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Method Article
This protocol describes a reproducible workflow integrating network pharmacology, untargeted metabolomics, and in vivo validation to elucidate the multi-component mechanisms of Liangxue Dihuang Decoction in a dextran sulfate sodium–induced mouse model of ulcerative colitis.
Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by persistent intestinal inflammation and metabolic dysregulation. Traditional Chinese medicine (TCM) formulas are widely used in clinical practice for inflammatory disorders; however, systematic experimental approaches to elucidate their multi-component and multi-target mechanisms remain limited. This article presents a reproducible experimental protocol integrating network pharmacology, untargeted metabolomics, and in vivo validation to investigate the anti-inflammatory effects of Liangxue Dihuang Decoction (LXDHD) in a dextran sulfate sodium (DSS)–induced mouse model of UC. The workflow includes preparation of the herbal decoction, DSS-induced colitis modeling, disease activity assessment, histopathological and hematological evaluation, serum cytokine measurement, LC–MS-based metabolomic profiling, and computational network analysis. Using this integrated approach, DSS-induced colitis was successfully established, and LXDHD treatment resulted in measurable improvements in the disease activity index, colon length, histopathological damage, inflammatory cytokine levels, and metabolic profiles. Network pharmacology and metabolomics analyses further demonstrated that this protocol enables the identification of key targets, pathways, and metabolites associated with therapeutic intervention. This visualized and standardized workflow provides a practical methodological framework for studying the pharmacological mechanisms of multi-component TCM formulas in inflammatory bowel disease and can be adapted to other herbal prescriptions and disease models.
Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by continuous mucosal inflammation of the colon1, leading to symptoms such as abdominal pain, diarrhea, rectal bleeding, and weight loss2. The incidence of UC has been increasing worldwide, imposing a substantial burden on healthcare systems and significantly impairing patients’ quality of life3. Although the etiology of UC remains incompletely understood, accumulating evidence indicates that dysregulated immune responses, excessive production of inflammatory cytokines, oxidative stress, and metabolic disturbances ....
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All animal experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine (Approval No. #2025DW-034-01; Approval Date: March 24, 2025). The preparation of Liangxue Dihuang Decoction was performed in accordance with the procedures described in the Chinese Pharmacopoeia27. The reagents, chemicals, and software used in the protocol are listed in the Table of Materials.
1. Preparation of Liangxue Dihuang Decoction (LXDHD)....
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Network pharmacology analysis of LXDHD in UC
Network pharmacology was applied to predict the potential therapeutic targets and molecular mechanisms of LXDHD in UC. A total of 211 compounds with favorable pharmacokinetic properties were screened from the TCMSP database, including 36 from Scutellaria baicalensis, 37 from Phellodendron chinense, 10 from Rehmannia glutinosa, 11 from Paeonia suffruticosa, 29 from Paeonia veitchii, 5 from.......
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This method presents a visualized, reproducible experimental workflow integrating network pharmacology, untargeted metabolomics, and in vivo validation to investigate the anti-inflammatory effects of TCM formulas in a DSS-induced mouse model of UC. Unlike conventional pharmacological studies that primarily focus on isolated mechanistic conclusions, this protocol emphasizes the robustness of the methodological framework itself while revealing critical biological insights. A major advantage of this integrated work.......
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All authors declare that they have no conflicts of interest related to this work.
This study was supported by the National Natural Science Foundation of China (81973769) and Xuzhou Medical University (ZX202406). The funding organizations had no involvement in study design, data collection and analysis, decision to publish, or manuscript preparation.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 5-Aminosalicylic Acid (5-ASA) | Sigma-Aldrich | A3537 | Positive control drug for ulcerative colitis; administered at 100 mg/kg by oral gavage. |
| Acetonitrile (LC-MS grade) | Merck | 100030 | Used for metabolomics sample preparation and LC-MS mobile phase. |
| AlphaFold Protein Structure Database | EMBL–European Bioinformatics Institute | https://alphafold.ebi.ac.uk/ | Database used to obtain predicted three-dimensional structures of target proteins. |
| Angelica sinensis (Dang Gui) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 1.6 g). |
| CB-Dock2 | CADD Lab | https://cadd.labshare.cn/cb-dock2/ | Web-based platform used for molecular docking and prediction of ligand–protein binding modes. |
