Exploring the Mechanism of Guben Pingchuan Granule in the Treatment of Bronchial Asthma

Summary

This protocol investigates the mechanism of action of Guben Pingchuan granules in bronchial asthma using network pharmacology, molecular docking, and in vivo. validation.

Abstract

This study investigates the mechanism of action of Guben Pingchuan granules in the treatment of bronchial asthma using network pharmacology, molecular docking, and experimental validation. Active ingredients and corresponding targets were identified using TCMSP, HERB, and SymMap databases. Bronchial asthma-related targets were collected from GeneCards, OMIM, TTD, DrugBank, and PharmGKB. Overlapping targets were identified, and component–target and protein–protein interaction (PPI) networks were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed with false discovery rate (FDR) correction. Molecular docking was conducted to evaluate binding affinity, followed by in vivo validation. A total of 121 active components and 234 drug-related targets were identified, yielding 155 overlapping targets. Core targets included TP53, AKT1, TNF, and IL6. KEGG analysis demonstrated significant enrichment of the PI3K–Akt signaling pathway, which was selected for experimental validation. Molecular docking revealed a strong binding affinity between core components and AKT1. In ovalbumin-induced asthmatic rats, Guben Pingchuan granule reduced serum IL-4 and IL-10 levels, increased FoxP3 expression in lung tissue, and upregulated PI3K and AKT mRNA levels. These findings indicate that Guben Pingchuan granule alleviates bronchial asthma by modulating the PI3K–Akt. signaling pathway, regulating inflammatory cytokines, and enhancing regulatory T cell function.

Introduction

Bronchial asthma is a heterogeneous chronic respiratory disease characterized by airway inflammation, airway hyperresponsiveness, mucus hypersecretion, and airway remodeling¹. Its pathogenesis involves complex interactions among immune cell activation, inflammatory mediators, and multiple signaling pathways. Clinically, asthma presents with recurrent wheezing, dyspnea, chest tightness, and cough. Airway inflammation is a central mechanism underlying disease onset and progression, characterized by increased secretion of pro-inflammatory cytokines such as IL-6, IL-4, IL-17, and TNF-α., infiltration of inflammatory cells including eosinophils and neutrophils, and structural changes such as airway smooth muscle proliferation, basement membrane thickening, and mucus gland hyperplasia^2˒3. These pathological processes ultimately lead to airway obstruction and impaired airflow.

Asthma pathophysiology is regulated by multiple interacting signaling pathways rather than a single mechanism. The PI3K–Akt signaling pathway plays a key role in modulating immune balance, particularly the Treg/Th17 axis, by regulating FoxP3 expression. This pathway also interacts with IL-17 and TNF signaling pathways to amplify inflammatory responses and airway remodeling, contributing to disease heterogeneity and treatment resistance^3,4. Epidemiological data indicate a continuous global increase in asthma prevalence, with over 358 million affected individuals worldwide and approximately 45.7 million adult patients in China^5,6. Current treatment strategies primarily rely on inhaled β₂-agonists and glucocorticoids; however, these therapies target limited pathological mechanisms and may lead to adverse effects and glucocorticoid resistance in certain patients. Dysregulation of pathways such as PI3K–Akt. has been associated with reduced therapeutic responsiveness, highlighting the need for multi-target therapeutic approaches^7,4.

Traditional Chinese medicine (TCM) classifies asthma as “Xiao Syndrome,” which is associated with deficiencies in lung, spleen, and kidney function, combined with external pathogenic factors. TCM formulations have been widely used in asthma management due to their multi-component, multi-target characteristics^8,9. Unlike single-target pharmacological agents, TCM compounds exert therapeutic effects by coordinating the regulation of multiple biological pathways, aligning with the complex pathophysiology of asthma¹⁰. Network pharmacology provides a systems-level approach for analyzing interactions among drug components, targets, and disease pathways, enabling identification of key bioactive compounds and molecular mechanisms¹¹. Molecular docking further complements this approach by evaluating the binding affinity between small molecules and target proteins.

