compound used in traditional Chinese ...

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Discover how Qiangzhifang exerts antidepressant effects in a chronic restraint stress rat model through network pharmacology and molecular docking analysis of HIF-1 and JAK-STAT pathways.

Network Pharmacology and Validation of the Antidepressant Mechanisms of Qiangzhifang in a Chronic Restraint Stress-induced Depression Rat Model (Video) | JoVE Journal

Network Pharmacology and Validation of the Antidepressant Mechanisms of Qiangzhifang in a Chronic Restraint Stress-induced Depression Rat Model

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