Network pharmacology and experimental validation identify the targets of Marsdenia tenacissima in neutrophilic asthma treatment

Ethnopharmacological relevance: Neutrophilic asthma (NA) refers to an asthma phenotype characterized by significantly elevated levels of neutrophils in induced sputum samples. It is typically associated with a poor response to glucocorticoid therapy and an increased frequency of disease exacerbations. Factors such as pathogen infections and oxidative stress are believed to be associated with the progression of the disease. Marsdenia tenacissima (Roxb.) Wight et Arn (MT), a traditional Chinese medicinal herb, has been extensively utilized for centuries in the treatment of airway inflammatory disorders, particularly asthma. Despite its prolonged clinical application, the specific molecular mechanisms responsible for its therapeutic effects remain inadequately elucidated.

Aim of the study: This study aims to reveal the molecular mechanisms underlying the therapeutic effects of MT in NA by an integrated approach of network pharmacology combined with in vivo and in vitro experiments.

Material and methods: We constructed a mouse model of NA using house dust Mite (HDM) and lipopolysaccharide (LPS). The therapeutic effects of MT on NA were assessed through pulmonary function tests, histology, and enzyme-linked immunosorbent assay (ELISA). Using network pharmacology, we screened the key compounds of MT for treating NA and their potential targets, which were validated through molecular docking and molecular dynamics simulations. Using in vitro cytotoxicity assay, ELISA, real-time quantitative polymerase chain reaction (qPCR), transwell assay, Western blot (WB), cell thermal shift assay (CETSA), and in vivo experiments, we verified the therapeutic effects and potential mechanism of the key compound.

Results: The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the intersection between MT's targets and asthma targets revealed that MT acts on multiple biological processes and pathways related to NA. We demonstrated that MT alleviates asthma symptoms, neutrophil infiltration, and the release of inflammatory cytokines in vitro. Employing network pharmacology, we identified 17β-Tenacigenin B as the key compound of MT, targeting interleukin (IL)-6 and Janus kinase (JAK)1, thereby modulating the JAK/STAT and its downstream PI3K/Akt/mTOR pathway. Molecular docking, molecular dynamics simulations, and CETSA demonstrated stable binding between this compound and its targets. The therapeutic effects of the key compound and the key protein within these pathways were validated in vivo and in vitro. Furthermore, we confirmed the therapeutic effects of MT in NA by suppressing the IL-6/JAK/STAT pathway through the administration of LMT-28, the IL-6 Inhibitor.

Conclusions: This research revealed that MT treats NA through the IL-6/JAK/STAT and PI3K/Akt/mTOR pathways. It is worth noting that this is the first time that network pharmacology has been comprehensively used to explore the mechanism of MT in treating asthma. This finding advances our understanding of the pharmacological mechanisms underlying MT and lends support to its usage as a treatment agent for NA.

IL-6/JAK/STAT; Marsdenia tenacissima; Neutrophilic asthma; PI3K/AKT/mTOR.