Guben Pingchuan granule is a traditional Chinese medicinal formulation composed of eight herbal components, including Ephedra sinica (Ephedra Herb), Psoralea corylifolia fruit, Prunus armeniaca seed (Apricot seed), Pseudostellaria heterophylla root, Perilla frutescens fruit, Eriobotrya japonica leaf (Loquat leaf), Lilium brownii bulb, and Epimedium brevicornum. This formulation is traditionally used to strengthen vital energy, resolve phlegm, and relieve wheezing. Previous clinical studies have demonstrated its therapeutic efficacy in bronchial asthma, with higher response rates compared to conventional treatments¹².

To elucidate the underlying mechanism of action, an integrated approach combining network pharmacology, molecular docking, and in vivo experimental validation is applied. Network pharmacology is used to identify active components and potential targets associated with bronchial asthma. Molecular docking is employed to evaluate interactions between key compounds and target proteins. Experimental validation in an animal model is subsequently conducted to assess the regulatory effects on critical signaling pathways, particularly the PI3K–Akt. pathway. This approach aims to provide mechanistic insights into the multi-target therapeutic effects of Guben Pingchuan granule and to support its application in the treatment of bronchial asthma.

Protocol

All animal procedures were approved by the Animal Ethics Committee of Liaoning University of Traditional Chinese Medicine (Approval No.: 21000042023058) and performed in accordance with institutional guidelines. The research tools used in this protocol are listed in the Table of Materials.

1. Databases and software

1. Access the following databases and software: TCMSP, HERB, SymMap, PubChem, STRING, GeneCards, OMIM, TTD, DrugBank, PharmGKB, DAVID, UniProt, and Cytoscape (version 3.9.1).

2. Acquisition of component targets

1. Retrieve chemical constituents of Ephedra Herb, Apricot seed, Pseudostellaria root, Perilla fruit, Loquat leaf, Lilium species, and Epimedium from the TCMSP database¹³. Retrieve compounds of Psoralea from the HERB and SymMap databases.
2. Screen active compounds using oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18)^14,15.
3. Predict target genes using TCMSP. Standardize all targets using the UniProt database by selecting Homo sapiens. Remove duplicate and invalid targets, including non-human entries, unannotated proteins, and obsolete identifiers.

3. Acquisition of bronchial asthma targets

1. Search GeneCards, OMIM, TTD, DrugBank, and PharmGKB using the keyword “bronchial asthma.” Set the GeneCards relevance score to >4. Merge all retrieved targets and remove duplicates.

4. Identification of intersection targets

1. Import drug targets and disease targets into an online Venn analysis tool. Identify overlapping targets and record the total number for subsequent analysis.

5. Construction of the component–target network

1. Import intersection targets into Cytoscape 3.9.1. Construct the component–target network. Adjust node size and color based on degree values.

6. Construction of protein–protein interaction (PPI) network

1. Upload intersection targets to the STRING database. Select Homo sapiens and set the confidence score to ≥0.900. Export the network and import it into Cytoscape.
2. Calculate topological parameters (degree centrality, betweenness centrality, and closeness centrality). Identify core targets using median thresholds and degree >20.

7. GO and KEGG enrichment analysis

1. Upload overlapping targets to the DAVID database or analyze using the R package clusterProfiler.
2. Perform Gene Ontology (GO) enrichment analysis for biological process, cellular component, and molecular function categories.
3. Perform KEGG pathway enrichment analysis. Apply false discovery rate (FDR) correction and set the significance threshold to FDR < 0.05.
4. Visualize the top enriched terms using bar plots or bubble plots.

8. Molecular docking

1. Download 2D structures of core compounds from PubChem. Generate 3D structures using ChemOffice and save as mol2 files.
2. Download protein structures from the RCSB PDB database. Remove water molecules and ligands using PyMOL.
3. Perform molecular docking using AutoDock Vina. Define grid box parameters and run docking simulations.
4. Record binding energies (kcal/mol) and key interacting residues. Visualize docking results using molecular modeling, visualization, and simulation platforms.

