BACKGROUND: Acute lung injury (ALI) has received considerable attention in the field of critical care as it can lead to high mortality rates. Polygala tenuifolia, a traditional Chinese medicine with strong expectorant properties, can be used to treat pneumonia. Owing to the complexity of its composition, the main active ingredient is not yet known. Thus, there is a need to identify its constituent compounds and mechanism of action in the treatment of ALI using advanced technological means.
PURPOSE: We investigated the anti-inflammatory mechanism and constituent compounds with regard to the effect of P. tenuifolia Willd. extract (EPT) in lipopolysaccharide (LPS)-induced ALI in vivo and in vitro.
METHODS: The UHPLC-Q-Exactive Orbitrap MS technology was used to investigate the chemical profile of EPT. Network pharmacology was used to predict the targets and pathways of action of EPT in ALI, and molecular docking was used to validate the binding of polygalacic acid to Toll-like receptor (TLR) 4. The main compounds were determined using LC-MS. A rat model of LPS-induced ALI was established, and THP-1 cells were stimulated with LPS and adenosine triphosphate (ATP) to construct an in vitro model. Pathological changes were observed using hematoxylin and eosin staining, Wright-Giemsa staining, and immunohistochemistry. The expression of inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) was determined using enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, and western blotting. The LPS + ATP-induced inflammation model in THP-1 cells was used to verify the in vivo experimental results.
RESULTS: Ninety-nine compounds were identified or tentatively deduced from EPT. Using network pharmacology, we found that TLR4/NF-κB may be a relevant pathway for the prevention and treatment of ALI by EPT. Polygalacic acid in EPT may be a potential active ingredient. EPT could alleviate LPS-induced histopathological lung damage and reduce the wet/dry lung weight ratio in the rat model of ALI. Moreover, EPT decreased the white blood cell and neutrophil counts in the bronchoalveolar lavage fluid and decreased the expression of genes and proteins of relevant inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) in lung tissues. It also increased the expression of endothelial-type nitric oxide synthase expression. Western blotting confirmed that EPT may affect TLR4/NF-κB and NLRP3 signaling pathways in vivo. Similar results were obtained in THP-1 cells.
CONCLUSION: EPT reduced the release of inflammatory factors by affecting TLR4/NF-κB and NLRP3 signaling pathways, thereby attenuating the inflammatory response of ALI. Polygalacic acid is the likely compounds responsible for these effects.
