ETHNOPHARMACOLOGICAL RELEVANCE: Chronic obstructive pulmonary disease (COPD) is a frequently encountered respiratory disease. Its clinical symptoms are characterized by long-term coughing, expectoration, and other airway symptoms, resulting in significant harm. Linggan Wuwei Jiangxin Decoction (LGWWJXD) has the effects of warming the lungs, transforming fluid retention, and relieving cough and asthma, and is a classic prescription for treating COPD.
AIM OF THE STUDY: This study aims to determine the mechanism of action of LGWWJXD in preventing and treating COPD.
MATERIALS AND METHODS: COPD rat models were established using a combination of exposure to cigarette smoke (CS), hypothermia, and lipopolysaccharide (LPS). Model rats were administered LGWWJXD. Histological changes in lung tissues were detected via hematoxylin-eosin (H&E) staining combined with transmission electron microscopy. Quantification of inflammatory factors in the bronchoalveolar lavage fluid, serum and cell culture medium were determined using enzyme-linked immunosorbent assays. The active ingredients of LGWWJXD and its components that could enter the systemic circulation were identified using UPLC-MS/MS. Metabolomic analysis characterized distinctive metabolites and associated pathways, and transcriptomics was used to identify differential genes and determine differential enrichment pathways. Core genes were identified through WGCNA combined with three machine learning algorithms. The interactions between cell subtypes was investigated through immune cell infiltration analysis and single-cell RNA sequencing. Molecular docking and molecular dynamics simulations were performed to screen key genes and core compounds. Subsequently, BEAS-2B cells were stimulated with CS and LPS to establish a COPD cell model, aiming to elucidate the mechanisms underlying the therapeutic effects of the key compounds. Finally, the aforementioned results were integrated to systematically explore the potential mechanism of LGWWJXD in the treatment of COPD. Finally, it was verified through western blotting and RT-PCR.
RESULTS: LGWWJXD alleviated lung inflammation in rats, reduced pathological lung damage to help improve lung function, and reduced cigarette smoke-LPS-low temperature COPD. Metabolomics results showed that the levels of most differential metabolites were normal after LGWWJXD intervention, with arginine biosynthesis and purine metabolism being the main pathways. Transcriptomic analysis revealed that the MAPK signaling pathway plays a pivotal role in the therapeutic effects of LGWWJXD. WGCNA and machine learning algorithms identified two key genes, Bmal1 and Per2. The integration of metabolomics and transcriptomics results revealed both to stem from inflammatory factor release. inflammatory factors. Molecular docking, molecular dynamics simulation, western blotting, and RT-PCR collectively validated the modulation of circadian rhythm-related genes Bmal1, Per2, and Nr1d1 by the potential active components of LGWWJXD, including Asarinin, 6-Shogaol, and Isoliquiritigenin, to inhibit the p38/JNK MAPK signaling pathway and alleviate COPD. In vitro, after intervention of BEAS-2B cells with different concentrations of Asarinin, the levels of TNF-α, IL-1β and IL-6 could be substantially diminished. Meanwhile, we found that the mechanism by which Asarinin exerts its anti-inflammatory effect is to up-regulate the expression of Bmal1 protein, thereby inhibiting the p38/JNK MAPK signaling pathway and thus suppressing the release of inflammatory factors.
CONCLUSIONS: LGWWJXD reduced inflammation in rat lungs, protected against apoptosis, and reduced pathological lung damage to help improve lung function. The potential active ingredients in LGWWJXD, including Asarinin, 6-Shogaol, and Isoliquiritigenin, may regulate the expression of circadian rhythm-related genes, inhibited the p38/JNK MAPK pathway, and jointly suppressed the inflammatory response in combination with arginine biosynthesis and purine metabolism pathways. It lays a scientific foundation for the clinical treatment of COPD with LGWWJXD.