9. Experimental validation

1. Animals
   1. Obtain 48 male Sprague–Dawley rats (9 weeks old, 220–240 g). House animals at 20–25 °C with 55%–60% humidity under standard conditions.
2. Drugs and reagents
   1. Prepare Guben Pingchuan granules composed of Ephedra Herb, Psoralea, Apricot seed, Pseudostellaria root, Perilla fruit, Loquat leaf, Lilium, and Epimedium.
   2. Prepare dexamethasone, ovalbumin (OVA), aluminum hydroxide, ELISA kits, RNA extraction kits, and antibodies as listed in the Table of Materials.
3. Instruments
   1. Prepare standard laboratory equipment, including a microplate reader, electrophoresis system, paraffin microtome, and imaging system.
4. Animal grouping and treatment
   1. Randomly assign rats into six groups (n = 8): blank control group, model group, dexamethasone group (0.3 mg/kg), low-dose Chinese medicine group, medium-dose Chinese medicine group, and high-dose Chinese medicine group.
   2. On days 1, 7, and 14, administer 1 mL intraperitoneal injection of 10% OVA + 10% Al(OH)₃ to rats in the model group, low-dose Chinese medicine group, medium-dose Chinese medicine group, high-dose Chinese medicine group, and dexamethasone group for sensitization.
   3. Administer 1 mL intraperitoneal injection of 0.9% physiological saline to rats in the blank control group on days 1, 7, and 14.
   4. From days 15 to 21, perform nebulized inhalation stimulation once daily for 20 min. Administer 5% OVA nebulized inhalation to rats in the model group, low-dose Chinese medicine group, medium-dose Chinese medicine group, high-dose Chinese medicine group, and dexamethasone group.
   5. Administer 0.9% physiological saline nebulized inhalation to rats in the blank control group from days 15 to 21.
   6. Administer treatments by oral gavage once daily from days 22 to 36 according to the assigned treatment group.
   7. Monitor body weight and general condition daily throughout the experiment. The experimental timeline is summarized in Table 1.
5. Histopathology
   1. Collect lung tissue. Fix, embed, and section samples. Perform hematoxylin–eosin staining.
   2. Perform Masson staining to evaluate collagen deposition.
6. RT-PCR analysis
   1. Extract total RNA from lung tissue.
   2. Homogenize frozen lung tissue under ultra-low-temperature conditions. After weighing the sample, quickly transfer it into a pre-cooled mortar containing liquid nitrogen and grind it into powder.
   3. Transfer the powdered sample into a centrifuge tube. Add 1 mL TransZol UP and 0.2 mL RNA extraction reagent to every 50–100 mg of tissue. Shake the tube at room temperature for 5 min.
   4. Centrifuge the sample at 1000 × g. and 2–8 °C for 15 min. Transfer the colorless aqueous phase into a new centrifuge tube.
   5. Add 1 mL TransZol UP to 0.5 mL isopropanol. Incubate the mixture at room temperature for 10 min.
   6. Centrifuge the sample at 1000 × g for 10 min. Add 1 mL of 75% ethanol and centrifuge again for 5 min.
   7. Discard the supernatant and air-dry the precipitate at room temperature for 5 min. Dissolve the precipitate in 50–100 µL of RNA solution, then incubate for 10 min.
   8. Store the sample at −70 °C for long-term preservation.
   9. Synthesize cDNA and perform quantitative PCR to measure PI3K and AKT expression.
7. ELISA
   1. Thaw serum samples at 4 °C. Measure IL-4 and IL-10. levels using ELISA kits according to manufacturer instructions.
8. Immunohistochemistry
   1. Fixed lung tissue with 4% paraformaldehyde, embedded in paraffin, sliced, dewaxed, and hydrated.
   2. Perform antigen retrieval, blocking, and antibody incubation. Stain according to the FoxP3 immunohistochemistry kit, DAB staining solution, and positive expression appears as brownish yellow or dark brown staining.
   3. Observe and photograph under a microscope, and use Image J software for quantitative statistical analysis of FoxP3 expression.
9. Statistical analysis
   1. Test data normality using the Shapiro–Wilk test and variance homogeneity using Levene’s test.
   2. Analyze data using one-way ANOVA or nonparametric tests as appropriate. Express data as mean ± standard deviation.
   3. Define p < 0.05 as statistically significant. ★★p < 0.01 and ★p < 0.05 vs. control group; ◆◆p < 0.01 and ◆p < 0.05 vs model group; ##p < 0.01 and #p < 0.05 vs dexamethasone group.

