Protopine ameliorates OVA-induced asthma through modulatingTLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis

Background: Chronic airway inflammation and hyperresponsiveness are characteristics of asthma. The isoquinoline alkaloid protopine (PRO) has been shown to exert anti-inflammatory effects, but its mechanism of action in asthma is not known.

Purpose: Investigate the protective properties of PRO upon asthma and elucidate its mechanism.

Study design and methods: The effects of PRO in asthma treatment were assessed by histology, biochemical analysis, and real-time reverse transcription-quantitative polymerase chain reaction. Then, we integrated molecular docking, western blotting, cellular experiments, immunohistochemistry, immunofluorescence analysis, flow cytometry, and metabolomics analysis to reveal its mechanism.

Results: In vivo, PRO therapy reduced the number of inflammatory cells (eosinophils, leukocytes, monocytes) in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of IgG and histamine. Molecular docking showed that PRO could DOCK with the proteins of TLR4, MyD88, TRAF6, TAK1, IKKα, and TNF-α. Western blotting displayed that PRO inhibited the TLR4/NF-κB signaling pathway. PRO regulated expression of the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3) inflammasome, gasdermin D, Caspase-1, and drove Caspase-1 inactivation to affect inflammatory responses by inhibiting the NLRP3 inflammasome. In vitro, 24 h after treatment with PRO, cell activity, as well as levels of Reactive Oxygen Species (ROS) and interleukin (IL)-1β and IL-18, decreased significantly. Immunofluorescence staining showed that PRO decreased expression of TLR4 and MyD88 in vitro. PRO decreased nuclear translocation of NF-κB p65. Twenty-one potential biomarkers in serum were identified using metabolomics analysis, and they predominantly controlled the metabolism of phenylalanine, tryptophan, glucose, and sphingolipids.

Conclusion: PRO reduced OVA-induced asthma. The underlying mechanism was associated with the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated Pyroptosis.

Metabolomics; Molecular docking; NLRP3 inflammasome; Protopine; TLR4/NF-κB pathway.