Network Pharmacology and Experimental Research of β-Sitosterol in Treating Chronic Obstructive Pulmonary Disease via the Glucocorticoid Receptor

Summary

β-sitosterol alleviated COPD and potentiated the efficacy of dexamethasone, with the glucocorticoid receptor mediating this effect. Additionally, β-sitosterol reduced inflammation, oxidative stress, and cellular senescence in both CSE-stimulated bronchial epithelial cells and cigarette smoke-induced COPD mice.

Abstract

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disorder characterized by persistent inflammation, oxidative stress, and progressive lung function decline. The therapeutic potential of β-sitosterol in COPD remains unclear. This study aimed to investigate the protective effects of β-sitosterol against COPD and elucidate its underlying mechanism. Database screening identified the glucocorticoid receptor (GR) as a potential target of β-sitosterol. A cigarette smoke-induced COPD mouse model and a cigarette smoke extract (CSE)-stimulated BEAS-2B cell model were established. Pulmonary function, lung injury-related markers, inflammation, oxidative stress, and cellular senescence were assessed using (RT-qPCR), DCFH-DA fluorescent probe assays, and senescence detection kits. The effects of β-sitosterol alone or in combination with dexamethasone (DEX) were also evaluated. β-sitosterol significantly improved pulmonary function, normalized lung injury-related markers, and attenuated inflammation, oxidative stress, and cellular senescence in both CSE-stimulated BEAS-2B cells and cigarette smoke-exposed mice. These protective effects were accompanied by increased GR expression. Moreover, β-sitosterol enhanced responsiveness to DEX, and combined treatment with β-sitosterol and DEX produced greater improvements in pulmonary function, lung injury markers, inflammation, reactive oxygen species levels, and cellular senescence than DEX treatment alone. Collectively, these findings suggest that β-sitosterol alleviates COPD-associated pathological changes, at least in part, by upregulating GR expression and enhancing the therapeutic efficacy of DEX, highlighting its potential as an adjuvant strategy for COPD treatment.

Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease characterized by persistent airway inflammation, alveolar damage, and irreversible airflow obstruction that impairs respiratory function and reduces quality of life¹. Its prevalence increases significantly with age, and the risk for people aged sixty-five and over is about five times that of people under forty². This demographic trend is associated with increased disease incidence, mortality, and disability³. Epidemiological projections indicate that by 2030, COPD may become the fourth leading cause of death worldwide, accounting for about 5% of all deaths^4,5,6. At present, the identified risk factors include genetic susceptibility, pulmonary dysplasia, particulate matter exposure, chronic bronchitis, and respiratory infection⁷.

Among the risk factors, cigarette smoke is the most important pathogenic factor of COPD⁸. It contains thousands of chemicals that can cause bronchial epithelial damage, promote the infiltration of inflammatory cells, and lead to remodeling of lung tissue⁹. Long-term exposure can induce persistent and irreversible epithelial injury, promote the release of a large number of pro-inflammatory chemokines, significantly increase (ROS) production, and induce cell apoptosis¹⁰. Many studies have shown that the increased levels of pro‑inflammatory chemokines such as interleukin‑8 (IL-8) and tumor necrosis factor‑α (TNF‑α), together with excessive ROS production and cellular senescence, are closely related to the severity of COPD^11,12. This highlights the need for agents that can effectively and safely modulate these key pathogenic factors to exert protective and therapeutic effects in COPD.

Phytosterols are natural bioactive compounds ubiquitously present in plant cell membranes. Among them, β‑sitosterol is the most abundant and is widely distributed in lipid‑rich plant‑derived foods such as vegetables, nuts, seeds, and olive oil^13,14,15,16. Studies have confirmed that administration of β‑sitosterol at doses up to 1000 mg/kg in rats and mice is not associated with cytotoxicity or genotoxicity¹⁷. Moreover, research has shown that β‑sitosterol exhibits antioxidant properties through its effects on antioxidant enzymes and human estrogen receptors¹⁸.