Table 1: Experimental timeline. Schedule of sensitization, challenge, and treatment procedures. Please click here to download this Table.

Results

Screening of active ingredients
A total of 121 active components were identified from the TCMSP database after filtering for target activity. These included 23 components from Ephedra Herb, 7 from Psoralea, 19 from Apricot seed, 8 from Pseudostellaria root, 16 from Perilla fruit, 18 from Loquat leaf, 7 from Lilium, and 23 from Epimedium. (Table 2). Sixteen common components were identified, including luteolin, quercetin, β-sitosterol, kaem...

Discussion

Bronchial asthma is a common disease in the respiratory department¹⁶. It is an obstructive airway disorder caused by allergens and airway hyperresponsiveness¹⁷. It has a high clinical incidence, is prone to recurrence, and is difficult to cure, significantly affecting patients' health and quality of life. The 2025 Global Initiative for Asthma (GINA) reports that inhaled glucocorticoid therapy is effective for asthma¹⁸; however, long-term use may ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
qPCR Kit	Novogene	NQ-001, 2×1mL	Detection of relative mRNA expression of target genes
aluminum hydroxide Al(OH)3	Shanghai Yubo Biotechnology Co., LTD	YB-AL001, 500g	Immunological adjuvant to enhance antigen immune response
BCA protein Concentration Test Kit	Fode Biotechnology Co., LTD	FD2001, 100 pieces per box	Total protein quantification to standardize sample loading
Chemiluminescence imaging system	Tanon	5200Multi	WB band imaging, gel and blot image collection and analysis
Color Pre-stained Protein Marker	Biolaibo Technology Co., LTD	BL005, 10-180kDa, 500μL	Protein molecular weight reference for electrophoresis experiments
Enhanced RNA Extraction Kit	Whole Golden Branch	ET111-01v2, 50 pieces per box	Extraction and purification of total RNA from tissues and cells
Enzyme-linked immunosorbent assay reader	U.S. Biotek Company	Epoch/BioTek	Quantitative detection of cytokines and protein contents in samples
FOXP3 primary antibody	Biyun Tian Biotechnology Co., LTD	AG5002, 50ul/piece	Targeted binding to FOXP3 protein for immunoassay
Gel Rapid Preparation Kit	Shanghai Yamei	PG212, 30 sets	Rapid preparation of separating gel and stacking gel for electrophoresis
masson Staining Kit	Solabao Biotechnology Co., LTD	G1340, 100 tablets	Specific staining for tissue collagen fibers and fibrosis observation
OVA	Beijing Boaotoda Technology Co., LTD.	OVA-10g, ≥98%	Classical allergen for establishing allergic animal models
primary Antibody Dilution solution	Biyun Tian Biotechnology Co., LTD	P0256-100ml	Dilute primary antibody, maintain activity and reduce non-specific binding
Rat IL-10ELISA Kit	Andy Bio	E-30649, 96T	Quantitative detection of rat IL-10 anti-inflammatory factor
Rat IL-4ELISA Kit	Andy Bio	E-30623, 96T	Quantitative detection of rat Th2 inflammatory factor IL-4
Reverse Transcription Kit	Baori Medical Technology Co., LTD	E047-01A	Reverse transcription of RNA into cDNA for subsequent gene analysis
Saturated Oil Red O Kit	Solabao Biotechnology Co., LTD	G1261, 50mL×2 bottle	Specific staining and detection of tissue lipid droplets
Shangyang Anti-Rabbit Secondary Antibody	Proteintech Biological Co., LTD	SA00001-2, 1mL	Bind to rabbit primary antibody for WB color reaction
TBST Buffer	Wuhan Sewell Biotechnology Co., LTD	G2150, 1L / bottle	Buffer for membrane washing and antibody dilution in Western blot
Transfer Electrophoresis System	Shanghai Jingcanshi Precision Machinery Co., Ltd.	PowerPac Basic	Used for protein electrophoresis and Western blot membrane transfer
Ultra-low temperature refrigerator	Zhongke Meiling	MDF-382	Low-temperature cryopreservation of reagents, protein samples and tissues
Vertical electrophoresis tank	BIO-RAD	JY-ZY5	Vertical electrophoretic separation of protein and nucleic acid samples