| Citrus aurantium (Zhi Ke) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.5 g). |
| Compound Discoverer Software | Thermo Fisher Scientific | Version 3.3 | Used for LC–MS data processing and compound identification. |
| CTAn Software | Bruker | Version 1.17 | Used for Micro-CT 3D image reconstruction and bone erosion analysis. |
| Cytoscape | Cytoscape Consortium | Version 3.8.0 | Software used for network construction and visualization. |
| Dextran Sulfate Sodium (DSS) | MP Biomedicals | 160110 | Molecular weight 36,000–50,000 Da; used to induce ulcerative colitis in mice. |
| EDTA-K2 Anticoagulant Tubes | Sinopharm Chemical Reagent Co., Ltd. | N/A | Used for peripheral blood collection. |
| Eosin Staining Solution | Biosharp | N/A | Used for cytoplasmic counterstaining in H&E staining. |
| Ethanol | Chengdu Cologne Chemicals Co., LTD | 64-17-5 | Used for tissue dehydration. |
| Formic acid (LC-MS grade) | Merck | 5.33002 | Used for LC-MS mobile phase preparation. |
| Glycyrrhiza uralensis (Gan Cao) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.5 g). |
| GraphPad Prism | GraphPad Software | Version 9.0 | Used for statistical analysis and graphical presentation. |
| Hematology Analyzer | Mindray | BC-2600vet | Three-part veterinary hematology analyzer for blood routine analysis. |
| Hematoxylin Staining Solution | Biosharp | BL700B | Used for H&E staining of colon tissue sections. |
| High-speed Centrifuge | Hitachi | CT15E/CT15RE | Used for serum and sample preparation. |
| IL-10 ELISA Kit (Mouse) | MEIMIAN | MM-0176M2 | Used to assess serum IL-10 levels. |
| IL-17 ELISA Kit (Mouse) | Abclonal | RK00039 | Used to measure serum IL-17 levels. |
| IL-1β ELISA Kit (Mouse) | Abclonal | RK00006 | Used for detection of serum IL-1β levels. |
| IL-6 ELISA Kit (Mouse) | Abclonal | RK00008 | Used to quantify serum IL-6 levels. |
| ImageJ (Fiji) Software | National Institutes of Health (NIH) | Version 1.53c | Open-source software used for image processing and quantification. |
| Leica Application Suite X (LAS X) | Leica Microsystems | Version 3.7 | Imaging software used for capturing histopathological slides. |
| Methanol (LC-MS grade) | Merck | 106035 | Used for metabolomics sample preparation and LC-MS mobile phase. |
| Mice (Male C57BL/6) | Anokang Biotechnology Co., Ltd. | N/A | Experimental animals (aged 6–8 weeks, 20–25 g) used for DSS-induced UC model. |
| mzCloud Database | Thermo Fisher Scientific | https://www.mzcloud.org/ | Database used for MS/MS spectral matching. |
| Optical Microscope | Leica | DM500 | Used for histopathological observation. |
| Paeonia lactiflora (Chi Shao) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.6 g). |
| Paeonia suffruticosa cortex (Mu Dan Pi) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.5 g). |
| Paraformaldehyde (4%) | Sinopharm Chemical Reagent Co., Ltd. | N/A | Used for fixation of colon tissues. |
| Pathological Microtome | Leica | RM2016 | Used for preparation of paraffin tissue sections. |
| Phellodendron chinense (Huang Bo) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.5 g). |
| Platycladus orientalis (Ce Bai Ye Tan) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.7 g). |
| Q Exactive High-Resolution Mass Spectrometer | Thermo Fisher Scientific | Q Exactive | Orbitrap-based mass spectrometer for LC–MS analysis. |
| R Software | R Foundation for Statistical Computing | Version 4.2.1 (https://www.r-project.org/) | Software used for GO and KEGG enrichment analyses. |
| Rehmannia glutinosa (Sheng Di Huang) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 2.1 g). |
| Sanguisorba officinalis (Di Yu Tan) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.6 g). |
| Saposhnikovia divaricata (Fang Feng) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.3 g). |
| Scutellaria baicalensis (Huang Qin) | Jiangsu Province Hospital of Chinese Medicine | Batch: 2409001 | Constituent herb of LXDHD (Dose: 0.5 g). |
| Sodium pentobarbital (1%) | Sigma-Aldrich | P3761 | Used for animal anesthesia prior to sample collection. |
| SoftMax Pro Software | Molecular Devices | Version 7.1 | Data acquisition and analysis software used with the microplate reader for ELISA. |
| STRING Database | STRING Consortium | https://string-db.org/ | Database used for protein–protein interaction analysis. |
| TCMSP Database | TCMSP Platform | https://tcmsp-e.com/ | Database used to screen active compounds and predict drug targets. |
| TNF-α ELISA Kit (Mouse) | Abclonal | RK00027 | Used to determine serum TNF-α concentrations. |
| Ultimate 3000 RS LC System | Thermo Fisher Scientific | Ultimate 3000 RS | UHPLC system coupled with mass spectrometry. |
| Ultrasonic Cleaner | Kunshan Ultrasonic Instruments Co., Ltd | KQ3200E | Used for ultrasonic extraction of LXDHD. |
| UniProt Database | UniProt Consortium | https://www.uniprot.org/ | Database used for gene and protein name standardization. |
| Vortex Mixer | Beijing PowerStar Technology Co., Ltd | LC-Vortex-P1 | Used for sample mixing. |
| Welch AQ-C18 Column (150 × 2.1 mm, 1.8 μm) | Welch Materials | AQ-18-15021 | Column used for separation of metabolites and herbal components. |
| Xylene | Chengdu Cologne Chemicals Co., LTD | 1330-20-7 | Used for tissue clearing during histological processing. |