β‑sitosterol displays a broad spectrum of pharmacological activities, including anti‑inflammatory¹⁹, antipyretic²⁰, analgesic²¹, and antidiabetic effects¹³. It has also shown therapeutic potential in various disease models, such as colitis and intestinal inflammation²², and possesses anti‑atherosclerotic²³ and immunomodulatory properties²⁴. Additionally, it has a favorable safety profile. Given these characteristics, β‑sitosterol has attracted considerable interest as a potential drug candidate. However, its precise role and underlying mechanisms in COPD remain largely unclear.

Therefore, the present study aimed to investigate the potential therapeutic effects and underlying mechanisms of β‑sitosterol in COPD. Specifically, a cigarette smoke extract (CSE)‑induced BEAS‑2B cell injury model was used to evaluate whether β‑sitosterol could effectively alleviate CSE‑mediated cellular damage, thereby identifying a novel candidate molecule for COPD therapeutic strategies.

Protocol

The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology guidelines for experimental and clinical studies²⁵. All animal experimental protocols were approved by the Animal Ethics Committee of The Second Affiliated Hospital, Zhejiang University School of Medicine (Approval No. 20230102), and all experiments were performed in accordance with the guidelines of the Animal Ethics Committee. The study was conducted in agreement with the ARRIVE guidelines.

Screening of key components and targets via network pharmacology

Active components of Fritillaria cirrhosa D. Don, Lonicera japonica Thunb., Glehnia littoralis F. Schmidt ex Miq., and Adenophora stricta Miq. were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP; accessed 12 March 2024). Candidate compounds were screened according to the following ADME criteria: oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, and Caco-2 permeability ≥ −0.4²⁵. Compounds meeting all three criteria were retained as candidate active ingredients for subsequent analyses.

The canonical Simplified Molecular Input Line Entry System (SMILES) structure of β-sitosterol was obtained from PubChem (accessed 18 March 2024) and imported into SwissTargetPrediction (accessed 21 March 2024) for target prediction. Homo sapiens was selected as the target species. Potential targets were predicted based on 2D and 3D structural similarity between β-sitosterol and known ligands in the database. Predicted targets with a probability greater than 0 were retained, and duplicate entries were removed.

COPD-associated genes were retrieved from the Therapeutic Target Database (TTD; accessed 25 March 2024) and the Comparative Toxicogenomics Database (CTD; accessed 28 March 2024) using the keyword “chronic obstructive pulmonary disease”. After removal of duplicate genes, the COPD-associated genes were intersected with the predicted β-sitosterol targets to identify candidate targets potentially involved in the therapeutic effects of β-sitosterol against COPD.

Molecular docking of β‑sitosterol and GR

The three-dimensional structure of β-sitosterol in SDF format was retrieved from the PubChem database (accessed 2 June 2026), while the crystal structure of the glucocorticoid receptor (GR) was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB; accessed 3 June 2026). After standard preparation of the ligand and receptor structures, molecular docking was performed using the CB-Dock2 online platform (accessed 3 June 2026). The prepared ligand and receptor files were uploaded to the platform, and docking calculations were conducted using the default parameters.

Functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

All predicted target genes of β-sitosterol were subjected to GO functional enrichment and KEGG pathway enrichment analyses. Enrichment outcomes, including significantly enriched biological processes, cellular components, molecular functions, and signaling pathways, were sorted and stored in Supplementary File 1 for subsequent bioinformatic analysis.

Animal modeling and grouping

The sample size was set at six animals per group based on a previous study that performed an a priori power analysis using G*Power software (α = 0.05, power = 0.80) and demonstrated that n = 6 was sufficient to detect biologically relevant differences under comparable experimental conditions²⁶. Male C57BL/6 mice aged 6–8 weeks (20 ± 2 g) were randomly assigned to five groups: control group, COPD model group, COPD + dexamethasone (DEX) group, COPD + β-sitosterol group, and COPD + DEX + β-sitosterol group. Mice were housed under standard laboratory conditions (25 °C, 12 h light/dark cycle) with free access to standard chow and water and were allowed to acclimatize for 1 week before experimentation.

