Computer-Aided Active Ingredients Screening and Validation of Dong Medicine for Fever and Cough Exploration

Summary

Here, we constructed a multi-dimensional network of "component-target-pathway-disease", integrating network pharmacology screening, molecular docking, and LPS-induced mouse model validation. By establishing clear key parameters, it enables the efficient identification of the active components of Tong ethnic medicine, Lengyuxiao Tang, and the verification of a repeatable process.

Abstract

To systematically screen and verify the active components in the Dong ethnic medicine Lengyuxiao Tang (LYXT; Semen Pharbitidis, Verbena officinalis L., and Zingiber officinale Roscoe) for treating fever and cough, this scheme established a methodology process integrating network pharmacology prediction and experimental verification. Firstly, using the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), with an oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as criteria, the key active components of LYXT were screened, and the related targets for fever and cough were collected from multiple disease databases. Subsequently, the "component-target-disease" interaction network was constructed, and, through topological analysis and gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment, the core action targets (CYP3A4, POR) and key signaling pathways (MAPK pathway) were identified. Molecular docking technology was used to verify the binding ability of key components to core targets. Finally, a chronic bronchitis mouse model was established by intranasal instillation of lipopolysaccharide (LPS), and the in vivo anti-inflammatory efficacy of the predicted main active components was verified. The results showed that components such as β-carotene and saparenol, screened by network pharmacology, exhibited varying degrees of anti-inflammatory effects in animal experiments, and their mechanisms may involve inhibition of the MAPK/p38 signaling pathway. This scheme provides a repeatable example for the screening and mechanism research of active components in traditional ethnic medicines.

Introduction

Dong medicine is an ethnomedical system within the framework of Traditional Chinese Medicine (TCM), practiced by the Dong ethnic group in Guizhou, Hunan, and Guangxi. It represents their cumulative empirical knowledge, developed over generations. This system integrates herbal medicine, acupuncture, bone-setting, massage, dietary therapy, and spiritual practices. Its distinct characteristics arise from the group's historical geographical isolation and oral tradition, which fostered a unique, practice-based pharmacopeia adapted to the local environment and cultural beliefs.

Persistent fever, a condition that develops when the body cannot ....

Protocol

The animal experiment program has been reviewed by the Experimental Animal Ethics Committee of Lanzhou University (D2024-292). It conforms to the principles of animal protection, animal welfare, and ethics, as well as the relevant provisions of the national experimental animal welfare ethics.

1. Preparation of the database and software

1. Active ingredient screening and target gene collection
   1. Access the TCM system pharmacology database and analysis platform (TCMSP) (https://tcmsp-e.com/) and perform separate searches using the Latin names of each herb in the LYXT formulation, namely Semen Pharbitidis, <....

Results

The TCMSP database was used to collect 409 active LYXT ingredients. Among them, 58 ingredients came from Verbena officinalis L., 86 from Semen Pharbitidis, and 265 active ingredients came from Zingiber officinale Roscoe. Twenty-six key active ingredients were screened according to the index, OB ≥ 30.1% and DL ≥ 0.12 for Verbena officinalis L., OB ≥ 60%, and DL ≥ 0.13 for Zingiber officinale Roscoe, OB ≥ 16.93%, and DL ≥ 0.50 for Semen Pharbitidis (Table 1).

