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 worldwid....

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 <....

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