For COPD model establishment, mice were placed in a whole-body cigarette smoke exposure chamber (63 cm × 55 cm × 45 cm) and exposed to mainstream cigarette smoke for 12 weeks. Each cigarette contained 10 mg tar, 0.9 mg nicotine, and 10 mg carbon monoxide. Smoke exposure was conducted twice daily (5 days/week) at 9:30 a.m. and 3:00 p.m., with smoke generated from 18 cigarettes delivered into the chamber for 90 min per session²⁷.

To evaluate the therapeutic effects of the interventions, drug administration was initiated after 8 weeks of cigarette smoke exposure and continued for the remaining 4 weeks. Beginning at week 9, mice in the respective treatment groups received daily intragastric administration of DEX (1 mg/kg)²⁸ or β-sitosterol (50 mg/kg)^29,30 1 h before cigarette smoke exposure. Previous pharmacokinetic studies have reported an oral bioavailability of approximately 4% for β-sitosterol in rodents³¹. In addition, several mouse studies have employed doses ranging from 20 to 50 mg/kg³². Therefore, a dose of 50 mg/kg was selected to ensure adequate systemic exposure while remaining within the range commonly used in preclinical studies.

Mice in the control group were exposed to filtered room air, whereas mice in the model and treatment groups received an equivalent volume of normal saline as a vehicle. At the end of the experimental period, mice were deeply anesthetized with isoflurane until loss of pedal reflex and euthanized by exsanguination via abdominal aorta blood collection. The thoracic cavity was immediately opened, and intact bilateral lung tissues were carefully excised, rinsed with ice-cold phosphate-buffered saline (PBS), and placed on ice. For bronchoalveolar lavage fluid (BALF) collection, the trachea was cannulated and lavaged with cold sterile PBS, which was gently instilled and withdrawn repeatedly to collect lavage fluid. Lung tissues and BALF samples were maintained on ice and subsequently processed for downstream analyses.

BEAS‑2B cell culture

BEAS-2B human bronchial epithelial cells were maintained in Dulbecco's Modified Eagle Medium/F12 (DMEM/F12) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under 5% CO₂ at 37 °C.

CSE preparation and BEAS‑2B cell exposure

BEAS‑2B cells were washed with phosphate-buffered saline and seeded into 24‑well plates in triplicate at a density of 5 × 10⁴ cells per well. After 12 h of culture, cells reached approximately 60% confluency and were then exposed to CSE. CSE was prepared freshly as previously described, with minor modifications³³. Briefly, one unfiltered commercial cigarette was continuously combusted, and mainstream smoke was bubbled into 10 mL of serum‑free medium using a vacuum pump at a constant flow rate of 1.05 L/min (approximately −11 kPa negative pressure) for approximately 5 min until the cigarette was completely burned. The obtained solution was first subjected to pre-filtration to remove large particulate impurities, then sterilized via a 0.22 µm sterile microporous filter to eliminate residual particles; the resultant stock solution was defined as 100% CSE. All freshly prepared CSE was used for cell treatment immediately after filtration without storage. Cells were stimulated with medium containing 2.5% CSE. After 24 h of CSE treatment, the culture supernatants were collected and stored at -40 °C for subsequent analysis³⁴.

Quantitative real-time polymerase chain reaction (RT‑qPCR)

Total RNA was extracted from BEAS-2B cells using a total RNA extraction kit and reverse-transcribed into cDNA using a reverse transcription kit according to the manufacturers’ instructions. RT-qPCR was subsequently performed using a real-time PCR detection system and a SYBR Green-based qPCR master mix. All RT-qPCR experiments were conducted with three biological replicates and three technical replicates per sample to ensure data reliability.