Discussion

This paper successfully identified 26 active ingredients and 207 potential targets of LYXT, a traditional Dong medicine decoction used for fever and cough. Key findings include the identification of CYP3A4 as a critical target protein through network topology analysis, and the experimental validation of LYXT, beta-carotene and spathulenol as active compounds with significant anti-inflammatory effects in an LPS-induced lung inflammation model. These findings were achieved through an integrated approach combining network p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-IL-18	Proteintech, Wuhan, China	27095-1-AP	Rabbit polyclonal, 1:500
Anti-IL-1β	Proteintech, Wuhan, China	66737-1-Ig	Mouse monoclonal, 1:500
Anti-p38	Proteintech, Wuhan, China	14064-1-AP	Rabbit polyclonal, 1:1000
Anti-p-p38	Proteintech, Wuhan, China	28796-1-AP	Rabbit polyclonal, 1:1000
Anti-TNF-α	Proteintech, Wuhan, China	80258-6-RR	Rabbit monoclonal, 1:500
Anti-β-actin	Proteintech, Wuhan, China	66009-1-Ig	Mouse monoclonal, 1:2000
AutoDock Vina	https://vina.scripps.edu/	Version 1.5.6	Molecular docking software
AutoDockTools	https://autodock.scripps.edu/	Version 1.5.6	Molecular docking preparation tool
BCA Protein Assay Kit	Thermo Fisher Scientific, Waltham, MA, USA	23225	For protein concentration determination
Beta-carotene	Sigma-Aldrich, St. Louis, MO, USA	83-46-5	High-purity standard, >95%
Beta-sitosterol	Sigma-Aldrich, St. Louis, MO, USA	83-46-5	High-purity standard, >95%
C57BL/6 mice	medical animal center of lanzhou university	-	Adult male, 25 ± 4 g
Cytoscape	https://cytoscape.org/	Version 3.8.1	Network visualization and analysis software
Dexamethasone	Sigma-Aldrich, St. Louis, MO, USA	D4902	Positive control
ECL Substrate	MilliporeSigma, Burlington, MA, USA	WBKLS0500	For Western blot detection
ELISA Kits
GraphPad Prism	https://www.graphpad.com/	Version 8.0 or higher	Statistical analysis and graphing software
Hematoxylin and Eosin (H&E) Staining Kit	Beijing Solarbio Science & Technology Co., Ltd, Beijing, China	G1120	For lung tissue staining
Histology
HRP-conjugated anti-mouse	Proteintech, Wuhan, China	SA00001-7G	Secondary antibody, 1:5000
HRP-conjugated anti-rabbit	Proteintech, Wuhan, China	SA00001-7H	Secondary antibody, 1:5000
IL-18 ELISA Kit	Beijing Solarbio Science & Technology Co., Ltd, Beijing, China	SEKH-0016	For measuring IL-18 in BALF and serum
IL-1β ELISA Kit	Beijing Solarbio Science & Technology Co., Ltd, Beijing, China	SEKM-0004	For measuring IL-1β in BALF and serum
ImageJ	https://imagej.nih.gov/ij/	Version 1.53	Image analysis software
Lipopolysaccharide (LPS)	Sigma-Aldrich, St. Louis, MO, USA	L2630	From Escherichia coli O111:B4
LYXT Raw Materials	Huaihua University	-	Powder mixture of Pharbitis nil (L.) Choisy, Verbena officinalis L., and Zingiber officinale Roscoe (5:3:2, w/w/w)
Other Materials
Pentobarbital sodium	Sigma-Aldrich, St. Louis, MO, USA	P3761	2% solution for anesthesia
PLIP	https://plip-tool.biotec.tu-dresden.de/	-	Protein-ligand interaction profiler
PVDF membranes	MilliporeSigma, Burlington, MA, USA	IPVH00010	For Western blot transfer
PyMOL	https://pymol.org/	Version 2.5	Molecular visualization software
R	https://www.r-project.org/	Version 4.0 or higher	Statistical computing and graphics software
Spathulenol	MedChemExpress, Monmouth Junction, NJ, USA	HY-N1205	High-purity standard, >95%
STRING	https://stringdb.org/	-	Search Tool for the Retrieval of Interacting Genes/Proteins
TCMSP	https://tcmsp-e.com/	-	Traditional Chinese Medicine Systems Pharmacology Database
Thiopental	Sigma-Aldrich, St. Louis, MO, USA	T102	For euthanasia
TNF-α ELISA Kit	Beijing Solarbio Science & Technology Co., Ltd, Beijing, China	SEKM-0034	For measuring TNF-α in BALF and serum
UniProt	https://www.uniprot.org/	-	Protein sequence and annotation database