Inhibition of the Il-17/Mapk/Nf-Κb Signaling Axis and Neutrophil Extracellular Traps (Nets) by Hanchuan Zupa Granule in Steroid-Resistant Asthma: Mechanisms and Novel Insights into Nets Inhibitors

Abstract

BackgroundSteroid-resistant asthma (SRA) is a subtype of asthma that exhibits a diminished response to conventional glucocorticoid therapy. There is currently a lack of specific treatment options for SRA. Hanchuan Zupa granule (HCZP), a classic prescription of Uyghur medicine, have a long history in treating asthma. However, whether it has therapeutic effects on SRA has not been reported.PurposeTo evaluate the therapeutic effects of HCZP on SRA, explore its mechanisms, and elucidate its material basis.MethodsWe established SRA mouse model and used dexamethasone as a negative control. The therapeutic effects of HCZP on SRA were evaluated including airway hyperresponsiveness, inflammatory cell infiltration, airway remodeling, and mucus secretion. Ultra Performance Liquid Chromatography-Quadrupole-Electrostatic Field Orbitrap High Resolution Mass Spectrometry (UPLC-QE-Orbitrap-MS) was employed to identified the blood components of HCZP. Based on the prototype components of HCZP, we utilized network pharmacology tools to predict the mechanisms and validated these in both in vivo and in vitro. Subsequently, we performed molecular docking and combined this with in vitro enzyme inhibition assays to explore potential NETs inhibitors.ResultsHCZP significantly reduced airway resistance, the number of neutrophil infiltrations, and alleviated goblet cell hyperplasia and mucus secretion in SRA mice. A total of 65 prototype compounds, 87 phase I metabolites, and 81 phase II metabolites were identified by UPLC-QE-Orbitrap-MS. Based on network pharmacology, HCZP treatment of SRA specifically involved the IL-17/MAPK/NF-κB signaling axis and NETs formation. And it was further confirmed in PMA-induced bone marrow-derived neutrophils and dHL-60 cells. Combined molecular docking with enzyme inhibition assays, 9 potential NETs inhibitors from HCZP were identified.ConclusionHCZP can reduce airway hyperresponsiveness, neutrophil-induced inflammation, and airway remodeling in SRA. The specific mechanisms involved the inhibition of the IL-17/MAPK/NF-κB signaling pathway and the NETs formation. These findings elucidated the material basis of HCZP against SRA and uncovered its underlying mechanisms. Furthermore, they highlighted the potential of specific compounds within HCZP to act as NETs inhibitors.

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Funding declaration: This project was financially supported by National Natural Science Foundation of China (82474186, 31000150), Hubei Provincial Natural Science Foundation Joint Fund Project (JCZRLH202500065) and Open Fund of Engineering Research Center of TCM Protection Technology and New Product Development for the Elderly Brain Health, Ministry of Education.

Conflict of Interests: All authors declare that they have no conflict of interest.

Ethical Approval: All procedures were examined and approved by the Institutional Animal Use and Care Committee at Huazhong University of Science and Technology (Animal experiment ethics No. 3424).

Keywords: Hanchuan Zupa granule, Steroid-resistant asthma, Neutrophil-induced inflammatory response, IL-17/MAPK/NF-κB signaling, NETs formation