Each qPCR reaction was performed in a total volume of 20 µL, containing 10 µL of SYBR Green qPCR master mix, 0.4 µL of forward primer, 0.4 µL of reverse primer, 2 µL of diluted cDNA template, and 7.2 µL of RNase-free water. The amplification conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. Relative gene expression levels were calculated using the 2^−ΔΔCt method, with GAPDH serving as the internal reference gene. The primer sequences used in this study are mentioned in Supplementary Table 1.

Intracellular reactive oxygen species (ROS) analysis

Intracellular ROS production in BEAS-2B cells was assessed using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and analyzed by flow cytometry. During flow cytometric acquisition, a standard forward scatter (FSC) and side scatter (SSC) gating strategy was applied to exclude cellular debris and select intact single cells. A total of 10,000 valid cellular events were acquired for each sample. The excitation and emission wavelengths were set at 488 nm and 525 nm, respectively. Flow cytometric data were subsequently analyzed to quantify intracellular ROS fluorescence intensity.

Malondialdehyde (MDA) level determination

Intracellular MDA content was measured using a lipid peroxidation assay kit according to the manufacturer's instructions. Briefly, treated BEAS-2B cells were harvested and lysed, then centrifuged at low temperature to collect the supernatant. The thiobarbituric acid (TBA) colorimetric reaction was then performed, and absorbance was measured at 532 nm using a microplate reader. Relative MDA levels were calculated according to the standard curve.

Cell senescence assessment

Cellular senescence was evaluated using a senescence detection assay based on senescence-associated β-galactosidase (SA-β-gal) activity. SA-β-gal-positive cells were quantified by flow cytometry. In parallel, the mRNA expression levels of the senescence-associated markers p53 and p21 were determined by RT-qPCR.

Cell proliferation assay

Cell proliferation was assessed using a colorimetric cell viability assay kit. Absorbance was measured at 450 nm using a microplate reader.

GR knockdown assay

Cell transfection was performed using a lipid-based transfection reagent. The following oligonucleotides were transfected: glucocorticoid receptor-targeting small interfering RNA (si-GR) and negative control small interfering RNA (si-NC). Candidate siRNA sequences targeting GR were designed using the online bioinformatics platform siDirect 2.0. Following transfection, cells were cultured for 48 h, and all siRNAs were used at a final concentration of 50 nM. Knockdown efficiency was verified by measuring GR mRNA expression using RT-qPCR.

Vehicle control groups were included in both β-sitosterol treatment and transfection experiments. For β-sitosterol intervention, cells treated with vehicle solvent alone served as the vehicle control group (denoted as CSE+0). Statistical analysis confirmed that no significant differences were observed between the vehicle control and blank control groups.

Statistical analysis

All quantitative results were expressed as mean ± standard deviation from three biologically independent in vitro experiments. Statistical analyses were performed using statistical analysis software. The Shapiro–Wilk test was used to examine the normality of the dataset distribution prior to statistical testing; Bonferroni post-hoc correction was adopted to adjust P values for multiple comparisons after analysis of variance (ANOVA). Comparisons between two groups were assessed by an unpaired Student's t-test, while one‑way or two‑way ANOVA was employed for multi‑group comparisons. A p‑value ≤ 0.05 was considered statistically significant.

Results

β-sitosterol was a potential natural product for treating COPD

Due to the reported curative effects of Fritillaria cirrhosa D. Don, dried flower of Lonicera japonica Thunb., dried root of Glehnia littoralis F. Schmidt ex Miq., and Adenophora stricta Miq. in treating pulmonary diseases^35,36,37,38, this study used th...

Discussion

COPD imposes a substantial global disease burden and remains a major challenge to worldwide health¹. Current management of COPD encompasses both pharmacological and non‑pharmacological interventions⁴². Among pharmacological treatments, glucocorticosteroids (GCs) are widely used in chronic inflammatory disorders. They act by binding to the GR and recruiting histone deacetylase 2 into transcriptional complexes to reverse histone acetylation⁴³. Ho...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BEAS-2B human bronchial epithelial cells	SunnCell, China	SC-0218	Human bronchial epithelial cell line for in vitro study
C57BL/6 mice	Vital River Laboratories, China	213	Animal model for COPD in vivo experiment
CB-DOCK2	China	https://cadd.labshare.cn/cb-dock2/php/index.php	for molecular docking simulation between ligand and receptor protein
Cell Counting Kit-8 (CCK-8)	Beyotime Biotechnology, China	C0038	Detection of cell viability and proliferation
CellEvent™ Senescence Green Detection Kit	Thermo Fisher, USA	C10851	Detection of cellular senescence
Cigarettes	Hongta, China	Not provided	Cigarette used for smoke exposure and CSE preparation
CTD	USA	https://ctdbase.org/	for retrieving COPD-related genes
DCFH-DA fluorescent probe	Sigma-Aldrich, USA	D6883-50MG	Detection of intracellular ROS level
Dexamethasone (DEX)	Yeasen, China	40323ES03	Glucocorticoid for combined intervention
DMEM/F12 medium	Gibco, USA	11330032	Basal medium for BEAS-2B cell culture
Fetal bovine serum (FBS)	Gibco, USA	10270106	Supplement for cell culture medium
GraphPad Prism	GraphPad Software, USA	9.3.1	Data statistical analysis and graph drawing
Isoflurane	RWD Life Science, China	R510-22-10	Inhaled anesthetic for animal euthanasia
Lipid Peroxidation MDA Assay Kit	Beyotime Biotechnology, China	S0131S	Detection of intracellular malondialdehyde (MDA) content
Lipofectamine 2000	Thermo Fisher, USA	11668030	Transfection reagent for siRNA delivery
Multimode microplate reader	Thermo Fisher, USA	VL0000D0	Detection of absorbance and fluorescence value
Navios EX Flow Cytometer	Beckman Coulter, USA	B80912	Analysis of ROS and senescent cells
Penicillin/streptomycin	HyClone, USA	SV30010	Antibiotics to prevent bacterial contamination
Power SYBR™ Green Master Mix	Thermo Fisher, USA	4367659	Fluorescent quantitative PCR amplification
PrimeScript™ RT reagent Kit	Takara, Japan	RR037Q	Reverse transcription for cDNA synthesis
PubChem database	USA	https://pubchem.ncbi.nlm.nih.gov/	for acquiring canonical SMILES sequence and downloading 3D SDF structural information of beta-sitosterol
QuantStudio Dx PCR Instrument	Thermo Fisher, USA	4479889	Real-time RT-qPCR detection
RCSB PDB	USA	https://www.rcsb.org/	to obtain GR crystal 3D structure file
RNA/Protein Isolation Kit	Beyotime Biotechnology, China	R0018S	Total RNA extraction from cell and lung tissue
si-GR	Designed in silico via siDirect 2.1	Custom synthesis	Guide: 5′-UUUUGAAUGGGUGUAGCAA-3′; Passenger: 5′-GCUUUCACCCAUUCAAAAGG-3′, knockdown for GR
si-NC	Thermo Fisher, USA	4390843	used as negative transfection control for cellular and in vivo assays
SwissTargetPrediction database	Switzerland	http://swisstargetprediction.ch/	for in silico prediction of potential drug-related protein targets
TCMSP database	China	https://www.tcmsp-e.com/	for herbal ingredient screening and target prediction
TTD	Singapore	https://db.idrblab.net/ttd/	for collection of validated COPD-associated disease genes
Whole-body smoke exposure chamber	Teague Enterprise, USA	TE-2	Establishment of cigarette smoke-induced COPD model
β-sitosterol	ChemicalBook, China	83-46-5	Active phytosterol for drug treatment